WO2013066028A1

WO2013066028A1 - Improved-safety entecavir-containing pharmaceutical composition and production method therefor

Info

Publication number: WO2013066028A1
Application number: PCT/KR2012/009005
Authority: WO
Inventors: 모세명; 이유진; 송세현
Original assignee: 대원제약주식회사
Priority date: 2011-11-01
Filing date: 2012-10-30
Publication date: 2013-05-10
Also published as: KR101439635B1; KR20130048165A

Abstract

The present invention relates to an improved-safety entecavir pharmaceutical composition and to a production method therefor. Provided are: an improved-safety entecavir pharmaceutical composition containing entecavir and cyclodextrin; and a production method therefor.

Description

[Correction 14.12.2012] according to Rule 26 약 Pharmaceutical composition containing entecavir with improved stability and preparation method thereof

The present invention relates to a pharmaceutical composition of entecavir with improved stability, and more particularly, to a pharmaceutical composition of entecavir with improved stability containing entecavir and cyclodextrin, and a method for preparing the same. Furthermore, the present invention provides a method for reducing the flexible material when granulating entecavir by the wet granulation method.

Entecavir (ETV) is a 2-amino-1,9-dihydro-9-[(1S, 3R, 4S) -4-hydroxy-3-hydroxymethyl-2-methylenecyclopentyl] -6H-purine -6-one (2-amino-1,9-dihydro-9-[(1S, 3R, 4S) -4-hydroxy-3- (hydroxymethyl) -2-methylenecyclopentyl] -6H-purin-6-one) It is well known for the treatment of chronic hepatitis B virus with selective activity against hepatitis B virus. Entercavir is a known substance disclosed in US Pat. No. 5,206,244 for its preparation and pharmaceutical use, and has been licensed for tablets and liquids from the US FDA.

On the other hand, in general, the method for producing a solid preparation such as tablets and capsules proceeds through the process of mixing, granulating, and compressing the main ingredient and the excipient. Mixing is a process for homogeneously mixing the main component and the excipient, and granules are a process of providing flowability for the compression process to facilitate compression and to prevent separation of the main component from the mixture. In addition, when the mixing of the main component is not homogeneous in the mixing step, it is possible to increase the mixing uniformity of the main component through the granulation step after the mixing step.

There are dry granulation method and wet granulation method in granulation process. Dry granulation method is a method of granulating by compressing a mixture of main ingredients and excipients in solid state without using a solvent, and wet granulation method is mainly a solvent in a mixture of main ingredients and excipients. It is a method of granulation by adding. Dry granulation method of the granulation process has the advantage of improving the stability of the drug unstable in a solvent such as water, but has a disadvantage of long working time. In contrast, the wet granulation method has the advantage of increasing the mixing uniformity of the main ingredients by dissolving the main ingredients in the solvent when the working time is short and the amount of the main ingredients is low. The process has the disadvantage of reducing the stability of the drug.

The granulation process is particularly important when the mixing is not performed smoothly or the amount of the main ingredient is small due to the difference in physical properties between the main ingredient and the excipient. For formulations containing relatively small amounts of the principal component relative to the amount of excipients, more specifically formulations containing low doses of the principal component with an excipient ratio of about 1/50 (w / w) or less, very important. In this case, by using the wet granulation method during the granulation process, the main component may be dissolved in a solvent and mixed with an excipient to ensure uniform mixing of the main component.

In the case of using the wet granulation method in preparing a pharmaceutical composition containing entecavir, a process of dissolving entecavir in a solvent to prepare a binding solution is required. However, when entecavir is present in a dissolved state in a solvent such as water, the formation of a flexible substance is increased, and thus stability is lowered when prepared as a binder liquid. In this invention, it is a 1st subject to increase the stability with respect to the solvent of entercavir and to reduce generation | occurrence | production of a flexible substance. Furthermore, in order to increase the efficiency of the process, it may be necessary to increase the temperature of the solvent in the preparation of the binder solution. At this high temperature, the generation of the flexible substance of entecavir is further increased. Accordingly, in the present invention, it is a second problem to improve the stability of enticavir even when using a solvent of high temperature to reduce the generation of the flexible substance.

In order to solve the above problems, the present invention provides a pharmaceutical composition with improved stability containing entecavir and cyclodextrin.

In the present invention, in the pharmaceutical composition, the ratio of entecavir and cyclodextrin is provided in a molar ratio of 1:01 or more provides a pharmaceutical composition.

Furthermore, in the present invention, in granulating entecavir by a wet granulation method, a method for reducing a flexible substance is provided by dissolving entecavir and cyclodextrin in a solvent to prepare a binder solution.

In addition, the present invention, (a) dissolving entecavir, cyclodextrin in water or other solvent to prepare a binder solution; (b) injecting a pharmaceutically acceptable excipient into the mixer and mixing; (c) injecting the binder solution prepared in step (a) into the mixer to prepare granules; (d) drying the granules; (e) mixing the dried granules with a pharmaceutically acceptable excipient to prepare a mixture; And (f) preparing the mixture in step (e) into tablets, capsules, powders, or granules, thereby providing a pharmaceutical composition comprising enticavir and cyclodextrin.

According to the present invention, it is possible to reduce the amount of the flexible material in the manufacturing process, it is possible to stabilize the active ingredient unstable to moisture.

Figure 1. Stability test results in the aqueous solution of entecavir according to the use of cyclodextrin

Figure 2. Stability test results in entecavir aqueous solution (room temperature) according to the amount of cyclotextine

Figure 3. Stability test results in an aqueous solution of entecavir (90 ℃) according to the amount of cyclodextrin used

4. Stability test results in the aqueous solution of entecavir according to the type of additive

5. Stability test results in the aqueous solution of entecavir according to the combined use of cyclodextrin and other additives

6. Stability test results of tablets containing cyclodextrins and tablets not containing cyclodextrins according to the present invention.

Entecavir (ETV) is 2-amino-1,9-dihydro-9-[(1S, 3R, 4S) -4-hydroxy-3-hydroxymethyl-2-methylenecyclopentyl] -6H-purin-6-one (2-amino-1,9-dihydro-9-[(1S, 3R, 4S) -4-hydroxy-3- (hydroxymethyl) -2-methylenecyclopentyl] -6H-purin-6 -one), which is well known for the treatment of chronic hepatitis B virus with selective activity against hepatitis B virus.

[Formula 1]

According to the U.S. Patent No. 5,206,244, the dose of entecavir may vary depending on the degree of infection, but it is preferable to set it at 1.0 to 50 mg / kg, which can be administered several times at appropriate intervals per day. It is. In the present invention, however, a pharmaceutical composition containing an administration dose of entecavir of 1 mg / day or less is provided as a preparation for once-daily administration, and thus a medicament containing a low dose of entecavir as an active ingredient. It focuses on the special problems that arise in pharmaceutical preparations. That is, when the daily dose of entecavir is 1 mg / day or less, for example, when the pharmaceutical composition is a tablet, even if the weight of the tablet is minimized to 50 mg, 1 mg per tablet (1/50) (w / w)), the main component is used so that the amount of the main component is very small compared to the tablet weight, so it is preferable to dissolve the main component in a solvent and granulate by wet granulation method.

However, according to the researches of the present inventors, when preparing a pharmaceutical composition containing entecavir through a wet granulation method, the stability of entecavir in the process of dissolving entecavir in a solvent decreases the flexibility of entecavir in the binder solution. I found out. This means that in order for pharmaceuticals to always exhibit a certain effect, the reduction of the content of the active ingredient in the manufacturing process, immediately after the manufacture and during the storage period of the product should be suppressed, as well as an increase in the decomposition products of the active ingredient, i.e., impurities or soft substances, during the same period. Considering that it is important to keep it to a minimum, it is obvious that this is a serious problem. In particular, entecavir contained as an active ingredient of the present invention is unstable against heat and moisture, and because it is used at a low content, it requires attention to handling, and in view of the fact that a highly stable formulation is required when formulating. It is clear that the problem of stability is more highlighted.

Thus, the present inventors confirmed that cyclodextrin plays a role of improving the stability of entecavir in the binding solution when a specific additive, ie, cyclodextrin, is added in preparing the binding solution by dissolving enticavir in a solvent. The invention has been reached, and this is the first technical feature of the present invention.

On the other hand, the inventors have confirmed that the flexible material increases significantly when entecavir is exposed to a solvent such as water for a long time. Therefore, it was confirmed that it is important to shorten the process time when preparing the granules by the wet granulation method, a method of increasing the temperature of the solvent may be considered. However, in the process of raising the temperature as described above, another problem may occur, in which the generation of the flexible material of entecavir is increased. In this regard, the present inventors have found that adding cyclodextrin to a solvent, there is no problem in the stability of entecavir even with a high temperature solvent, i. Is obtained as the second technical feature of the present invention.

Accordingly, a first technical feature of the present invention is to improve the stability of entecavir by adding cyclodextrin to a solvent in preparing a pharmaceutical composition of entecavir by using a wet granulation method, and a second technical feature is entecavir In preparing a pharmaceutical composition using a wet granulation method, the present invention relates to a method for improving the stability of entecavir even by using a solvent having a high temperature by adding cyclodextrin to a solvent.

Furthermore, it was found that the pharmaceutical composition of entecavir prepared according to the present invention greatly reduces the formation of the flexible substance not only in the manufacturing process but also in the stability test for the final product after manufacture, and thus the stability is improved. Pharmaceutical compositions of entecavir also fall within the scope of the present invention.

The cyclodextrin used in the present invention is a cyclic oligosaccharide having 6 to 12 glucoses each having α-1,4 glycosidic bonds, and is divided into α, β, and γ-cyclodextrins according to the number of glucose, and the structure thereof is as follows. Same as the formula (2).

[Formula 2]

[Revision 14.12.2012 under Rule 26]

Cyclodextrins are known to form inclusion complexes with organic compounds, and these properties are used mainly as solubilizers in the pharmaceutical field, but there is no report that can improve the stability of entecavir. In addition, the amount of cyclodextrin used in the present invention is preferably 1: 0.1 or more as molar ratio of entecavir: cyclodextrin, and may be used up to 1: 200, but depending on the type and amount of other excipients, the ratio of entecavir and cyclodextrin may be adjusted. You can adjust the enemy. Meanwhile, in the pharmaceutical composition of the present invention, a pharmaceutically acceptable excipient, a binder, a disintegrant, a lubricant, a controlled release additive, etc. may be used together with entecavir and cyclodextrin. Such materials include polyethylene glycol, polyvinylprolidone, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, guar gum, xanthan Gum, lactose, microcrystalline cellulose, calcium phosphate, dextrin, dextrose, dextrate, mannitol, sorbitol, sucrose, crospovidone, croscarmellose, sodium glycolate starch, pregelatinized starch and corn starch, magnesium stearate , Stearic acid, sodium stearyl fumarate, lactohydrate, sodium lauryl sulfate and the like. On the other hand, the inventors have found that the effect of reducing the soft matter of the cyclodextrin is not limited by the addition of the above materials. For example, additives such as PEG6000, PVP, Copovidone, HPMC, and HPC, when dissolved together with entecavir in a solvent, tend to affect the stability of entecavir and significantly increase the amount of analogues. It is possible to greatly reduce this by adding.

Furthermore, in the present invention, in granulating entecavir by a wet granulation method, a method for reducing a flexible substance is provided by dissolving entecavir and cyclodextrin in a solvent to prepare a binder solution. In addition, as described above, when heating the temperature of the solvent, for example, when using a solvent of 50 ~ 90 ℃ can reduce the flexible material of entecavir. Therefore, when entecavir is a solid preparation by the wet granulation method, when using the method of the present invention, it will be possible to implement to reduce the flexible material in various pharmaceutical compositions without being limited to a specific formulation.

Finally, the present invention provides a method for preparing a solid preparation of entecavir, specifically, (a) dissolving entecavir, cyclodextrin in water or other solvent to prepare a binder solution; (b) injecting a pharmaceutically acceptable excipient into the mixer and mixing; (c) preparing granules by adding the binding solution prepared in step (a) into the mixer; (d) drying the granules; (e) mixing the dried granules with a pharmaceutically acceptable excipient to prepare a mixture; And (f) provides a method for producing a solid formulation comprising enticavir and cyclodextrin consisting of the step of preparing the mixture in step (e) tablets, capsules, powders, granules. In the above method, in step (a), water may be used as a solvent, or other solvents such as alcohols such as methanol, ethanol, isopropyl alcohol, acetonitrile, and the like may be used. It is possible to dissolve entecavir and cyclodextrins while warming to 90 ° C, for example 60 ° C. In addition, the pharmaceutically acceptable excipient in step (b) may include lactose monohydrate, microcrystalline cellulose, crospovidone, povidone, and the drying in step (d) may be appropriately set and is not limited. For example, it can carry out at about 60 degreeC. The present invention will be described through the following examples. However, the following examples are not intended to limit the scope of the present invention as an exemplary embodiment of the present invention, various modifications may exist within the spirit of the present invention.

Experimental Example 1. Stability test in aqueous solution of entecavir depending on the use of β-cyclodextrin

In order to confirm the stability in the aqueous solution of entecavir according to the use of β-cyclodextrin was performed as follows. As shown in [Table 1] and [Fig. 1], water at room temperature was added to a sample obtained by using only entecavir alone, and a sample obtained by entecavir and β-cyclodextrin in a 1: 2 mol ratio. The degree of formation of the flexible substance was confirmed at.

When entecavir is present in water alone, it was confirmed that the formation of the flexible substance was about 2 times greater at 1 hour than when entecavir and β-cyclodextrin were present at a molar ratio of 1: 2. In addition, when entecavir alone was present in water for a long time (8 hours), the formation of analogue material increased by about 50% or more, and when entecavir and β-cyclodextrin were present in water for a long time at a molar ratio of 1: 2, As a result, the formation of lead substances increased about 30%.

Therefore, it can be seen that the entecavir and β-cyclodextrin mixtures according to the present invention improve the stability in the aqueous solution of entecavir.

Table 1

Example	Item (molar ratio)	ppm
Example	Item (molar ratio)	0 hr	1 hr	4 hr	8 hr
Control Example 1	ETV	0	579	732	879
Example 2	ETV: β-CD (1: 2)	0	243	279	319

(ppm: flexible material unit)

The analysis method of lead substance is as follows.

(1) Standard Solution Preparation

20 mg of entecavir was taken into a 100 ml volumetric flask and labeled with water.

(2) sample preparation

20 mg of entecavir is taken and placed in a 100 ml volumetric flask, which is labeled by adding water at room temperature to Comparative Example 1. 20 mg of entecavir and 144 mg of β-CD are taken into a 100 ml volumetric flask, and water is added at room temperature to make a sample.

The amount of analog in the aqueous solution of entecavir was analyzed under the following conditions. High performance liquid chromatography (HPLC) was used for quantitative analysis under the following conditions to calculate the content of lead substances.

40 column: 4.6 mm × 25 cm, 5 um L1 packing or similar column

Flow rate: 1.0 ml / min

Analysis wavelength: 254 nm

Injection volume: 10 ul

Mobile phase A: acetonitrile / water = 3/97

Mobile Phase B: Acetonitrile = 100

-The mobile phase concentration gradient is shown in [Table 2].

TABLE 2

time	Mobile phase A	Mobile phase B
0	100	0
8	100	0
50	77	23
75	13	83
75.1	100	0
90	100	0

Experimental Example 2. Stability test in aqueous solution of entecavir (room temperature) according to the amount of β-cyclodextrin

In order to determine the stability in the aqueous solution of entecavir according to the amount of β-cyclodextrin was used as follows. As shown in [Table 3] and [Fig. 2], the ratio of entecavir and β-cyclodextrin was 1: 0.01, 1: 0.05, 1: 0.1, 1: 0.25, 1: 0.5, 1: 1, 1: 2, Water was added at a mol ratio of 1: 3, 1: 4, 1; 5, 1:10, 1: 100, 1: 200, and water was added.

β-cyclodextrin and entecavir resulted in a significant decrease in the formation of lead substances at mol ratios of more than 1: 0.1, and it was confirmed that the reduction effect of lead substances was excellent at mol ratios of more than 1: 0.1.

Therefore, it can be seen that the entecavir and β-cyclodextrin mixtures according to the present invention have a great effect of improving the stability in the aqueous solution of entecavir when used in a ratio of 1: 0.1 or more.

TABLE 3

Example	Item	mol ratio	0 hr	1 hr
Example	Item	(ETV: β-CD)	0 hr	ppm	%
Control Example 1	ETV	One	0	579	-
Example 3	ETV: β-CD	1: 0.01	0	542	93.6
Example 4		1: 0.05	0	517	89.3
Example 5		1: 0.1	0	295	50.9
Example 6		1: 0.25	0	298	51.5
Example 7		1: 0.5	0	287	49.6
Example 8		1: 1	0	258	44.6
Example 2		1: 2	0	243	42.0
Example 9		1: 3	0	251	43.4
Example 10		1: 4	0	249	43.0
Example 10-1		1: 5	0	245	42.3
Example 10-2		1: 10	0	240	41.4
Example 10-3		1: 100	0	228	39.3
Example 10-4		1: 200	0	220	37.9

(ppm: unit of lead substance,%: rate of lead substance compared to CD not included)

The analysis method of lead substance is as follows.

(1) Standard Solution Preparation

(2) sample preparation

20 mg of entecavir and β-CD, respectively, 0.72, 3.6, 7.2, 18, 36, 72, 144, 21 6, 288 mg each were taken in a 100 ml volumetric flask and added with water at room temperature, respectively. , 4, 5, 6, 7, 8, 2, 9, 10 (Example 10 includes Example 10-1 to Example 10-4).

Experimental Example 3. Stability test in aqueous solution of entecavir (90 ° C) according to the amount of β-cyclodextrin used

In order to determine the stability in the aqueous solution of entecavir according to the amount of β-cyclodextrin was used as follows. As shown in [Table 4] and [Fig. 3], the ratio of entecavir and β-cyclodextrin was 1: 0.01, 1: 0.05, 1: 0.1, 1: 0.25, 1: 0.5, 1: 1, 1: 2, Water was added at a molar ratio of 1: 3 and 1: 4, and heated to 90 ° C., and then the formation degree of the flexible material was confirmed at 1 hour.

Table 4

Example	Item	mol ratio	0 hr	1 hr
Example	Item	(ETV: β-CD)	0 hr	ppm	%
Control Example 11	ETV	One	0	720	-
Example 12	ETV: β-CD	1: 0.01	0	626	86.9
Example 13		1: 0.05	0	572	79.4
Example 14		1: 0.1	0	394	54.7
Example 15		1: 0.25	0	391	54.3
Example 16		1: 0.5	0	385	53.5
Example 17		1: 1	0	381	52.9
Example 18		1: 2	0	379	52.6
Example 19		1: 3	0	382	53.1
Example 20		1: 4	0	379	52.6

The analysis method of lead substance is as follows.

(1) Standard Solution Preparation

(2) sample preparation

20 mg of entecavir was taken and placed in a 100 ml volumetric flask. Take 20 mg of entecavir and β-CD, 0.72, 3.6, 7.2, 18, 36, 72, 144, 216, 288 mg, respectively, put them in 100 ml flasks, add water, mark and warm to 90 ° C. Examples 12, 13, 14, 15, 16, 17, 18, 19, and 20 are assumed.

Experimental Example 4. Stability test in aqueous solution of entecavir according to the type of additive

In order to confirm the stability in the aqueous solution of entecavir according to the type of additive, the following experiment was performed. As shown in [Table 5] and [Fig. 4], β-CD, PEG6000, PVP, Copovidone, HPMC, HPC, and entecavir are taken as a mass ratio of 10: 1, and water is added and heated to 90 ° C. At 1 hour after the complete dissolution of the enticavir and the additives, the stability in the entecavir aqueous solution was evaluated through the degree of formation of the flexible substance.

Table 5

Example	Item	Mass ratio	0 hr	1 hr
Example	Item	(ETV: polymer)	0 hr	ppm	%
Control Example 11	ETV	One	0	720	-
Example 21	ETV + β-CD	1: 10	0	340	47.2
Comparative Example 22	ETV + PEG6000	1: 10	0	1760	244.4
Comparative Example 23	ETV + PVP	1: 10	0	1935	268.8
Comparative Example 24	ETV + Copovidone	1: 10	0	1253	174.0
Comparative Example 25	ETV + HPMC	1: 10	0	1776	246.6
Comparative Example 26	ETV + HPC	1: 10	0	787	109.3

The solution of Example 21 in which entecavir and β-CD were dissolved was found to have less formation of a flexible substance than the solution of Example 11 in which entecavir was dissolved alone. In addition, solutions No. 22, 23, 24, 25, and 26, which mixed polymers such as PEG6000, PVP, Copovidone, HPMC, and HPC and entecavir, were more flexible than the solution of β-cyclodextrin and entecavir. The results showed a marked increase.

Therefore, it can be seen that the use of β-cyclodextrin according to the present invention improves the stability in the aqueous solution of entecavir. The analysis method of lead substance is as follows.

(1) Standard Solution Preparation

(2) sample preparation

200 mg each of β-CD, PEG6000, PVP, Copovidone, HPMC, and HPC was added to a 100 ml flask, and 20 mg of entecavir was added thereto, followed by water and water. It is set as Example 22, 23, 24, 25, 26.

Experimental Example 5. Stability test in aqueous solution of entecavir in combination with β-cyclodextrin and additives

When using a combination of various additives and β-cyclodextrin, in order to check the stability in the aqueous solution of entecavir as shown in [Table 6] and [Fig. 5] polymers such as PEG6000, PVP, Copovi done, HPMC, HPC and β-cyclo Dextrin and entecavir are dissolved in water by heating to 90 ° C. in a mass ratio of 10: 10: 1, respectively. The degree of formation of the flexible material was confirmed at 1 hour.

Example 27, 28, 29, 30, 31 solution of PEG6000, PVP, Copovidone, HPMC, HPC mixed with β-cyclodextrin and entecavir, respectively, compared with PEG6000, PVP, Copovidone, HPMC, HPC and entecavir Example 22, 23, 24, 25, 26 It was confirmed that the generation of the flexible material compared to the solution.

Therefore, it can be seen that the present invention improves stability in aqueous solution of entecavir when β-cyclodextrin is used in combination with various additives.

Table 6

Example	Item	Mass ratio (ETV: polymer)	0 hr	1 hr
Example	Item	Mass ratio (ETV: polymer)	0 hr	ppm	%
Control Example 11	ETV	One	0	720	-
Example 21	ETV + β-CD	1:10	0	340	-
Comparative Example 22	PEG6000 + ETV	1:10	0	1760	-
Example 27	PEG6000 + ETV + β-CD	1:10:10	0	1282	72.8
Comparative Example 23	PVP + ETV	1:10	0	1935	-
Example 28	PVP + ETV + β-CD	1:10:10	0	528	27.3
Comparative Example 24	Copovidone + ETV	1:10	0	1253	-
Example 29	Copovidone + ETV + β-CD	1:10:10	0	863	68.9
Comparative Example 25	HPMC + ETV	1:10	0	1776	-
Example 30	HPMC + ETV + β-CD	1:10:10	0	1362	76.7
Comparative Example 26	HPC + ETV	1:10	0	787	-
Example 31	HPC + ETV + β-CD	1:10:10	0	519	65.9

The analysis method of lead substance is as follows.

(1) sample preparation

Take 200 mg of PEG6000, PVP, C opovidone, HPMC, and HPC into 100 ml flask, add 200 mg of β-CD and 20 mg of entecavir, add water, mark and warm to 90 ° C. It is set as 27, 28, 29, 30, and 31.

Experimental Example 6. Preparation and Stability Test of Tablets Containing Entercavir

Based on the amount of the drug substance shown in Table 7, (a) entecavir and β-cyclodextrin were dissolved in purified water while warming to about 60 ° C., and (b) lactose hydrate, microcrystalline cellulose, crospovidone, and povidone were mixed in the mixer. Adding and mixing, (c) adding the binder solution prepared in step (a) into the mixer to prepare granules, (d) drying the granules at 60 ° C., and (e) lowering the dried granules through a sieve. After sizing and mixing magnesium stearate to prepare a lubricant, (f) tableting the mixture prepared in step (e).

TABLE 7

Furtherance	Weight (mg)	weight(%)
Entecavir	1.0	0.3
Lactose Carb	Quantity	Quantity
Microcrystalline cellulose	Quantity	Quantity
β-cyclodextrin	7.7	1.9
Crospovidone	Quantity	Quantity
Povidone	Quantity	Quantity
Magnesium stearate	Quantity	Quantity
Purified water	Quantity	-
total	400.0	100.0

In order to test the stability of the tablet prepared as described above, the following experiment was performed. Example 32 containing β-cyclodextrin was prepared by the above-described drug substance fraction and the preparation method described above, and Comparative Example 33 was prepared by the same preparation method without using β-cyclodextrin in the above-described drug substance fraction. The stability under the accelerated condition (40 ℃, 75% relative humidity, 1 week) was evaluated by the degree of formation of the flexible material. The results are shown in [Table 8] and [FIG. 6].

Table 8

Example	Item	ppm (n = 3)	%
Example 32	Tablets Prepared Using β-cyclodextrin	1475 ± 144.0	-
Comparative Example 33	Tablets made without using β-cyclodextrin	2467 ± 202.3	167.2

(ppm: flexible material unit,%: flexible material generation rate compared to Example 32)

Tablet No. 32 prepared using β-cyclodextrin was found to produce less analogues than tablet No. 33 prepared without β-cyclodextrin. Therefore, it can be seen that the use of β-cyclodextrin according to the present invention improves the stability of the tablet containing entecavir.

Experimental Example 7. Preparation and long-term stability test of tablets containing entecavir

Based on the amount of the drug substance shown in Table 9, (a) entecavir and β-cyclodextrin are dissolved in purified water while warming to about 60 ° C., and (b) lactose hydrate, microcrystalline cellulose, crospovidone, and povidone are mixed in the mixer. Adding and mixing, (c) adding the binder solution prepared in step (a) into the mixer to prepare granules, (d) drying the granules at 60 ° C., and (e) lowering the dried granules through a sieve. After sizing and mixing magnesium stearate to prepare a lubricant, (f) the mixture prepared in step (e) was compressed and then coated with Opadry.

Table 9

Furtherance	Weight (mg)
Entecavir	One
Lactose Carb	Quantity
Microcrystalline cellulose	Quantity
β-cyclodextrin	7.7
Crospovidone	Quantity
Povidone	Quantity
Magnesium stearate	Quantity
Purified water	Quantity
Opadry	Quantity
total	412

The content of the formulation, the content of the individual flexible materials (%), and the content of the total flexible materials (%) under accelerated conditions of temperature and humidity for the film-coated tablet prepared by the above method were confirmed by the following method. After manufacturing the film-coated tablets HDPE packaging and stored for 6 months under accelerated conditions (40 ℃, 75% RH) conditions, the content (%) of the flexible material of each initial, 6 months was measured. Five tablets of each tablet were taken as a sample solution, placed in a 250 mL volumetric flask, and 150 mL of 0.01 mol / L hydrochloric acid was added, followed by shaking and mixing for about 30 minutes. 0.01 mol / L hydrochloric acid was added to the mark, and the mixture was filtered. Separately, 50 mg of entecavir was taken as a standard solution, and 50 mL or less of methanol was added to a 250 mL volumetric flask to dissolve. Then, 0.01 mol / L hydrochloric acid solution was added, followed by mixing and filtering. 10 mL of the filtered solution was collected and placed in a 100 mL volumetric flask, followed by addition of 0.01 mol / L hydrochloric acid solution, followed by mixing and filtration.

It was carried out under the following chromatograph method conditions (Table 10: instrument conditions), and the concentration gradient of the mobile phase is shown in Table 11 (mobile phase concentration gradient). The experimental results are shown in [Table 12] (content results) and [Table 13] (flexible material results).

Table 10

column	C18 (100 X 4.6 mm, 3)
Detector	Ultraviolet absorption spectrophotometer (wavelength 254 nm)
Mobile phase	A water: ACN: TFA (99.1: 1: 0.1) B water: ACN: TFA (70: 30: 0.1)
Flow rate	1.0 mL / min
Column temperature	30 ° C
Analysis time	35 minutes
Injection volume	75 μl

Table 11

Time (min)	Mobile phase A	Mobile phase B
0-3.5	100	0
3.5-21	100-69	0-31
21-24	69-51	31-49
24-27	51-0	49-100
27-28	0-100	100-0
28-35	100	0

Table 12

	Initial	6 months later
Content test result	98.2%	99.0%

Table 13

	Initial	6 months later
Individual lead materials	0.09%	0.05%
Total lead	0.39%	0.34%

From the above experimental results, the long-term stability of the tablet containing β-cyclodextrin and entecavir according to the present invention can be confirmed.

Experimental Example 8. Stability test in aqueous solution of entecavir (room temperature) according to the amount of α- or γ-cyclodextrin

In order to confirm the stability in the aqueous solution of entecavir according to the amount of α- or γ-cyclodextrin was used as follows. As shown in [Table 14], the ratio of entecavir and α- or γ-cyclodextrin was used in a mol ratio of 1: 0.01, 1: 0.1, 1: 0.5, 1: 1, 1: 2, 1: 3, and water was added thereto. The degree of formation of the analog was checked at 1 hour at room temperature.

From Table 14, it can be seen that the use of α- or γ- cyclodextrin improves the stability in the aqueous solution of entecavir.

Table 14

ETV: CD (mol ratio)	0 hr	1 hr (ppm)		1 hr (%)
ETV: CD (mol ratio)	0 hr	α-CD	γ-CD	α-CD	γ-CD
1: 0	0	371	371	-	-
1: 0.01	0	326	375	87.9	101.2
1: 0.1	0	328	316	88.4	85.2
1: 0.5	0	329	299	88.7	80.7
1: 1	0	323	300	87.1	80.8
1: 2	0	300	298	80.9	80.3
1: 3	0	300	295	80.9	79.4

Analysis of the flexible material was performed in the same manner as in Experiment 2.

Experimental Example 9. Stability test in aqueous solution of entecavir (90 ° C) according to the amount of α- or γ-cyclodextrin

In order to confirm the stability in the aqueous solution of entecavir depending on the amount of α- or γ- cyclodextrin was carried out as follows. As shown in [Table 15], the ratio of entecavir and cyclodextrin was used in mol ratios of 1: 0.01, 1: 0.1, and 1: 2, and water was added thereto. Confirmed.

From Table 15, it can be seen that the use of α- or γ- cyclodextrin improves the stability in the aqueous solution of entecavir.

Table 15

ETV: CD (mol ratio)	0 hr	1 hr (ppm)		1 hr (%)
ETV: CD (mol ratio)	0 hr	α-CD	γ-CD	α-CD	γ-CD
1: 0	0	371	371	-	-
1: 0.01	0	364	365	98.0	98.4
1: 0.1	0	339	336	91.4	90.6
1: 2	0	272	338	73.3	91.1

Analysis of the flexible material was carried out in the same manner as in Experiment 3.

Experimental Example 10. Stability test in aqueous solution of entecavir by the combination of α- or γ-cyclodextrin and additives

When using a combination of various additives and α- or γ- cyclodextrin, to confirm the stability in the aqueous solution of entecavir as shown in Table 16, polymers such as PEG6000, PVP, Copovi done, HPMC, HPC and α- or γ-cyclo Dextrin and entecavir are dissolved in water by heating to 90 ° C. in a mass ratio of 10: 10: 1, respectively. The degree of formation of the flexible material was confirmed at 1 hour.

From Table 16, a solution containing α- or γ-cyclodextrin and entecavir in PEG6000, PVP, Copovidone, HPMC, and HPC, respectively, was compared to a solution containing only PEG6000, PVP, Copovidone, HPMC, HPC, and entecavir. It was confirmed that less generation of.

Therefore, it can be seen that the present invention improves the stability in the aqueous solution of entecavir when the α- or γ-cyclodextrin is used in combination with various additives.

Table 16

Item	1hr (ppm)
ETV	371
PEG 6000 + ETV	1760
PEG 6000+ ETV + α-CD	1265
PEG 6000 + ETV + γ-CD	1442
PVP K-30 + ETV	1935
PVP + ETV + α-CD	738
PVP + ETV + γ-CD	1266
HPMC 2910 6cP + ETV	1776
HPMC + ETV + α-CD	1116
HPMC + ETV + γ-CD	1688
HPC L-type + ETV	787
HPC + ETV + α-CD	685
HPC + ETV + γ-CD	700
Copovidone + ETV	1253
Copovidone + ETV + α-CD	633
Copovidone + ETV + γ-CD	1092

(ppm: Leading substance unit)

Analysis method of the flexible material was carried out in the same manner as in Experiment 5.

There is provided a pharmaceutical composition containing entecavir at a low content and an increased stability thereof, and a method for reducing the flexible material when granulating entecavir by a wet granulation method.

Claims

A pharmaceutical composition containing entecavir and cyclodextrin and having improved stability for treating hepatitis B virus infection.
The pharmaceutical composition of claim 1, wherein the molar ratio of entecavir to cyclodextrin is at least 1: 0.1.
In granulating entecavir by a wet granulation method, a method for reducing a flexible substance, comprising dissolving entecavir and cyclodextrin in a solvent to prepare a binding solution.
(a) dissolving entecavir and cyclodextrin in a solvent to prepare a binding solution;

(b) injecting a pharmaceutically acceptable excipient into the mixer and mixing;

(c) injecting the binder solution prepared in step (a) into the mixer to prepare granules;

(d) drying the granules;

(e) mixing the dried granules with a pharmaceutically acceptable excipient to prepare a mixture; And

(f) A method for preparing a pharmaceutical composition comprising enticavir and cyclodextrin, which comprises the step of preparing the mixture in step (e) into tablets, capsules, powders or granules.