WO2023038202A1 - Sustained-release microsphere using biodegradable polymer, and method for preparing same - Google Patents

Abstract

The present invention relates to a sustained-release microsphere using a biodegradable polymer and a method for preparing same and, more particularly, to a microsphere containing entecavir and made of a biocompatible polymer and a method for preparing same. The microsphere containing entecavir according to the present invention exhibits a stable drug release rate for a long period of time and can maintain an effective concentration of entecavir in the blood for a certain period of time, thereby prolonging the administration cycle of a drug, increasing the patient's medication compliance, and alleviating the side effects caused by rapid initial release of a drug.

Description

Sustained-release microspheres using biodegradable polymer and method for preparing the same

This application claims priority to Republic of Korea Patent Application No. 10-2021-0119931 filed on September 8, 2021, and the entire specification is a reference in this application.

The present invention relates to sustained-release microspheres using a biodegradable polymer and a method for preparing the same, and more particularly, to microspheres containing entecavir and made of a biocompatible polymer and a method for preparing the same.

A variety of formulation techniques have been used to provide sustained and controlled release of pharmacologically active substances. Among them, the sustained-release microsphere injection formulation is an injection that is provided in the form of microspheres in which a pharmacologically active substance is encapsulated in a polymer matrix, and is designed so that the drug can be uniformly released from the microspheres when injected subcutaneously or intramuscularly.

In order for the release formulation using such sustained-release microspheres to substantially extend the administration cycle, it is necessary to increase the content of the drug encapsulated in the microspheres as well as to efficiently control the initial release.

Meanwhile, chronic hepatitis B virus (HBV) infection is estimated to affect 240 million people worldwide. The number of new cases of HBV infection has plummeted since the availability of a vaccine against it. However, some areas with very high HBV incidence still exist. Current treatments can control the viral load of HBV to a low level, but it is very difficult to completely eliminate the virus from the body, so hepatitis B requires long-term treatment.

Entecavir is a drug that exhibits very strong anti-HBV efficacy and shows low drug resistance (resistance of 1.2% or less after 5 years of administration), and is selected as a first-line drug for HBV infection. However, it is worth noting that anti-HBV medications must be taken regularly for at least 2 to 3 years until the requirements for termination of treatment are met. Cases of severe hepatitis B infection have been reported in patients who discontinued hepatitis B treatment, including entecavir treatment. Food intake significantly affects the absorption of entecavir from dispersible tablets. Specifically, taking entecavir with food delayed drug absorption (from 0.75 to 1 to 1.5 hours), reduced Cmax by 44% to 46%, and increased AUC (area under the concentration-time curve) by 18 to 20%. Decrease. Therefore, to increase the effectiveness of Entecavir, it must be taken daily on an empty stomach. However, taking Entecavir on an empty stomach causes side effects such as headache, nausea, vomiting, abdominal pain, diarrhea, and indigestion, thereby reducing the patient's compliance with the medication, and as a result, it is difficult to achieve a satisfactory therapeutic effect. In particular, in order to solve these problems, a sustained-release microsphere drug delivery technology that reduces the number of administrations by using an administration route other than oral can be used. However, when the microspheres are prepared by the W/O/W double emulsion method, which is generally used for the preparation of sustained-release microspheres containing Entecavir, there are problems in not only microsphere formation but also encapsulation rate and initial release inhibition.

Therefore, for the treatment of hepatitis B, it is necessary to develop an entecavir microsphere drug delivery system capable of high encapsulation rate and sustained release.

The present inventors have found that Entecavir has a problem in that it is not well encapsulated in microparticles, and the encapsulation rate or initial release rate when microspheres are prepared by the W/O/W double emulsion method commonly used for encapsulation of Entecavir polymer microspheres. After confirming that there is a problem, and researching to improve it, a polar aprotic solvent that can dissolve entecavir and a co-solvent of a solvent that dissolves biocompatible polymers are used to create an O/W single emulsion (O/W single emulsion). The present invention was completed by discovering that the encapsulation rate of Entecavir is significantly improved, rapid initial release is controlled, and a sustainable release formulation can be provided when the microspheres are prepared using the emulsion) method.

Accordingly, an object of the present invention is to provide microspheres containing 0.1 to 50% by weight of entecavir and made of a biocompatible polymer.

Another object of the present invention is (a) preparing a dispersed phase using at least one solvent of entecavir and a biocompatible polymer; (b) forming microspheres by putting the prepared dispersed phase into a continuous phase and stirring; And (c) to provide a method for producing the microspheres, comprising the step of removing the solvent.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hepatitis B containing the microspheres.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hepatitis B comprising the microspheres.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hepatitis B essentially consisting of the microspheres.

Another object of the present invention is to provide a use of the microspheres for preparing a preparation for preventing and treating hepatitis B.

Another object of the present invention is to provide a method for treating hepatitis B comprising administering an effective amount of the composition containing the microspheres to a subject in need thereof.

In order to achieve the above object of the present invention, the present invention provides microspheres containing 0.1 to 50% by weight of entecavir and made of a biocompatible polymer.

In order to achieve another object of the present invention, the present invention provides a dispersion phase comprising: (a) preparing a dispersed phase of entecavir and a biocompatible polymer using one or more solvents; (b) forming microspheres by putting the prepared dispersed phase into a continuous phase and stirring; and (c) removing the solvent.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating hepatitis B containing the microspheres.

In addition, the present invention provides a pharmaceutical composition for preventing or treating hepatitis B comprising the microspheres.

In addition, the present invention provides a pharmaceutical composition for preventing or treating hepatitis B essentially consisting of the microspheres.

In order to achieve another object of the present invention, the present invention provides the use of the microspheres for preparing a preparation for preventing and treating hepatitis B.

In order to achieve another object of the present invention, the present invention provides a method for treating hepatitis B comprising administering an effective amount of a composition containing the microspheres to a subject in need thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides microspheres containing 0.1 to 50% by weight of entecavir and made of a biocompatible polymer.

In the present invention, the entecavir includes free entecavir and pharmaceutically acceptable salts thereof, and refers to the compound represented by Formula 1 below, which exhibits a therapeutic effect on hepatitis B.

[Formula 1]

In the present invention, the "pharmaceutically acceptable salt" means a salt in a form that can be used pharmaceutically among salts in which cations and anions are bonded by electrostatic attraction. salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine It can be a salt with; Salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; A salt with a basic amino acid may be a salt with arginine, lysine, ornithine, and the like; A salt with an acidic amino acid may be a salt with aspartic acid or glutamic acid. For the purpose of the present invention, the pharmaceutically acceptable salt may be interpreted as an acid addition salt or base addition salt of Entecavir suitable for treatment of patients who are expected to develop hepatitis B or have the disease, but are not particularly limited thereto. does not

In one aspect of the present invention, the content of entecavir contained in the microspheres may be 0.1 to 50% by weight. If the content of Entecavir contained in the microspheres exceeds 50% by weight, the initial release amount of Entecavir in the body environment may be too high, resulting in a rapid increase in the blood concentration of the drug. If it is less than %, there may be a problem in that the administration becomes difficult because the ratio of the biocompatible polymer is relatively high and the dosage is excessive.

Preferably, the content of entecavir contained in the microspheres may be 0.1 to 45% by weight, more preferably 5 to 40% by weight, and most preferably 10 to 30% by weight.

The microspheres provided by the present invention mean that the entecavir is encapsulated in microspheres prepared using a biocompatible polymer, and in the present specification, they are simply referred to as entecavir-containing microspheres, entecavir microspheres, or microspheres. As long as entecavir is encapsulated in microspheres prepared using biocompatible polymers, all of them belong to the scope of the present invention without limitation on the type of biocompatible polymer used.

In the present invention, the "biocompatible polymer" refers to a polymer that does not induce high cytotoxicity and inflammatory reaction when administered in vivo, and has secured in vivo safety, and is also simply referred to as a polymer in the present specification. .

In the present invention, the biocompatible polymer may have an intrinsic viscosity of 0.1 to 1.9 dL/g. In the present invention, if the biocompatible polymer has an intrinsic viscosity of less than 0.1 dL/g, the decomposition of the polymer is too fast, and it may be difficult to continuously release entecavir for a desired time. It is too slow, and the release of entecavir is low, so the drug may not be effective.

In the present invention, the biocompatible polymer may be included in an amount of 50 to 99.9% by weight, preferably 55 to 99.9% by weight, more preferably 60 to 95% by weight, and most preferably 70 to 90% by weight based on the total weight of the microspheres. there is. In the present invention, if the biocompatible polymer is included in less than 50% by weight, the distribution of Entecavir is relatively increased, and there may be problems with initial excessive release or inability to maintain the drug effect for a desired period. The amount to be administered may become too large, making administration difficult or impossible.

In one aspect of the invention, the microspheres can provide encapsulated entecavir in a controlled or extended release form. The controlled or extended release form may be understood as the same meaning as "sustained release", "controlled release" or "delayed release".

In one aspect of the present invention, the microspheres can be characterized in that the release of entecavir encapsulated in an in vivo or in vitro environment lasts for 24 hours or longer, preferably for 1 to 12 weeks. there is. In particular, the entecavir contained in the microspheres is 30% by weight or less, preferably 20% by weight or less, more preferably 10% by weight or less within 24 hours after entering the use environment (eg, after administration to the human body). Preferably, less than 5% by weight is released, and the remainder can be characterized in that it is continuously released after 24 hours, preferably for a period of 1 to 12 weeks.

In one aspect of the present invention, the release of entecavir encapsulated in the microspheres in vivo or in vitro can be characterized in that the release lasts for 24 hours or longer, preferably for 1 to 5 weeks. there is. In particular, the entecavir contained in the microspheres is 30% by weight or less, preferably 20% by weight or less, more preferably 10% by weight or less within 24 hours after entering the use environment (eg, after administration to the human body). Preferably, less than 5% by weight is released, and the remainder can be characterized in that it is continuously released after 24 hours, preferably for a period of 1 to 5 weeks.

In one aspect of the present invention, the microspheres containing Entecavir are prepared by solvent evaporation or solvent extraction through an emulsion, more preferably, an oil-in-water (O/W) emulsion containing a biocompatible polymer, Entecavir, and a dispersing solvent. It may be produced by an O / W type solvent evaporation method of preparing and aggregating them into fine particles.

More specifically, in order to prepare an O/W emulsion and aggregate it into polymer fine particles, an O/W emulsion containing a biocompatible polymer, entecavir, and a dispersing solvent is first prepared.

A conventional method known in the art may be used for the preparation of the O/W type emulsion. More specifically, for the preparation of the O/W type emulsion, a dispersed phase containing a biocompatible polymer and entecavir is added to a dispersion solvent can be manufactured

These entecavir-containing polymer microparticles are prepared by coagulating emulsion into microparticles by solvent evaporation or solvent extraction, or by ammonolysis or hydrolysis. In the case of the ammonolysis process, the addition of ammonia, and in the case of the hydrolysis process, the addition of acid or base causes ammonolysis or hydrolysis reaction to convert the water-insoluble organic solvent into a water-soluble solvent. included

The solvent evaporation method is not limited thereto, but, for example, the method described in U.S. Patent Nos. 6,471,996, 5,985,309, and 5,271,945, etc., after dispersing or dissolving a drug in an organic solvent in which a polymer compound is dissolved, followed by dispersing medium such as water After emulsifying to prepare an O/W type emulsion, the organic solvent in the emulsion is diffused into a dispersion medium to evaporate the organic solvent through an air/water interface, thereby forming entecavir-containing polymer microspheres.

The solvent extraction method includes a conventional solvent extraction method used in the preparation of entecavir-containing polymeric microspheres, such as effectively extracting the organic solvent in emulsion droplets using a large amount of solubilizing solvent.

In addition, a method of simultaneously applying a solvent evaporation method and a solvent extraction method, for example, methods described in U.S. Patent Nos. 4,389,840, 4,530,840, 6,544,559, 6,368,632 and 6,572,894, etc. are included.

Aggregation by the ammonelysis process induces ammonialysis by adding ammonia to an O/W emulsion containing a water-insoluble organic solvent, as in the method described in Korean Patent No. 918092, for example, and converting the water-insoluble organic solvent into a water-soluble solvent. It shows how to agglomerate microparticles by converting to .

Aggregation by the hydrolysis process is a base such as NaOH, LiOH, KOH in an O/W emulsion containing a water-insoluble organic solvent, such as the method described in Korean Patent Application Nos. 2009-109809 and 2010-70407. Alternatively, hydrolysis, which is a kind of hydrolysis reaction of esters, is induced by adding an acid solution such as HCl or H2SO4, and the water-insoluble organic solvent is converted into a water-soluble solvent to aggregate fine particles.

The present invention also comprises the steps of (a) preparing a dispersed phase using at least one solvent of entecavir and a biocompatible polymer; (b) forming microspheres by putting the prepared dispersed phase into a continuous phase and stirring; and (c) removing the solvent.

Step (a) is a step of preparing a dispersed phase containing entecavir and a biocompatible polymer. In step (a), Entecavir is used in an amount of 0.1 to 50 parts by weight, preferably 0.1 to 45 parts by weight, more preferably 0.1 to 40 parts by weight, and most preferably 0.1 to 30 parts by weight, based on 100 parts by weight of the biocompatible polymer. It can be dispersed or dissolved in parts. The type of solvent used for preparing the organic phase is not particularly limited, but dichloromethane, chloroform, and the like may be used.

In addition, the polymer compound of step (a) is polylactic acid, polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, from the group consisting of polyanhydrides, polyorthoesters, copolymers of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalate, polyhydroxybutyrate, polyamino acids, copolymers of lactic acid and amino acids and mixtures thereof may be selected, most preferably polylactide-co-glycolide (PLGA).

In the step (a), the solvent is not particularly limited as long as it can dissolve the biocompatible polymer, and may preferably be a non-aqueous solvent. In the present invention, non-limiting examples of the non-aqueous solvent include dichloromethane, chloroform, acetonitrile, dimethyl sulfoxide, dimethylformamide, ethyl acetate and the like, but are not limited thereto.

In one aspect of the present invention, the solvent in step (a) may include at least one polar aprotic solvent as a co-solvent. The polar aprotic solvent may be selected from the group consisting of ether-based solvents, ketone-based solvents, amide-based solvents, cyan-based solvents and sulfoxide-based solvents. Preferably, the polar aprotic solvent is an ether-based solvent such as diethyl ether, dipropyl ether, dibutyl ether, butyl ethyl ether, tetrahydrofuran, or the like, or a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, or cyclohexanone. amide-based solvents such as N-methyl-2-pyrilidinone, 2-pyrilidinone, N-methylformamide, N,N-dimethylformamide, cyan-based solvents such as acetonitrile and benzonitrile, and dimethyl sulfoxide , methyl phenyl sulfoxide, diphenyl sulfoxide, p-tolyl sulfoxide, 4-chlorophenyl sulfoxide, methyl p-tolyl sulfoxide, butyl sulfoxide and the like.

In the present invention, the solvent in step (a) includes a polar aprotic solvent as a co-solvent. Dimethyl sulfoxide is used as the polar aprotic solvent. The dimethylsulfoxide content of the dispersed phase in step (a) may be 10 to 90%, preferably 20 to 80%, more preferably 30 to 70%, and most preferably 40 to 60%. .

In addition, dimethyl sulfoxide, a polar aprotic solvent, and methylene chloride, an organic solvent, are used as solvents to prepare the dispersed phase in step (a). When the ratio of the solvent is methylene chloride to 5, the ratio of dimethyl sulfoxide is 1 to 9, preferably 2 to 8, more preferably 3 to 7, and most preferably 4 to 6.

Step (b) is a step of solidifying the microspheres by dispersing the dispersed phase prepared in step (a) in an external continuous phase to prepare an emulsion solution (O/W).

In the step (b), a hydrophilic polymer may be included as a surfactant, and the type thereof is not particularly limited, and a dispersed phase containing Entecavir and a biocompatible polymer may form a stable liquid droplet dispersed phase in the external continuous phase. Anything that can help with this can be used. The hydrophilic polymer is preferably methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyvinyl alcohol, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene sorbitan fatty acid ester and polyoxyethylene It may be selected from the group consisting of castor oil derivatives and mixtures thereof, and most preferably may be polyvinyl alcohol.

In step (b), the external continuous phase may be a 0.1 to 5% (w/w) aqueous solution of a hydrophilic polymer, in which case the molecular weight of the hydrophilic polymer may be 10,000 to 30,000, and the degree of hydrolysis may be 80 to 90%. .

In the step (b), the dispersed phase containing the entecavir and the biocompatible polymer prepared in the step (a) is drop-by-drop or by using an in-line mixer to form an external continuous phase containing the hydrophilic polymer. and vigorously stirred to prepare an emulsion solution (O/W). In this process, the entecavir is encapsulated into biocompatible polymeric microspheres.

Then, in the step (c), the solvent may be removed, and the desired microspheres may be obtained after conventional filtration and washing. That is, a step of washing the obtained microspheres with an organic solvent such as ethanol may be included to enhance the initial release suppression effect, if necessary.

In one aspect of the present invention, the weight of Entecavir encapsulated in the microspheres obtained according to the preparation method may be 30% or more of the weight of Entecavir dissolved in step (a). That is, the encapsulation rate of entecavir in microspheres may be 30% or more.

In another aspect of the present invention, the weight of Entecavir encapsulated in the microspheres obtained according to the manufacturing method may be 50% or more of the weight of Entecavir dissolved in step (a). That is, the encapsulation rate of entecavir in microspheres may be 50% or more.

In another aspect of the present invention, the weight of Entecavir encapsulated in the microspheres obtained according to the manufacturing method may be 70% or more of the weight of Entecavir dissolved in step (a). That is, the encapsulation rate of entecavir in microspheres may be 70% or more.

In another aspect of the present invention, the weight of Entecavir encapsulated in the microspheres obtained according to the manufacturing method may be 90% or more of the weight of Entecavir dissolved in step (a). That is, the encapsulation rate of entecavir in microspheres may be 90% or more.

The present invention also provides a pharmaceutical composition for preventing or treating hepatitis B containing the microspheres.

The pharmaceutical composition according to the present invention may contain the microspheres alone or may be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may further contain an excipient or diluent. Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

A pharmaceutically acceptable carrier may further include, for example, a carrier for parenteral administration. The carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, and the like in addition to the above components. Reference may be made to other pharmaceutically acceptable carriers and agents known in the art.

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration can be

The pharmaceutical composition of the present invention can be formulated as a preparation for parenteral administration according to the administration route as described above.

In the case of preparations for parenteral administration, they may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in prescriptions generally known to all pharmaceutical chemists.

The total effective amount of the composition of the present invention can be administered to the patient in a single dose or by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per kg of patient body weight per day. However, the dose of the pharmaceutical composition is determined by considering various factors such as the formulation method, administration route, and number of treatments as well as the patient's age, weight, health condition, sex, severity of disease, diet, and excretion rate. Therefore, considering this point, those skilled in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it exhibits the effects of the present invention.

The present invention provides the use of the microspheres for preparing a preparation for preventing and treating hepatitis B.

The present invention provides a method for treating hepatitis B comprising administering an effective amount of a composition comprising the microspheres to a subject in need thereof.

The 'effective amount' of the present invention refers to an amount that exhibits an effect of improving, treating, preventing, detecting, diagnosing, or suppressing or reducing the disease of hepatitis B when administered to a subject, and the 'subject' refers to an animal, preferably may be mammals, especially animals including humans, and may be cells, tissues, organs, etc. derived from animals. The subject may be a patient in need of the effect.

The 'treatment' of the present invention refers comprehensively to improving hepatitis B or the symptoms of hepatitis B, which may include curing, substantially preventing, or improving the condition of hepatitis B, Alleviating, curing or preventing one or most of the symptoms resulting from the disease, but is not limited thereto.

In this specification, the term "comprising" is used in the same meaning as "including" or "characterized by", and in the composition or method according to the present invention, specifically mentioned It does not exclude additional components or method steps not specified. Also, the term "consisting of" means excluding additional elements, steps or components not separately described. The term "essentially consisting of" means that in the scope of a composition or method, in addition to the described materials or steps, materials or steps that do not substantially affect the basic characteristics thereof may be included.

Entecavir-containing microspheres according to the present invention exhibit stable drug release for a long period of time and can maintain an effective concentration of Entecavir in the blood for a certain period of time, thereby extending the drug administration cycle, increasing patient compliance, and rapidly taking drugs. Side effects caused by early release can be alleviated.

1 is a result of observing the shape of the microspheres prepared in Example 1 and Comparative Example 1 of the present invention with a scanning electron microscope (Scanning electron microscopy, SEM).

Figure 2 shows the experimental results of evaluating the initial release rate of Entecavir of the microspheres prepared in Example 1 and Comparative Example 2.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1: Preparation of microspheres containing entecavir

Dissolve 0.35 g of Entecavir in 5.82 g of DMSO, dissolve 1.4 g of a biocompatible polymer (Resomer RG 504H, I.V.: 0.45-0.63, Durect 8A, I.V.: 0.7-0.9) in 5.82 g of methylene chloride, and mix them using a homogenizer. The dispersed phase was completed. As the continuous phase, an aqueous solution of 3% polyvinyl alcohol (molecular weight: 1,3000-23,000) was used. 1,000ml of the continuous phase was put into a production tank to be maintained at 25° C., and the prepared dispersed phase was injected using an in-line mixer to prepare microspheres. After stirring at 1,000 rpm, the organic solvent was removed for 24 hours. After washing the prepared microspheres several times with water for injection, residual polyvinyl alcohol was removed and the microspheres were lyophilized.

Comparative Example 1: Preparation of microspheres containing entecavir

After dissolving 0.35 g of Entecavir and 1.4 g of a biocompatible polymer (Resomer RG 504H, I.V.: 0.45-0.63) in 14.55 g of methylene chloride, they were mixed using a homogenizer to complete the dispersed phase. As the continuous phase, an aqueous solution of 0.5% polyvinyl alcohol (molecular weight: 1,3000-23,000) was used. 1,000 ml of the continuous phase was put into a production tank and set to 25° C., and the prepared dispersed phase was injected using an in-line mixer to prepare microspheres, but it was confirmed that microspheres were not formed.

Experimental Example 1: Microsphere shape measurement

In order to observe the shape of the microspheres prepared in Example 1 and Comparative Example 1, they were analyzed by scanning electron microscopy (SEM). After fixing about 10 mg of the microspheres on an aluminum stub, the surface of the microspheres was observed using an SEM. All images were observed with a 10 KeV electron beam at a magnification of about 500X.

The results of measuring the morphology of microspheres are shown in FIG. 1 .

It was confirmed that the microspheres prepared in Example 1 were spherical without pores, but the microspheres according to the method of Comparative Example 1 were not observed because no spherical shape was formed.

Experimental Example 2: Measurement of entecavir loading in microspheres

About 20 mg of the microspheres prepared in Example 1 was taken and placed in a 25 mL volumetric flask, followed by Sonication with 2.5 mL of acetonitrile (manufacturer: Honeywell) for 10 minutes, distilled water was added to about 90% of the volume of the volumetric flask, and then Sonication was performed for 10 minutes . Thereafter, the sample was leveled with distilled water and filtered through a 0.45um syringe filter. This solution was put in a vial and detected with a UV detector using HPLC (equipment name: Agilent). The column packing was L1, the inner diameter was 4.6 mm x 150 mm, and the thickness was 4 μm.

The results for this are shown in Table 1 below.

	Enclosure rate (%)	Actual loading (%)
Example 1	93.56	14.48

Microspheres according to the method of Comparative Example 1 were not formed, so the encapsulation rate and loading amount could not be measured.

Experimental Example 3: Measurement of residual solvent amount

About 200 mg of the microspheres prepared in Example 1 were taken and placed in a 5 mL volumetric flask, and sonication was performed with 3 mL of an internal standard solution of toluene for 10 minutes. After adjusting the mark with toluene internal standard solution, 4 mL was taken, put into a head space vial, sealed, and detected with a spark ionization detector using Agilent 8890 gas chromatograph/Head Space 7697A equipment. The mobile phase was He, and the column was G43 0.53 mm x 30 m, 3 μm film thickness.

The results for this are shown in Table 2 below.

	Residual DCM amount (ppm)	Residual DMSO amount (ppm)
Example 1	70	2,406

It was confirmed that the entecavir microspheres prepared according to the present invention were suitable for the residual solvent amount of 600 ppm or less, which is the standard value of DCM, and 5,000 ppm or less, which is the standard value of DMSO.

Experimental Example 4: In vitro release test and initial release rate evaluation

About 10 mg of the microspheres prepared in Example 1 was put into a vial, and 8 mL of the release solution was stirred at 100 rpm and maintained at 37 ° C. In order to measure the release amount at a certain time, after centrifugation, the supernatant was taken and filtered through a 0.45um syringe filter. This solution was put in a vial and detected with a UV detector using HPLC (equipment name: Agilent). The column packing was L1, the inner diameter was 4.6 mm x 150 mm, and the thickness was 4 μm.

As shown in Figure 2, it was confirmed that only 3% of the microspheres prepared in Example 1 was released within 24 hours, and the release continued for more than 20 days.

Since microspheres according to the method of Comparative Example 1 were not formed, the release rate could not be measured.

The entecavir-containing microspheres according to the present invention exhibit stable drug release for a long period of time and can maintain an effective concentration of entecavir in the blood for a certain period of time, thereby extending the drug administration cycle, increasing patient compliance, and rapidly taking drugs. Side effects caused by the initial release can be reduced, so industrial applicability is high.

Claims (14)

1. Microspheres containing 0.1 to 50% by weight of Entecavir and made of a biocompatible polymer.
2. The microspheres according to claim 1, wherein the microspheres release entecavir for 24 hours or longer.
3. The microspheres according to claim 1, wherein the entecavir contained in the microspheres is released in an amount of 30% by weight or less within 24 hours after entering the use environment.
4. The method of claim 1, wherein the biocompatible polymer is polylactic acid, polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester , polyanhydrides, polyorthoesters, copolymers of lactic acid and caprolactone, polycaprolactone, polyhydroxyvalate, polyhydroxybutyrate, polyamino acids, copolymers of lactic acid and amino acids, and mixtures thereof Microspheres, characterized in that selected from.
5. The microspheres according to claim 1, wherein the microspheres are prepared by an oil-in-water (O/W) solvent evaporation method or a solvent extraction method containing a biocompatible polymer, entecavir, and a dispersion solvent.
6. (a) preparing a dispersed phase of entecavir and a biocompatible polymer using one or more solvents;

   (b) forming microspheres by putting the prepared dispersed phase into a continuous phase and stirring; and

   (c) a method for producing microspheres according to any one of claims 1 to 5, comprising the step of removing the solvent.
7. The method of claim 6, wherein in step (a), entecavir is dispersed or dissolved in an amount of 0.1 to 50 parts by weight based on 100 parts by weight of the biocompatible polymer.
8. The method of claim 6, wherein the solvent in step (a) includes a polar aprotic solvent as a co-solvent.
9. The method according to claim 6, further comprising washing the microspheres obtained after step (c).
10. The method according to claim 6, wherein the weight of Entecavir encapsulated in the microspheres obtained by the method is 30% or more of the weight of Entecavir dissolved in step (a).
11. A pharmaceutical composition for preventing or treating hepatitis B comprising microspheres according to any one of claims 1 to 5.
12. The pharmaceutical composition according to claim 11, wherein the composition is for injection.
13. Use of the microspheres according to any one of claims 1 to 5 for the manufacture of a formulation for prophylaxis and treatment of hepatitis B.
14. A method for treating hepatitis B comprising administering to a subject in need thereof an effective amount of a composition comprising microspheres according to any one of claims 1 to 5.

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