KR20230153973A - Pharmaceutical composition comprising high amount of an active ingredient derived from herbal substances - Google Patents

Abstract

One aspect of the present disclosure relates to a preparation containing, as an active ingredient, a high content of herbal extracts of Uryeongseon, Brachialis root, and Herbal extract useful for treating or improving arthritis, etc. The formulation according to one aspect of the present disclosure contains a high content of the active ingredient and is small in size, providing excellent medication convenience, and has a release pattern and release property that shows an excellent arthritis treatment effect even when taken twice a day. Another aspect of the present disclosure relates to a preparation that contains 280-320 mg of herbal extracts of Euryeongseon, Brachialis root, and Herbal extracts of the herb, is taken twice a day, has a specific release pattern, and is excellent in treating arthritis.

Description

Pharmaceutical composition comprising high amount of an active ingredient derived from herbal substances}

The present disclosure relates to a preparation containing herbal extracts of Uryeongseon, Brachiorrhea root, and Herbaceous root. In particular, the present disclosure relates to a formulation that can be taken twice a day, and has a release pattern that shows excellent drug efficacy while containing a high dose.

Osteoarthritis, also called degenerative arthritis, is the most common form of arthritis in adults. The number of patients with osteoarthritis is rapidly increasing as the aging society progresses, leading not only to a decline in the quality of life of individual patients but also to social and economic losses.

Arthritis is a disease that causes inflammation in the joints, and there are many types. The most common form of arthritis is osteoarthritis, and others include rheumatoid arthritis, gout, and psoriatic arthritis. Each type of arthritis has different causes, but the phenomenon of destruction of cartilage tissue is the same.

Degenerative arthritis (osteoarthritis) is the most frequently occurring chronic joint disease among arthritis. In degenerative arthritis, the cartilage tissue surrounding the ends of the bones gradually wears away, causing inflammation and severe pain within the joint, and the bone under the cartilage becomes abnormally hard. Therefore, the cartilage, which acts as a buffer for friction caused by joint movement, wears away, causing severe pain and movement disorders when moving the joint.

Despite many studies to control degenerative arthritis, a fundamental treatment based on the pathological cause of the disease has not yet been developed. To date, in addition to surgical methods, non-steroidal analgesics and anti-inflammatory drugs (NSAIDs) and disease-relieving osteoarthritis treatment drugs (Disease) have been used to control degenerative arthritis. Drug therapy for anti-inflammatory and pain relief, such as -modifying osteoarthritis drug (DMOAD), is mainly used. In addition, cartilage protective agents such as hyaluronic acid, glucosamine, and chondroitin have been developed and are commercially available, but their therapeutic effects on cartilage protection and regeneration induction have not been established.

Meanwhile, complex herbal extracts of Uryeongseon, Brachialis root, and Hagochoe are known to have anti-inflammatory, analgesic, joint-protective, and immunosuppressive effects, and are widely used as a treatment for osteoarthritis and rheumatoid arthritis.

These herbal extracts can be manufactured into tablets containing a high content, but if the dosage is simply increased to increase the efficacy, an increase in side effects may occur. In addition, the currently commercially available preparations containing complex herbal extracts of Uryeongseon, Brachiorrhea root, and Hagocho are required to be taken three times a day, making them less convenient to take.

Therefore, in order to prevent side effects caused by high-dose preparations and increase the convenience of administration, delayed-release preparations can be considered. However, the present inventors confirmed through experiments that there are many unexpected problems in the case of complex herbal extract preparations of Uryeongseon, Brachylus root, and Hagocho. This was revealed.

Korean Patent No. 0180567 Korean Patent No. 0483707

Therefore, the problem that the present disclosure aims to solve is to contain a high content of the extracts of the euphorbia gland, brachiorrhea root, and hagochoe as active ingredients, while being easy to take due to the small size of the preparation, and has a release pattern that can exhibit desirable medicinal effects, taking it twice a day. Possible formulations, especially tablets, are provided.

In order to solve the above problem, one aspect of the present invention includes 280-320 mg of herbal extracts of Uryeongseon, Guaru root, and Hagocho as active ingredients, and the content of the active ingredient is the total weight of the mixture before coating or empty capsule filling. It is 40 to 90% by weight (preferably, 50-90% by weight, more preferably 60-90% by weight) and includes ethylcellulose, ethyl methacrylic acid copolymer, or a mixture thereof to control release. Provided are preparations, particularly tablets, characterized in that.

The present inventors attempted to manufacture a twice-daily formulation containing high doses of herbal extracts of Uryeongseon, Brachiocephalic hernia, and Herbal extracts, but unexpected problems were revealed as a result of the research. Specifically, it was possible to delay or control the release using various binders, but in this case, it was difficult to completely elute all the contained extracts. In additional studies, delayed release and good release amount could be achieved by reducing the relative content of the extract by using relatively more excipients and disintegrants. However, in this case, the size of one preparation to be taken was too large, making it inconvenient to take. In other words, due to the specific physical properties of the herbal extracts of Wiryeongseon, Brachiocephalic herb, and Herbal Medicine extract, the product contains a high content of Herbal Medicinal Extracts of Wiryeongseon, Brachiocephalic herb, and Herbal Medicine extract at a high weight percent relative to the weight of the preparation, while providing a good release pattern and high release properties at the same time. It was not easy to achieve.

The present inventors completed one aspect of the present invention by confirming that these contradictory problems can be solved by using ethylcellulose, ethyl methacrylic acid copolymer, or a mixture thereof as a binder.

As a herbal extract of Wiryeongseon, Gwanrugeun, and Hedgehog herb of the present invention, the extract of 'Wiryeongseon, Hwaneurogeun, and Hedgehog herb 30% ethanol extract (40→1)', which is in the form of a yellow-brown, hygroscopic powder, can be used, and this extract For example, the herbal extracts of Wiryeongseon, Gwanrugeun and Hagocho may be herbal extracts prepared according to the method described in Korean Patent Nos. 0180567, 0483707 (WO2002-094301 A1), etc.

Among the two binder components of the present invention, ethylcellulose is more preferable, and therefore, a preferred embodiment of the present invention contains 280-320 mg of herbal extracts of Wiryeongseon, Gwanrugeun, and Hagochoe as active ingredients, and the active ingredient The content is 40 to 90% by weight (preferably, 50-90% by weight, more preferably 60-90% by weight) relative to the total weight of the uncoated tablet before coating, and is characterized by controlling the release by including ethylcellulose. , especially tablets.

Such ethylcellulose can be used to have a viscosity of 3-22 mPa.s when a 5% by weight solution is prepared with a mixed solvent of toluene/ethanol 80:20 (volume ratio) and the viscosity is measured at room temperature.

The present inventors sought to develop a preparation that contains 280-320 mg of herbal extracts of Uryeongseon, Brachialis root, and Herbal extracts and is to be taken twice a day. Accordingly, preparations with various release patterns (rates) were manufactured and evaluated. As described later, when a specific release pattern is used, the treatment effect for arthritis (e.g., osteoarthritis (degenerative joint disease), rheumatoid arthritis, etc.) is significantly superior. It came out. Typically, in the case of a preparation administered twice a day, the sustained release is generally controlled so that the drug ingredient is released over a long period of time, similar to Example 4-4 described later. However, very specifically, as shown in FIGS. 4 to 10, for the purpose of treating arthritis with the herbal extracts of Uryeongseon, Brachiocephalicum, and Herbal Medicine Extracts, the release of the medicinal ingredients must be slightly delayed (see FIG. 4) and taken three times a day. The arthritis treatment effect increased compared to the formulation.

That is, one aspect of the present disclosure is to provide a dissolution pattern that shows a desirable arthritis treatment effect in a preparation containing 280-320 mg of herbal extracts of Uryeongseon, Brachialis root, and Herbaceous herb, and taken twice a day. The dissolution pattern that can achieve the excellent arthritis treatment effect of the present invention is the paddle test method at 37 ± 0.5 ℃ and 50 rpm speed, and when dissolution is tested in 900 ml of water as a medium, the release of rosmarinic acid is 40 to 70% in 45 minutes. % (more preferably 45 to 65%, even more preferably 45 to 60%), 75% or more (preferably 80% or more) in 90 minutes, and 80% or more (preferably 85%) in 120 minutes. above).

Additives other than the binders or release retardants mentioned above or described below may be used if such a release pattern can be achieved. In other words, the scope of the present invention is not limited by the type of specific additives described above or below as long as it is possible to achieve a specific release pattern according to the present disclosure. The specific additives described above or below are merely intended to achieve more preferred aspects of the present disclosure.

In other words, another aspect of the present invention is a preparation containing 280-320 mg of the extract of 'Uryeongseon, Gwanlugeun, and Coriander root 30% ethanol extract (40→1)' as an active ingredient, and is a preparation to be taken twice a day, When dissolving in 900 ml of water using the paddle test method at 37 ± 0.5 ℃ and 50 rpm, the release of Rosmarinic Acid was 40 to 70% in 45 minutes and 75% or more in 90 minutes (more preferably (40 to 70% in 45 minutes, 75% or more in 90 minutes, and 80% or more in 120 minutes), and preparations for treating arthritis, especially tablets. Here, preferably, the content of the extract of 'Wiryeongseon·Guarugeun·Hagocho 30% ethanol extract (40→1)' is 40 to 90% by weight (preferably) relative to the total weight of the mixture before coating or empty capsule filling. Preferably, 50-90% by weight, more preferably 60-90% by weight.

In a preferred embodiment of the present invention, the formulation for treating arthritis is a tablet or capsule formulation containing 280-320 mg of herbal extracts of Euryeongseon, Brachialis root, and Herbaceae as active ingredients, and is a formulation to be taken twice a day, and the dosage is 37± When dissolution was tested under the conditions of 900 ml of water using the paddle test method at 0.5 ℃ and 50 rpm speed, the release of rosmarinic acid was 45-65% in 45 minutes, more than 80% in 90 minutes, and more than 85% in 120 minutes (more Preferably, 45 to 60% in 45 minutes, 80% or more in 90 minutes, and 85% or more in 120 minutes. Here, preferably, the content of the extract of 'Wiryeongseon·Guarugeun·Hagocho 30% ethanol extract (40→1)' is 40 to 90 weight compared to the total weight of the capsule filling excluding the weight of the uncoated tablet or empty capsule before coating. % (preferably 50-90% by weight, more preferably 60-90% by weight).

Preferably, a preferred embodiment of the present invention is a tablet containing 280-320 mg of the extract of 'Wiryeongseon, Gwalugeun, and Coriander root 30% ethanol extract (40→1)' as an active ingredient, and the content of the active ingredient is the same as that of the uncoated tablet before coating. It is 40 to 90% by weight (preferably, 50-90% by weight, more preferably 60-90% by weight) relative to the total weight, is a tablet to be taken twice a day, and is administered at 37 ± 0.5 ° C. with a paddle speed of 50 rpm ( When dissolving in a 900 ml water medium using the paddle test method, the release of rosmarinic acid is 40-70% in 45 minutes and 75% or more in 90 minutes (more preferably 40-70% in 45 minutes, 75% in 90 minutes). % or more, more preferably 80% or more in 120 minutes, more preferably 45 to 65% in 45 minutes, 80% or more in 90 minutes, and 85% or more in 120 minutes), and the release control is performed using ethylcellulose, methacrylic A tablet comprising an acid ethyl acrylic acid copolymer or a mixture thereof (more preferably, ethylcellulose) is provided.

One aspect of the present invention is a tablet containing 280-320 mg of herbal extracts of Wiryeongseon, Guaru root and Hagocho as active ingredients, and the content of the active ingredient is 40 to 90% by weight relative to the total weight of the uncoated tablet before coating (preferably, 50-90% by weight, more preferably 60-90% by weight), is a tablet to be taken twice a day, and is tested for dissolution in 900ml water using the paddle test method at 37±0.5°C and 50rpm speed. , the release of rosmarinic acid is 40-70% at 45 minutes, 75% or more at 90 minutes, and 80% or more at 120 minutes (more preferably 45-65% at 45 minutes, 80% or more at 90 minutes, and 120 minutes or more). 85% or more), and provides a tablet characterized in that the release is adjusted using ethylcellulose. The content of ethylcellulose is preferably 0.1-10% by weight, more preferably 0.4-5% by weight, and even more preferably 0.8-3% by weight, based on the total weight of the uncoated tablet before coating.

In one aspect of the present invention, the tablet may optionally further include pharmaceutically acceptable excipients, adsorbents, disintegrants, lubricants, etc.

In one aspect of the present invention, various materials such as polyvinylpyrrolidone, ethylcellulose, ethyl methacrylic acid copolymer, hydroxypropylmethylcellulose, hydroxypropylcellulose, and acrylic acid copolymer can be used. there is. However, in terms of reducing the size of the tablet and achieving good release properties, ethylcellulose, ethyl methacrylic acid copolymer, or a mixture thereof is preferred. The release control material is preferably 0.1-10% by weight, more preferably 0.4-5% by weight, and even more preferably 0.8-3% by weight, relative to the total weight of the uncoated tablet before coating.

Microcrystalline cellulose, lactose, mannitol, etc. can be used as the excipients. The content of these excipients is preferably 5-40% by weight, more preferably 10-35% by weight, and 15-30% by weight relative to the weight of the bare tablet before coating. is even more desirable.

As the adsorbent, light anhydrous silicic acid (including hydrophobic light anhydrous silicic acid), magnesium aluminum silicate, etc. can be used. The content of such adsorbent is preferably 0.5-9% by weight, more preferably 1-7% by weight, based on the weight of the bare tablet before coating. Preferred, 2-5% by weight is even more preferred.

As the disintegrant, croscarmellose sodium, sodium starch glycolate, crospovidone, low-substituted hydroxypropyl cellulose, etc. can be used. However, in terms of being able to achieve good release properties while reducing the size of the tablet, croscarmellose sodium, low-substituted hydroxypropyl cellulose, or a mixture thereof is preferable. When croscarmellose sodium, low-substituted hydroxypropyl cellulose, or a mixture thereof is used, good release properties are achieved even under conditions where the content of the active ingredient is increased. Among croscarmellose sodium or low-substituted hydroxypropyl cellulose as a disintegrant, croscarmellose sodium is particularly more preferable for the purpose of the present invention. The content of this disintegrant is preferably 1-10% by weight, more preferably 1-8% by weight, and even more preferably 2-5% by weight, based on the weight of the bare tablet before coating.

As the lubricant, stearyl fumarate, magnesium stearate, stearic acid, talc, etc. can be used. The content of such lubricant is preferably 0.1-4% by weight, more preferably 0.2-2% by weight, based on the weight of the bare tablet before coating. do.

In one aspect of the invention, tablets according to the invention may also be coated for various purposes, such as protection from external impacts, appearance, and control of release. For example, the uncoated tablet can be coated using a coating composition containing a film former, a plasticizer, an anti-adhesion agent, etc., and the content of the coating is preferably 1-15% by weight based on the total weight of the uncoated tablet before coating. Can be coated in an amount of 3-10% by weight. As such a film coating composition (product), various coating base products sold by Colorcon Company containing polyvinyl alcohol as a basic coating material can be used.

A preferred embodiment of the present invention is a tablet containing 280-320 mg of herbal extracts of Uryeongseon, Guaru root, and Hagocho as active ingredients, and the content of the active ingredient is 40 to 90% by weight (preferably 40 to 90% by weight) relative to the total weight of the uncoated tablet before coating. , 50-90% by weight, more preferably 60-90% by weight), is a tablet to be taken twice a day, and is eluted in 900ml of water using the paddle test method at 37±0.5°C and 50rpm speed. During the test, the release of rosmarinic acid is 40-70% at 45 minutes, 75% or more at 90 minutes, 80% or more at 120 minutes (more preferably 45-65% at 45 minutes, 80% or more at 90 minutes, and 120% or more). 85% or more per minute), the release is adjusted using ethylcellulose, and a tablet is provided, characterized in that it contains croscarmellose sodium as a disintegrant.

The formulation according to the present invention is prepared by producing granules by a wet granulation method using a solvent such as water, ethanol, or isopropanol, or a dry granulation method using compression force, and then mixing the granules with post-mixing components such as a disintegrant and lubricant. It can be manufactured as tablets or capsules. The preparation according to the present invention can be manufactured into tablets by a direct compression method without a granule manufacturing process. However, wet granulation tablets may be preferable in terms of maintaining the release pattern without deviation to achieve one of the several purposes of the present invention.

Accordingly, one aspect of the present invention includes the steps of preparing granules using herbal extracts of Wiryeongseon, Gwanrugeun, and H. aquifolia, an adsorbent, a binder, optionally an excipient, and optionally a disintegrant; Preparing tablets by post-mixing the granules prepared above with pharmaceutically acceptable additives such as lubricants and disintegrants and compressing them into tablets; and optionally coating the tablet.

One aspect of the present disclosure provides a preparation that contains a high content of herbal extracts of Uryeongseon, Brachiorrhea root, and Herbal extracts of Prickly pear, and can be taken twice a day. The formulation according to the present disclosure has a specific release pattern and exhibits excellent arthritis treatment effects even when taken twice a day. The formulation of one aspect according to the present disclosure also contains a high content of herbal extracts of euphorbia, brachialis root, and hagchoi, and thus is small in size and has excellent medication convenience.

The following drawings attached to this specification serve to further understand the technical idea of the present invention along with the contents of the above-described invention, and therefore, the present invention should not be construed as limited only to the matters described in such drawings.
Figure 1 is a graph showing the elution pattern in pH 1.2 medium for each type of binder.
Figure 2 is a graph showing the dissolution pattern in pH 6.8 medium for each ethylcellulose viscosity.
Figure 3 is a graph showing the dissolution patterns in water of examples and comparative examples according to the present invention.
Figure 4 is a graph showing the dissolution patterns in water of preparations with various dissolution patterns.
Figure 5 shows the results of evaluating the effects of agents with various dissolution patterns on gait after meniscectomy and anterior cruciate ligament resection.
Figure 6 shows the results of evaluating the effect of agents with various dissolution patterns on arthritis, showing the results of a weight bearing test (Incapacitance test).
Figure 7 shows the results of an ELISA analysis evaluating the effect of agents with various dissolution patterns on cytokines in arthritis.
Figure 8 shows the results of area histochemical staining analysis evaluating the effect of agents with various dissolution patterns on biomarkers in arthritis.
Figure 9 shows the results of evaluating the effects of agents with various dissolution patterns as OARSI scores through histopathological analysis.
Figure 10 shows the results of immunohistochemical analysis, showing the results of Hematoxylin & Eosin (H&E) and Safranin-O staining of the joint surface, cartilage, and synovium after completion of the test.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the field to which the present invention pertains.

Example 1: Confirmation of dissolution patterns using various binders

Tablets containing 30% ethanol extract (40 → 1) extracts of Wiryeongseon, Gwarugeun, and Hedgehog herb were prepared using the contents and ingredients listed in Table 1 below. The extract, crospovidone, hydrophobic light anhydrous silicic acid, and binder were mixed. Afterwards, magnesium stearate was added, mixed, and tableted.

Ingredients (unit: mg) Example
1-1 Example
1-2 Example
1-3 Example
1-4 Example
1-5 Example
1-6 Herbal extract 300 300 300 300 300 300 Hydrophobic light anhydrous silicic acid 15 15 15 15 15 15 polyvinylpyrrolidone 10.5 Ethylcellulose 10.5 Methacrylic acid ethyl acrylic acid copolymer 10.5 Hydroxypropylmethylcellulose 10.5 Hydroxypropylcellulose 10.5 Acrylic acid copolymer 10.5 Crospovidone 20 20 20 20 20 20 Magnesium stearate 2.5 2.5 2.5 2.5 2.5 2.5 tablet total weight 348 348 348 348 348 348

Polyvinylpyrrolidone: Kollidon K30, viscosity approximately 5.5~8.5 cps (20℃, 10% w/v)

Methacrylic acid ethyl acrylic acid copolymer: Eudragit L100-55, viscosity approximately 50~200cps

Hydroxypropylmethylcellulose: Metolose, viscosity about 100cps (20℃/2% w/v)

Hydroxypropylcellulose: Nisso HPC-L, viscosity approximately 6~10cps (20℃/2% w/v)

Acrylic acid copolymer: Carbomer 971, viscosity approximately 4000~11000 (0.5%w/w)

Experimental Example 1: Elution evaluation by binder type

The tablets prepared in Example 1 were tested for dissolution according to the dissolution test method of the 12th edition of the Korean Pharmacopoeia. The paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0 ± 0.5°C. Elution was performed at 900 ml at pH 1.2. To improve the stability of the sample solution, it was analyzed after pretreatment with acetonitrile. The dissolution test results are shown in Table 2 and Figure 1 below. The dissolution rate was measured by measuring the content of rosmarinic acid, the active ingredient in herbal medicine extract, over time. The content of rosmarinic acid is reported in J. Sep. Sci. It was measured similarly to the method described in 2015, 0, 1-8 (“Quality evaluation of Salvia miltiorrhiza Bge. by ultra high performance liquid chromatography with photodiode array detection and chemical fingerprinting coupled with chemometric analysis”).

Time (minutes) Example
1-1 Example
1-2 Example
1-3 Example
1-4 Example
1-5 Example
1-6 5 3 22 21 5 10 4 10 10 38 38 8 23 8 15 19 47 45 10 34 12 30 37 62 59 18 62 30 45 47 73 70 27 77 47 60 52 80 77 37 83 63 90 60 92 86 56 84 72 120 66 97 94 66 85 76 180 76 100 98 76 85 80 240 81 101 101 80 85 82

As shown in Table 2, Examples 1-1, 1-4, and 1-6 showed a large dissolution delay effect, and Examples 1-2 and 1-3 showed similar dissolution delay effects. Examples 1-5 had little dissolution delay effect.

However, in the case of the remaining examples except for Examples 1-2 and 1-3, it was confirmed that the dissolution amount did not reach about 80% even when the release was evaluated for a very sufficient time, so it was found that it was practically impossible to use. This is presumed to be due to the combination of the physical properties of the specific extract used in the present invention and the unique physical properties of the polymer. For example, it is thought to be related to the function of preventing gelation of the herbal extract, etc., but the present invention It is not limited to these theoretical mechanisms.

The methacrylic acid-ethylacrylic acid copolymer used in Examples 1-3 showed a desirable release pattern in an acidic medium, but there was a problem in that the release pattern was different in different pH media, making it difficult to control the release, and as a result, Example 1 It was less desirable than -2 ethylcellulose.

The present inventors also performed various evaluations and determined that in a pH 1.2 medium, the dissolution pattern of Examples 1-2 or 1-3 is preferable as the dissolution pattern of a preparation containing 300 mg tablets taken twice. Therefore, in this respect, Binders other than Examples 1-2 and 1-3 also have the problem of delaying release too much.

As a result, surprisingly, ethylcellulose and ethyl acrylic acid copolymer of methacrylate are preferred as release-controlling ingredients for a preparation that contains 300 mg of herbal extracts of Uryeongseon, Brachialis root, and Herbal extracts and is taken twice a day, rather than the commonly used HPMC. , ethylcellulose was more preferable.

Example 2: Confirmation of delayed dissolution pattern using ethylcellulose

Tablets containing 30% ethanol extract (40 → 1) extracts of Wiryeongseon, Gwarugeun, and Coriander root were prepared using the contents and ingredients shown in Table 3 below. First, a binding solution was prepared by dissolving ethyl cellulose in isopropyl alcohol, and then the extract and hydrophobic light anhydrous silicic acid were added to the binding liquid to prepare wet granules. Crospovidone and magnesium stearate were mixed into the wet granules prepared above and compressed into tablets.

Ingredients (unit: mg) Example 2-1 Example 2-2 Example 2-3 Example 2-4 Herbal extract 300 300 300 300 Hydrophobic light anhydrous silicic acid 15 15 15 15 Ethylcellulose 4cps 20 Ethylcellulose 7cps 20 Ethylcellulose 10cps 20 Ethylcellulose 20cps 20 Crospovidone 15 15 15 15 Magnesium stearate 2 2 2 2 tablet total weight 352 352 352 352

Experimental Example 2: Elution evaluation by viscosity of ethylcellulose in pH 6.8 medium

The dissolution of Examples 2-1 to 2-4 was evaluated according to the dissolution test method of the 12th edition of the Korean Pharmacopoeia. The paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0 ± 0.5°C. The elution was performed at 900 ml at pH 6.8. To improve the stability of the sample solution, it was analyzed after pretreatment with acetonitrile. The dissolution test results of rosmarinic acid are shown in Table 4 and Figure 2 below.

Time (minutes) Example 2-1 Example 2-2 Example 2-3 Example 2-4 5 12 10 9 6 10 13 12 14 11 15 17 17 17 16 30 27 25 25 25 45 34 33 32 33 60 43 39 39 39 90 65 60 52 56 120 92 85 72 78 180 96 95 94 92 240 96 95 95 93

As shown in Table 4, the formulation of the present invention using ethylcellulose showed a good release pattern even at pH 6.8, and was able to exhibit a release close to 100% even at pH 6.8.

In addition, it was confirmed that the dissolution rate slowed down from Example 2-1 to 2-4, which was consistent with the increase in viscosity of ethylcellulose.

Example 3 and Comparative Example 1: Evaluation of dissolution according to various components

Tablets were prepared according to the contents and ingredients listed in Table 5 below. Example 3 was prepared by wet granulation of herbal extracts of Wiryeongseon, Gwanrugeun, and Hagochoe, hydrophobic light anhydrous silicic acid, and ethyl cellulose. Microcrystalline cellulose, croscarmellose sodium, and stearyl fumarate were mixed into the wet granules prepared above and compressed into tablets. The core prepared in this way was coated using a coating agent. Comparative Example 1 was prepared by dry mixing the herbal extracts of Wiryeongseon, Gwanru root, and H. perilla, light anhydrous silicic acid, corn starch, microcrystalline cellulose, and sodium starch glycolate. Magnesium stearate was mixed with the mixture prepared above and compressed into tablets. The core prepared in this way was coated using a coating agent.

Ingredients (unit: mg) Example 3 Comparative Example 1 Herbal extract 300 200 microcrystalline cellulose 95 113 corn starch 50 Light anhydrous silicic acid 10 Hydrophobic light anhydrous silicic acid 10 Ethylcellulose 4cps 8 Croscarmellose Sodium 13 Sodium starch glycolate 25 Magnesium stearate 2 Stearyl fumarate 2 Coating mechanism 28 30 tablet total weight 456 430

Experimental Example 3: Dissolution evaluation of Example 3 and Comparative Example 1

The dissolution patterns of Example 3, Comparative Example 1, and Comparative Example 2 were evaluated according to the dissolution test method of the 12th edition of the Korean Pharmacopoeia. The paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0 ± 0.5°C. Elution was performed in 900 ml of purified water. To improve the stability of the sample solution, it was analyzed after pretreatment with acetonitrile and pH 1.2 buffer. The dissolution test results of rosmarinic acid are shown in Table 6 and Figure 3 below.

Time (minutes) Example 3 Comparative Example 1 5 5 6 10 14 24 15 20 39 30 40 67 45 57 81 60 71 82 90 88 84 120 94 85 180 98 86 240 99 86

As shown in Table 6, the formulation according to the present invention showed a good release pattern even in water. The present inventors performed various evaluations and found that when the dissolution medium was water, the dissolution pattern was the same as in Example 3 (e.g., 40 to 70% in 45 minutes, 75% or more in 90 minutes, and more preferably 85% in 120 minutes). % or more) was found to be desirable, but it was not easy to achieve the above dissolution pattern. For example, the dissolution results of Comparative Example 1 show that although the initial dissolution rate was increased by using more excipients and disintegrants and lowering the extract content, the final dissolution rate was not high.

Example 4: Evaluation of drug efficacy according to dissolution pattern

With the prescription shown in Table 7 below, 30% ethanol extract (40 → 1) tablets of Wiryeongseon, Gwarugeun, and Hedgehog herbacea with various dissolution patterns were manufactured and then the effect of the dissolution pattern on drug efficacy was evaluated. 200mg immediate-release tablets, 300mg immediate-release tablets, 300mg delayed-release tablets, and 300mg sustained-release tablets were manufactured. First, a binding solution was prepared by dissolving the binder in a suitable solvent among water, ethanol, isopropyl alcohol, or a mixed solvent thereof. Afterwards, extract, light anhydrous silicic acid, and excipients were added to the binding solution to prepare wet granules. The wet granules prepared above were mixed with a disintegrant and a lubricant and compressed into tablets. Afterwards, film coating was performed using a conventional method.

Ingredients (unit: mg) Example
4-1 Example
4-2 Example
4-3 Example
4-4 200mg immediate-release tablet 300mg immediate-release tablet 300mg delayed-release tablet 300mg extended-release tablet Gwanoru root, Uryeongseon, Hagocho 30% ethanol x (40:1) 200 300 300 300 Light anhydrous silicic acid (fluidizing agent) 10 15 20 20 Corn starch (binder) 50 Povidone K30 (Binder) 5 Ethylcellulose (binder) 4 4 Microcrystalline cellulose PH102 (excipient) 113 85 115 115 Kollidon SR (Westernized topical) 50 Sodium starch glycolate (disintegrant) 25 Crospovidone (disintegrant) 15 Croscarmellose sodium (disintegrant) 25 10 10 Magnesium stearate (lubricant) 2 5 Sodium stearyl fumarate (lubricant) 5 5 film coating 30 27 29 30 tablet total weight 430 477 483 534

Experimental Example 4-1: Dissolution evaluation

The dissolution of 200mg immediate-release tablets, 300mg immediate-release tablets, 300mg delayed-release tablets, and 300mg extended-release tablets was evaluated in the same manner as in Experimental Example 3. That is, the paddle method was used, the stirring speed was 50 rpm, and the elution temperature was 37.0 ± 0.5°C. Elution was performed in 900 ml of purified water. The dissolution results of rosmarinic acid are summarized in Table 8 and Figure 4 below.

Time/dissolution % (n=6) Example 4-1 Example 4-2 Example 4-3 Example 4-4 0 0 0 0 0 5 3 6 3 2 10 24 21 9 5 15 45 36 15 7 30 85 71 31 12 45 98 93 49 16 60 100 102 66 20 90 102 106 88 29 120 104 106 95 38 180 105 105 100 57 240 106 106 103 71 360 106 105 105 89

When looking at the time when the average dissolution rate reached 80%, it was confirmed to be 30 minutes for the ‘200mg immediate-release tablet’, 45 minutes for the ‘300mg immediate-release tablet’, 90 minutes for the ‘300mg delayed-release tablet’, and 360 minutes for the ‘300mg sustained-release tablet’. Therefore, each dosage form is judged to have characteristics of immediate-release tablets, delayed-release tablets, and sustained-release tablets, respectively.

Experimental Example 4-2: Evaluation of osteoarthritis treatment effect

Experimental animals, test substances and test groups

The effectiveness of osteoarthritis treatment was evaluated using the Beagle dog meniscectomy and anterior cruciate ligament resection (ACLT) models. As test materials, the 200mg immediate-release tablet, 300mg immediate-release tablet, 300mg delayed-release tablet, and 300mg sustained-release tablet prepared in Example 4 were used. As a positive control group, Pfizer's Celecoxib preparation (manufacturing (lot) number: EF6274, capsule) was used. As a negative control group (vehicle), tablets with the prescription shown in Table 9 below were used.

Ingredients (unit: mg) Vehicle (placebo) Microcrystalline cellulose PH102 (excipient) 319 Corn starch (excipient) 47 Sodium starch glycolate (disintegrant) 9 Magnesium stearate (lubricant) 5 film coating 29 tablet total weight 409

The composition of the test group was as shown in Table 10 below.

army gender number of animals
(number of animals) animal number Meniscectomy administered substance Number of administrations dosage
(mg) dosage
(affection) G1 M 4 1-4 N G2 M 4 5-8 Y Vehicle b.i.d. N/A 1T G3 M 4 9-12 Y Celecoxib q.d. 200 1T G4 M 4 13-16 Y 200mg immediate-release tablet t.i.d. 200 1T G5 M 4 17-20 Y 300mg immediate-release tablet b.i.d. 300 1T G6 M 4 21-24 Y 300mg delayed-release tablet b.i.d. 300 1T G7 M 4 25-28 Y 300mg extended-release tablet b.i.d. 300 1T

It was administered orally to a Beagle dog (Xi’an Dilepu Biology & Medicine Co., Ltd) according to the number of administrations in Table 9 above, and administered for 8 weeks. Specifically, the animal opened its mouth in a natural position in the breeding box, placed the test substance on the inside of the tongue, closed its mouth, and then gently stroked the larynx to swallow. After confirming that the patient had swallowed, approximately 10 mL of water was administered using a syringe.

Meniscectomy and anterior cruciate ligament resection

Before surgery, the area around both knees of the animal was removed using a clipper. The animal was anesthetized, and the area to be incised was widely disinfected using Povidone and 70% alcohol. Next, an incision was made in the skin of the right knee. The surrounding tissues were bluntly dissected to expose the articular surface of the distal femur. A defect window was created on the medial articular surface, and the anterior cruciate ligament was resected. Wound closure was performed using 4-0 nylon. For the left knee, only anterior cruciate ligament resection was performed.

One week after meniscectomy, artificial exercise (30 minutes/day) was performed on the animal once per day for 7 days.

Observation and inspection items

(1) Gait evaluation

A gait evaluation was performed to evaluate the effectiveness of the formulations prepared in Example 4 on an osteoarthritis animal model. The test was conducted twice a week before and after administration of the test substance. Gait evaluation was performed according to the evaluation criteria in Table 11 below, and images were taken using a digital camera.

Score Parameters 0 No observable lameness One Intermittent, mild weight-bearing lameness with little, if any, change in gait 2 Moderate weight-bearing lameness—obvious lameness with noticeable gait change 3 Severe weight-bearing lameness - "toe-touching" only 4 Non-weight-bearing

The results are shown in Figure 5. As shown in Figure 5, the results of the 300mg delayed-release tablet (G6) were most favorable. In particular, compared to the 300mg immediate-release tablet (G5) and 300mg sustained-release tablet (G7), only the 300mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

(2) Incapacitance test

The effect of the formulations prepared in Example 4 on weight bearing in an osteoarthritis animal model was confirmed. Weight bearing on the hind limb was measured using a paw weight meter (Incapacitance tester (1029-S, Linton instrumentation, USA). In this test, beagle dogs with osteoarthritis underwent anterior cruciate ligament resection compared to the left hindlimb that underwent anterior cruciate ligament resection only. Since more severe pain occurred in the right hind limb where the and medial meniscectomy were performed simultaneously, the beagle stood relying on the left hind limb, and as a result, the weight load on the right hind limb was measured to be relatively light compared to the weight load on the left hind limb. The weight (g) of both feet was measured while the dog's stomach was not in contact with the device sensor, and using the measured weight of each foot, the weight bearing ratio (%) was calculated using the following equation 1. The weight bearing was carried out by the feet. With the force of supporting and pressing, in normal cases, the weight of both feet is balanced and the weight bearing ratio (%) of one foot is at the level of 50%. However, as the pain becomes worse due to osteoarthritis, the weight bearing ratio (%) of the hind limb caused by osteoarthritis decreases.

[Calculation Formula 1]

Weight bearing ratio (%) = [Weight of the hind limbs causing osteoarthritis / (Weight of both hind limbs)] × 100

Before and after administration of the test substance, the level of hindlimb weight bearing distribution ratio was measured using an Incapacitance tester once a week for 8 weeks. The results are shown in Figure 6.

As shown in Figure 6, the results of the 300mg delayed-release tablet (G6) were most favorable. In particular, compared to the 300mg immediate-release tablet (G5) and 300mg sustained-release tablet (G7), only the 300mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

(3) ELISA analysis

To confirm the activity of biomarkers related to the development of arthritis, three types of cytokines, IL-1β, MMP-3, and TNF-α, were analyzed as biomarkers in joint fluid using joint fluid collected on the day of autopsy. The analysis was performed using a commercially available ELISA kit. The results are shown in Figure 7.

One of the influential mechanisms causing osteoarthritis is increased production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, as well as collagenase and stromelysin. The secretion of MMPs such as these increases, causing damage to joint cartilage. In other words, when osteoarthritis develops, the expression of MMP-3, MMP-9, and MMP-13 increases, and this increase in MMPs is known to worsen degenerative arthritis by damaging the collagen matrix that makes up cartilage. there is. In addition, it has been established that ADAMTS5 and MMP13 play a critical role in the progression of degenerative arthritis in the decomposition of aggrecan and type II collagen, representative cartilage extracellular matrices.

There are several causes of arthritis progression. Among them, the most important cause is the activity of macrophages. When macrophages existing in synovial fluid are activated by PAMPs, DAMPs, and inflammasomes, they are polarized into M1 macrophages, which play a role in inflammation. These M1-macrophages secrete inflammatory cytokines (e.g., IL-1B, IL-6, TNF-α), growth factors, MMPs (e.g., MMP1, MMP2, MMP3, MMP13), and TIMP, and as a result, Inflammation. Cartilage breakdown and osteophyte formation occur. These cytokines, MMPs, and secretory proteins maintain the activity of macrophages in the autocrine form in the M1 form, and M1-macrophages control chondrocytes including signaling of TGF-β, JNK, Akt, NF-κB, and beta-catenin. By altering signaling pathways, it promotes the degradation of extracellular matrix (ECM) components. By acting as a DAMP, the decomposed ECM stimulates macrophage activation and polarization into M1-macrophages, resulting in repeated inflammation and cartilage degradation.

As a result of checking the levels of the main cytokines (IL-1β, TNF-α) and MMPs (MMP3) secreted by macrophages that cause arthritis in the joint fluid through ELISA test, G3, G4, and G5 compared to the induced group (G2) , the level of IL-1β was significantly decreased in G6 and G7, and the level of TNF-α was significantly decreased in the test groups G3, G4, G5, and G6 compared to the induced group (G2). The level of MMP3 in joint fluid was significantly decreased in test groups G3, G4, G5, G6, and G7 compared to the induced group (G2). As shown in Figure 7, the results of the 300mg delayed-release tablet (G6) were most favorable. In particular, compared to the 300mg immediate-release tablet (G5) and 300mg sustained-release tablet (G7), only the 300mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result. Based on these results, it is believed that the levels of inflammatory cytokines and MMP3 were reduced, helping to alleviate arthritis according to the ELISA test.

(4) Immunohistochemical staining

In addition, to confirm the inhibitory effect of MMP-13 and the production effect of Collagen type II as a biomarker related to the development of arthritis, analysis of Collagen type II and MMP-13 was performed using immunohistochemical staining (IHC) markers.

Joint fluid was collected on the day of necropsy, the animal was euthanized, the defect was visually observed, and photographs were taken. The defect was extracted and fixed in 10% neutral buffered formalin solution. The collected joint fluid was stored in an ultra-low temperature freezer set to -70°C or lower until analysis. The fixed tissue went through general tissue processing procedures such as decalcification, trimming, dehydration, paraffin embedding, and sectioning to prepare a specimen for histopathological examination. Afterwards, Hematoxylin & Eosin (H&E), Safranin-O, and immunohistochemical staining (Collagen type II and MMP-13) were performed, and histopathological changes were performed using an optical microscope (Olympus BX53, Japan). Observe and perform immunohistochemical staining analysis using an Image analyzer (Zen 2.3 blue edition, Carl Zeiss, Germany).

For immunohistochemical staining, MMP13 (R&D System, MAB511-500) and Collagen II (abcan, ab34712) are used as primary antibodies. For MMP13, DAKO, Envision+System-HRP labeled polymer anti-mouse (K4001) ), For Collagen II, DAKO, Envision+System-HRP labeled polymer anti-rabbit (K4003) was used as a secondary antibody. After staining with DAB, counterstaining was performed with Hematoxylin.

The results are shown in Figure 8.

As shown in Figure 8, 300mg delayed-release tablet (G6) showed good results. In particular, compared to the 300mg immediate-release tablet (G5) and 300mg sustained-release tablet (G7), only the 300mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

(5) Histopathological analysis experiments

After H&E staining, among the OARSI score items, cartilage structure and chondrocyte levels were confirmed on the joint surface, and the levels of synovial lining, inflammatory cell infiltration, and synovial hyperplasia were confirmed in the joint space. After Safranin-O staining, the level of Proteoglycan in the joint surface was confirmed among the OARSI score items. The corresponding evaluation method was scored by referring to The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the dog (Osteoarthritis Cartilage. 2010 Oct;18 Suppl 3:S66-79.).

The results are shown in Figures 9 and 10. As shown in Figure 9, 300mg delayed-release tablet (G6) showed good results. In particular, compared to the 300mg immediate-release tablet (G5) and 300mg sustained-release tablet (G7), only the 300mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

Additionally, as shown in Figure 10, as a result of Safranin-O staining, in the osteoarthritis-induced group, normal cartilage tissue was destroyed by the induction, and the level of damage to the proteoglycan tissue increased. On the other hand, in the drug administration group, stained proteoglycan tissue was widely distributed around the synovial membrane. In H&E staining, compared to the cartilage tissue of the normal group, the osteoarthritis-induced group showed deeper cracks and erosion of the joint surface, and it was confirmed that the level of damage to the joint surface was reduced in the drug-administered group. Compared to the joint synovial tissue of the normal group, the group with induced osteoarthritis had an increased level of villi due to increased damage due to excessive infiltration of synovial cells around the joint, and in the drug-administered group, it was confirmed that this infiltration of cartilage and bone was relatively reduced. Additionally, 300mg delayed-release tablet (G6) showed good results. In particular, compared to the 300mg immediate-release tablet (G5) and 300mg sustained-release tablet (G7), only the 300mg delayed-release tablet (G6) with a specific dissolution pattern showed good results, which was a very specific result.

Claims (10)

It is a tablet or capsule formulation that contains 280-320 mg of herbal extracts of Uryeongseon, Brachialis root, and Herbaceae as active ingredients, and is taken twice a day.
When dissolving in 900 ml of water using the paddle test method at 37 ± 0.5 ℃ and 50 rpm, the release of rosmarinic acid was 40 to 70% in 45 minutes, more than 75% in 90 minutes, and more than 80% in 120 minutes. Preparations for treating arthritis. The formulation according to claim 1, wherein the content of the herbal extracts of the active ingredients, such as Uiryeongseon, Gwanrugeun, and Hagocho, is 40 to 90% by weight based on the total weight of the capsule filling excluding the weight of the uncoated tablet or empty capsule before coating. The preparation according to claim 1, wherein the preparation is a tablet. The method according to any one of claims 1 to 3, wherein when the formulation is tested for dissolution in 900 ml of water using a paddle test method at 37 ± 0.5° C. and a speed of 50 rpm, the release of rosmarinic acid is 45% in 45 minutes. ~65%, ≥80% at 90 minutes, and ≥85% at 120 minutes. The formulation according to any one of claims 1 to 3, wherein the formulation contains ethylcellulose, ethyl methacrylic acid copolymer, or a mixture thereof to control release. The formulation according to claim 5, wherein the formulation contains ethylcellulose to control release. The active ingredient contains 280-320 mg of herbal extracts of Uryeongseon, Gwanoru root and Hagochoe, and the content of the active ingredient is 40 to 90% by weight of the total weight of the mixture before coating or empty capsule filling, ethylcellulose, methacrylic A formulation characterized by controlled release comprising an acid ethyl acrylic acid copolymer or a mixture thereof. The formulation of claim 7, wherein the formulation comprises ethylcellulose. The tablet according to claim 7 or 8, wherein the content of the active ingredient is 50 to 90% by weight based on the total weight of the uncoated tablet before coating or the mixture before filling the empty capsule. The formulation according to claim 7 or 8, wherein the formulation is a tablet.