WO2002069962A1

WO2002069962A1 - Compositions and preparations of silymarin complex with the improved bioavailability

Info

Publication number: WO2002069962A1
Application number: PCT/KR2002/000291
Authority: WO
Inventors: Chang-Hui Koo; Dong-Young Choi; Il-Yong Song
Original assignee: Bukwang Pharmaceutical Company Ltd.
Priority date: 2001-03-05
Filing date: 2002-02-25
Publication date: 2002-09-12
Also published as: KR20020071235A; KR100385366B1

Abstract

Disclosed is a novel composition and manufacturing method of silymarin complex with enhanced bioavailability which is applied for the prevention and treatment of liver diseases and a method of preparing therefor. The composition of the invention is prepared by adding tromethamine and poloxamer 407 in certain amounts which promote the absorption of water-insoluble silymarin in the digestive tract, thereby improving water solubility and enhancing bioavailability thereof.

Description

COMPOSITIONS AND PREPARATIONS OF SI YMARIN COMPLEX WITH THE IMPROVED BIOAVAILABILITY

Technical Field

The present invention relates to a composition of a silymarin preparation with enhanced bioavailability which is applied for the prevention and treatment of liver diseases.

Background Art

Silymarin is a substance extracted from the medicinal herb with the scientific name Carduus marianus Linne (Silybum marianum). The medicinal benefits of the plant have been known to the West, including ancient Greece since before Christ. After this substance was introduced to Korea in the 1970s as an agent for treating liver disorders, many pharmaceutical agents containing it as a main ingredient have been marketed. Although a preparation containing silymarin as a main ingredient has been already applied for the treatment of liver diseases, the main ingredient, silymarin, is a water-insoluble chemical. As a result, upon its oral administration the absorption rate in the gastrointestinal tract is low, providing only about 20 to 40 % bioavailability. Moreover, such an insolubility of silymarin restricts the formulation thereof to just tablets, capsules and suspensions, that is, the application is very limited. Thus, it is an important task to achieve improved solubility of silymarin, so enhancing its bioavailability. The invention is directed to the enhancement of bioavailability of silymarin by improving its solubility. This is accomplished by compounding silymarin with tromethamine and poloxamer 407 in certain amounts, thus manufacturing a silymarin preparation.

Silymarin preparations are currently used as a supportive therapy for the treatment of liver diseases such as toxic liver damages, chronic inflammatory liver disease and liver cirrhosis. The main ingredient of the preparations, silymarin, comprises 4 structural isomers of silybin, isosilybinin, silicristin and silidianin. They are poorly soluble or insoluble in water, so the rate of absorption thereof in the body is very low, thus having a disadvantage upon oral administration. Therefore, many trials to enhance bioavailability of silymarin preparations have been conducted. In the past, attempts have been made to convert silymarin into derivatives with improved solubility by treating or reacting with appropriate chemicals. For example, polyethyleneglycol and ethanol are mixed and then added with polysorbate with the aim of developing a preparation with improved bioavailability, which has been disclosed in Korea Patent No. 0138574. Such a method, however, fails to provide a considerable increase in the solubility of silymarin. Further, there is a shortcoming that the volume of the preparation gets large. Recently, another method applied for patent in which silymarin was dispersed in a surfactant, cosurfactant and oil to make an auto-emulsified microemulsion type of soft capsule preparation has been known (Korea Pat. Laid-open No. 2001-8804). However, such a microemulsion type of preparation may cause irritation to people suffering gastric disorders such as ulcers and gastritis, due to the use of a large quantity of surfactant. Adverse effects such as diarrhea and abdominal pain may be caused. Also, the large volume of the preparation can be uncomfortable during oral administration. Moreover, in the above Korea Pat. Laid-open No. 2001-8804, when conducting a comparative test for bioavailability in an animal model, the procedure of collecting blood directly from the animal's heart was employed to assess the concentration of drug in the blood, different from a procedure of cannulation of the carotid artery, which is widely used in assessing the concentration of drugs in blood.

Contrary to the preparations in the prior art, the silymarin preparation of the invention has advantages over the above encountered problems. In the invention, the silymarin preparation provides high bioavailability and improved solubility without conjugation with exogenous compounds. A large quantity of surfactants is not used in the manufacture of the preparation, thereby minimizing undesirable side effects. Also, the preparation provides the convenience of reducing the frequency of drug administration. Further, when assessing the concentration of drug in the blood, the invention employs a procedure of cannulation of the carotid artery. This method is generally used and can minimize experimental errors due to individual variations, producing accurate results.

Disclosure of the Invention

The present invention is directed to enhance bioavailability of water-insoluble silymarin. For bolstering its absorption into body, a whole chemical structure of silymarin compound was considered. Silymarin extracted from the Silybum marianum plant mainly contains components shown in Fig. 1, which are different structural isomers with the same molecular weight, including silybin, silicristin and silidianin. Some extracts may include silandrin and silihermin shown in Fig. 1. The main component is silybin. It has three acidic moieties (that is, resorcinol, maltol and guaiacol; see Fig. 1) with phenolic hydroxyl groups, in terms of its structure. In the conventional method of the prior art, those acidic moieties can conjugate with alkaline substance so as to greatly increase the solubility, thus enhancing bioavailability, while the conjugated substances are neutralized by acidic gastric juice upon oral administration, returning to their insoluble forms. Thus, in terms of conjugation to silymarin's structure, a suitable alkaline substance is required. The inventors found that when 3 to 4 equivalents of the alkaline substance, tromethamine (Fig. 1) is employed, the solubility is not significantly reduced in gastric juice and the bioavailability is enhanced, thus the invention was accomplished. Such a use of tromethamine to enhance the solubility and bioavailability of silymarin differs from the methods of the prior art, in which a strong alkaline substance is used for neutralization, and an exogenous derivative compound, for example, succinic acid, is added to produce an ester compound. Further, in the invention, large amounts of surfactants are not used, unlike the common method in the prior art. That is, the present invention is directed to a silymarin preparation manufactured by the addition of agents which promote absorption of water-insoluble silymarin to the body, in certain amounts, thereby enhancing bioavailability thereof, increasing convenience of the drug administration and avoiding adverse effects.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 shows the structures of main components of silymarin and related substances;

Fig. 2 is a graph showing bioavailability of a silymarin preparation of the invention in comparison with a conventional preparation in SD rats, based on a time-course curve of silybin concentration in the blood; Fig. 3 is a graph showing bioavailability of a silymarin preparation of the invention in comparison with a conventional preparation in SD rats, based on a time-course curve of isosilybinin concentration in the blood; and, Fig. 4 is a graph showing bioavailability of a silymarin preparation of the invention in comparison with a conventional preparation in SD rats, based on a time-course curve of total silybin concentration in the blood.

Best Mode for Carrying Out the Invention

Example 1 : Manufacture of a silymarin preparation of the invention

Silymarin containing more than 34.3 % silybin was mixed with tromethamine (listed in U.S. Pharmacopeia) in a weight ratio of 1 : 1, and the mixture was mixed with 2.5 % poloxamer 407 by weight, relative to the weight of the mixture. The resulting mixture was dissolved in ten equivalents of 95 % ethanol, relative to the weight of the resulting mixture. The solvent was removed under reduced pressure. The residue was vacuum-dried at less than 40 °C under reduced pressure and the product was ground. Experimental example 1 : Dissolution rates of the capsule preparations in water, artificial gastric juice, artificial intestinal juice and phosphate buffer at pH 4.0

A conventional silymarin preparation (A) and the silymarin preparation manufactured according to Example 1 (B) were respectively measured out at amounts corresponding to 60 mg of total silybin. Each preparation was packed into a hard gelatin capsule. Such preparations were subjected to a dissolution test using a paddle apparatus (sinker) with a stirring speed of 50 rpm at 37 C , pursuant to the Korea Pharmacopeia (7 rϊi revised edition) II paddle method. As dissolution media, 900 ml each of artificial gastric juice (hydrochloride buffer at pH 1.2), artificial intestinal juice (50mM phosphate buffer at pH 6.8), phosphate buffer at pH 4.0 (50mM phosphate buffer) and water were employed. After the dissolution test began, respective dissolution media were collected in an amount of about 5 ml each, at 5, 10, 15, 30, 45, 60, 90 and 120 min. These media were filtered. At every collection time, about 3 ml each of the filtrates was returned to the original media, and only about 2 ml each of the filtrates were employed as respective samples for the analysis, and the calculation of dissolution rates was adjusted accordingly. The respective samples were quantified with the aid of high performance liquid chromatography. The column used was a stainless steel column of 4.6 mm in internal diameter and 250 mm in length, packed with octadecylsilyl silica gel (used in liquid chromatography) with a particle size of 5 μm. For the mobile phase, a mixture of acetonitrile and 10 mM phosphate buffer, pH 3.0 (mixing ratio 45 : 55) was used. The flow rate was 1.0 ml per minute. The analysis was performed at 288 nm using a UV/NIS detector. The samples and columns were maintained at a temperature of 40 °C . The respective samples were injected in amounts of 20 μl. In the column, the retention time of silybin and isosilybinin were 5.1 and 5.5 min, respectively. Both preparations of A and B showed steep increases in dissolution rates during the first about 30 min, followed by little change after 45 min. Considering the dissolution rates through 2 hr, the preparation (B) according to the invention exhibited a dissolution rate approximately 1.8 to 4.3-fold higher than that of the conventional preparation (A).

Table 1 : Dissolution profiles

Experimental example 2: Bioavailability of the preparations in SD rats

A conventional silymarin preparation and the silymarin preparation manufactured according to Example 1 and were respectively measured out at amounts corresponding to 43 mg of total silybin. Each preparation was suspended in a 1 ml solution of 1 % sodium carboxymethyl cellulose, and the solution was orally administered to each rat. Prior to the administeration, SD rats were fasted for 24 hrs. The rats were anesthetized and cannulation of the carotid artery was surgically performed. The cannulated rats were given sufficient rest to minimize stress, followed by being orally administered with each drug. 9 rats were assigned to each experimental group (i.e., n = 9), to give precise statistics. With regard to the each rat, 0.2 ml of blood was collected immediately prior to the administration of the drug, and at 30, 60, 90, 120, 180, 240, 360 and 480 min post-administration, followed by centrifugation to give 100 ^ plasma, which served as a specimen for the analysis. For preparing a sample for chromatograpic analysis, the 100 μl specimen was mixed with 100 μi of 1 M sodium acetate buffer at pH 4.5 and 40 unit of β-glucuronidase, and the resulting solution was stirred at 60 rpm for more than 2 hrs, thereby thoroughly disassembling the conjugates in plasma to the free forms thereof. As an internal standard, 0.05 mg/ml of naringenin with a standard quality was added to 200 μl of 0.5 M phosphate buffer at pH 8.0. Each of the mixtures thus prepared was added with 1ml diethylether, followed by shaking. Then, the diethylether layer was removed to completely evaporate and dry under nitrogen gas. The residue was added with 1ml of a mobile phase, and employed as a sample for the analysis. The analysis was performed using a liquid chromatography method. The column used was a stainless steel column of 4.6 mm in internal diameter and 250 mm in length, packed with octadecylsilyl silica gel with a particle size of 5 μm. For the mobile phase, a mixture of acetonitrile and 10 mM phosphate buffer, pH 3.0 (mixing ratio 38.5 : 61.5) was used. The flow rate was 1.0 ml/min. The analysis was performed at 288 nm using a UN/NIS detector. The columns were maintained at a temperature of 40 ^°C . The respective samples were injected in amounts of 100 μl. In the column, the retention time of silybin, isosilybinin and the internal standard were approximately 5.8, 6.4 and 7.0 min, respectively. The bioavailability was evaluated on the basis of parameters such as AUC (the area under the time-blood concentration curve), Cma (the maximal plasma concentration of the drug), and T_max (the time to reach the maximal plasma concentration of the drug). The values of AUC were obtained using the trapezoidal rule. A statistical test of significance with respect to the differences in bioavailability between two preparations was performed using a paired-t test (one-tailed test).

Table 2: Bioavailability of the preparations in SD rats

As for AUC, the conventional preparation measured 2,211.8±588J μg ^• min/mL, while the preparation of the invention measured 2,912.5±651J μg ^• min/mL. That is, the preparation of the invention exhibited bioavailability approximately 1.32-fold higher than that of the conventional preparation (p = 0.0222, n = 9, see Table 2). As for C_ma_x, the conventional preparation and the preparation of the invention measured 9.1 ±3.7 and 23.8 ±7.7 g/mL, respectively. That is, the preparation of the invention exhibited a maximal plasma concentration approximately 2.63-fold higher than that of the conventional preparation (p = 0.00296, n = 9, see Table 2). As for T max, the conventional preparation and the preparation of the invention measured 93.3±23.6 and 36.7± 13.2 min, respectively. That is, it was observed that the time to reach the maximal plasma concentration of the preparation of the invention is approximately 1 hr earlier than that of the conventional preparation (Figs. 2 to 4).

Experimental example 3 : Acute toxicity To perform an acute toxicity test for the silymarin preparation manufactured according to Example 1 , male SD rats (a weight of 200 -240 g) were orally administered with a suspension of 1 % sodium carboxymethyl cellulose. The dosages according to the respective experimental groups were as follows: for group I, 4,878 mg/kg (silybin 1,000 mg/kg, n = 5); for group II, 7,317 mg/kg (silybin 1,500 mg/kg, n = 5); for group III, 9,756 mg/kg (silybin 2,000 mg/kg, n = 5); for group IN, 12,195 mg/kg (silybin 2,500 mg/kg, n = 5); and, for group N, 14,634 mg/kg (silybin 3,000 mg/kg, n = 5). The administered groups were all observed for 1 week. The rats were weighed to check for changes of body weights. On the last day of the experiment, the rats were autopsied for observing the changes to the internal organs with naked eyes. As for groups I and II, no death of the experimental animals or toxic effects thereon were found. However, for groups III, IN and N, the death rates increased with the increase of the dosages administered. Several other toxic effects, such as temporary weight loss and lethargy, and loss of appetite were observed. Thus, the highest dosage at which the drug is non-toxic is 7,317 mg/kg, revealed in group II, while the lowest dosage at which the drug is toxic is 9,756 mg/kg, revealed in group IN. The preparation of the invention exhibited an LD₅₀ of more than 9,756 mg/kg (silybin 2,000 mg/kg). This demonstrates that the silymarin preparation of the invention is no more toxic than the conventional silymarin preparation, and is safe upon oral administration thereof.

Preparative examples 1 to 4: Preparation of capsule p reparation containing silymarin

1)

Silymarin (more than 80.0 % content, containing 41.8 % silybin) 150 mg

Tromethamine 150 mg

Poloxamer 407 7.5 mg

Magnesium stearate typical

Talc typical

2)

Silymarin (more than 80.0 % content, containing 41.8 % silybin) 150 mg

Tromethamine 150 mg

Polysorbate 80 7.5 mg

Magnesium stearate typical

Talc typical

3)

Silymarin (more than 80.0 % content, containing 41.8 % silybin) 150 mg

Tromethamine 150 mg

Sodium lauryl sulfate 0.3 mg

Magnesium stearate typical

Talc typical

4)

Silymarin (more than 92.4 % content) 150 mg

Tromethamine 150 mg

Sodium lauryl sulfate 0.3 mg

Magnesium stearate typical

Talc typical The above ingredients were mixed according to a common method for making respective capsule preparations. Each mixture was packed with a hard gelatin capsule, thereby manufacturing a capsule preparation.

Industrial Applicability

As apparent from the above description, the present invention provides a composition of a silymarin preparation. The preparation is manufactured by the addition of tromethamine and poloxamer 407 in certain amounts which promote a safe absorption of water-insoluble silymarin in the digestive tract, thereby improving water solubility and enhancing bioavailability thereof. Such a preparation thus exhibits a high dissolution rate and an enhanced bioavailability in a SD rat model, compared to a conventional preparation. Further, as revealed in an acute toxicity test, the composition is no more toxic than the conventional preparation, and the composition has an LD₅₀ of more than 9,756 mg/kg in SD rats, demonstrating safety of the composition.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

Claims:

1. A composition of a silymarin preparation with enhanced bioavailability, in which silymarin is mixed with tromethamine which promotes absorption of silymarin into the body, at a weight ratio of 1 : 0.5 to 1 : 2, thereby enhancing bioavailability of water-insoluble silymarin.

2. The composition of the silymarin preparation as set forth in claim 1, wherein silymarin and tromethamine are mixed at a weight ratio of 1 : 1.

3. The composition of the silymarin preparation as set forth in claim 1 or claim 2, wherein the silymarin preparation is manufactured by mixing the mixture of silymarin and tromethamine with poloxamer 407 at an amount of 0.05 to 5 % by weight, relative to the weight of the mixture, then dissolving the resulting mixture in 95 % ethanol and drying the resultant at less than 40 °C under reduced pressure.

4. The composition of the silymarin preparation as set forth in claim 3, wherein the mixture of silymarin and tromethamine is mixed with sodium lauryl sulfate at an amount of 0.01 to 1.0 % by weight relative to the weight of the mixture, instead of poloxamer 407.

5. The composition of the silymarin preparation as set forth in claim 1 or claim 2, wherein the silymarin preparation is formulated as a hard capsule preparation.