KR20020071235A - Compositions and preparations of silymarin complex with the improved bioavailability - Google Patents

Abstract

PURPOSE: A novel composition prepared by adding a suitable amount of tromethamine and poloxamer 407 to poorly water soluble silymarin to increase water solubility and improve bioavailability is provided. Therefore, the composition is surprisingly improved in an elution rate and has an LD50 of 9,756mg/kg. CONSTITUTION: This silymarin formulation composition is obtained by mixing tromethamine and poloxamer 407 in a ratio of 1:1, solubilizing in 95% ethanol, and drying under reduced pressure at 40deg.C or less. In the process, 0.01 to 1.0% by weight sodium lauryl sulfate is also added instead of poloxamer 407. The silymarin formulation is a soft gelatin capsule.

Description

Compositions and preparations of silymarin complex with the improved bioavailability}

The present invention is directed to compositions that increase the bioavailability of the silymarin formulations used in the prevention and treatment of liver disease. Silymarin is derived from a herb named under the scientific name Carduus marianus Linne (Silybum marianum). This plant has long been known as an important drug in the West, including ancient Greece. Since it was introduced in Korea in the 1970s as an interstitial agent, many pharmaceuticals based on this ingredient are on the market. Although the formulation containing silymarin as a main ingredient has already been widely applied to treat liver disease clinically, its main ingredient, silymarin, is a poorly soluble substance that is almost insoluble in water, and its bioavailability is low due to its low absorption rate in oral administration. Only%. In addition, due to these poor solubility problems, the formulations applied are very limited to tablets, capsules and suspensions. Therefore, improving bioavailability by increasing solubility has become an important task in the development of this drug. An object of the present invention is to improve the bioavailability of the drug by increasing the water solubility by properly mixing, adding, and preparing tromethamine and poloxamer 407 to poorly soluble silymarin.

Silymarin preparations are currently used as adjuvant therapy for the treatment of liver diseases such as addictive liver disease, chronic hepatitis and cirrhosis. The main ingredient, silymarin, contains four structural isomers of sillybin: sillybin, isosilibinin, sililicristin, and sililidianin. The disadvantage is that the absorption in the body is very low when administered orally. Therefore, many attempts have been made to increase the bioavailability of the silymarin formulation. In the past, attempts have been made to induce the transformation of molecules such that they are treated with or reacted with suitable chemicals to convert them into derivatives having good water solubility. In the Republic of Korea Patent Publication (Registration No .: 0138574), polysorbate was added again after mixing polyethylene glycol and ethanol to develop a formulation for increasing bioavailability, but in this case, the solubility of silymarin does not increase significantly. There is a disadvantage in that the volume of the preparation is large. In addition, recently, a patent application has been filed for dissolving silymarin in a surfactant, a cosurfactant, and an oil. However, since the spontaneous microemulsion preparation in this patent publication uses a large amount of surfactant, it may cause further irritation to the gastrointestinal disorders such as gastritis and gastric ulcer, and the volume of the preparation is inconvenient to take and diarrhea. It may also cause undesirable side reactions such as abdominal pain. In addition, Japanese Patent Application Laid-Open No. 2001-8804 discloses a test method through cannular insertion of a carotid artery, which is generally used to measure blood levels of drugs in a bioavailability comparison test in experimental animals. Direct blood collection was used. In contrast, the present invention provides high bioavailability and water solubility without combining silymarin and an exogenous compound, and does not use a large amount of surfactant, thereby minimizing undesirable side effects and reducing the number of drug doses. It has the advantage of providing. In addition, the present patent is most commonly used to measure blood concentrations of drugs, and it is an experimental method to obtain accurate experimental results by minimizing experimental errors according to individual differences. Measured.

The present invention is a study for increasing the solubility of poorly soluble silymarin, and is a result of a careful study of the chemical structure of the silymarin compound in a direction for enhancing absorption in the body. Silymarin contains the main components as shown in Figure 1, that is, the structural isomers of the same molecular weight but different structures, Silybin, Silycristin, and Silydianin. Likewise, it may contain Silandrin and Silyhermin. In terms of the structure of the main component, silinine, it has a three-part acidic structure (Resorcinol, Maltol, Guaiacol, see Fig. 1) having a phenolic hydroxyl group, and the acidic structure has solubility when combined with an alkaline substance. It can increase greatly and increase bioavailability, but when it is administered in the body, it is neutralized with acidic gastric juice and returned to poor solubility. Therefore, in the present invention, by using 3 to 4 equivalents of the alkaline substance Tromethamine (Tromethamine, FIG. 1), the present invention was completed by finding that the solubility in gastric fluid does not change significantly and the bioavailability is high. The use of tromethamine is not made with exogenous derivatives such as neutralizing with strong alkaline substances or making ester compounds with succinic acid, and using water-soluble surfactants without the use of large amounts of surfactants as is commonly known. And inventions to increase bioavailability. That is, it is an invention that can increase the bioavailability, increase the convenience of taking and avoid the occurrence of side effects by adding and preparing an appropriate amount of the body absorption promoter in the silymarin raw material.

1 shows the structure of the main components of silymarin and related substances.

Figure 2 compares the bioavailability (blood concentration curve of silybin over time) in rats.

Figure 3 compares the bioavailability (blood concentration curves of isosilbin over time) in rats.

Figure 4 compares the bioavailability (blood concentration curve of total silybin over time) in rats.

Example 1 Preparation of Raw Materials for Silymarin Formulations of the Invention

Silymarin containing 34.3% or more of silybin and tromethamine (US Pharmacopoeia) were mixed 1: 1 by weight, and poloxamer 407 corresponding to 2.5% of the weight of the mixture was added and mixed. 95.0% ethanol corresponding to 10 times the weight of the mixture was added to dissolve and the solvent was distilled off under reduced pressure. The residue was dried under reduced pressure and vacuumed at 40 캜 or lower.

Experimental Example 1. Comparison of elution rate in water, artificial gastric juice, artificial intestine and pH 4.0

The raw material of the conventional formulation and the raw material of the silymarin formulation prepared according to Example 1 were each filled with 60 milligrams of total silicide and filled into gelatin hard capsules. Each formulation was tested for dissolution at a stirring rate of 50 rpm and a temperature of 37 ° C. using a sinker in accordance with the Korean Pharmacopeia (7 revisions) method 2 (paddle method). 900 ml of artificial gastric fluid (pH 1.2 hydrochloride buffer), artificial intestinal fluid (pH 6.8, 50 mM phosphate buffer), pH 4.0 buffer (50 mM phosphate buffer) and water were used. After the dissolution test was started, about 5 mL of the eluate was taken at 5, 10, 15, 30, 45, 60, 90, and 120 minutes, and then filtered. The first 3 mL was replenished in the elution vessel, and then about 2 mL of the filtrate was collected to obtain a sample solution. The dissolution rate was calculated by correcting it. The sample solution was quantified by high performance liquid chromatography. The column was filled with 5 micrometer octadecylated silica gel in a stainless steel tube having a diameter of 4.6 mm and a length of 250 mm, and a mixture of acetonitrile and pH 3.0, 10 mM phosphate buffer as a mobile phase (45:55). ) Was used. The flow rate was 1.0mL / min and the ultraviolet / visible detector was analyzed at 288nm, and the temperature of the sample solution and the column was 40 ° C. The test liquid injection amount was 20 µl. The holding time of sillybin and isosilbinin was 5.1 minutes and 5.5 minutes, respectively. In both A and B prescriptions, the dissolution rate increased rapidly until about 30 minutes after the start of dissolution, but there was little change in the dissolution rate after 45 minutes. As shown in Table 1, the dissolution rate of the new formulation improved from about 1.8 times to 4.3 times compared to the existing formulation.

Dissolution test result pH1.2 pH4.0 pH6.8 water 2-hour dissolution rate (%) Existing Formulation (A) 19.6 ± 1.5 15.8 ± 1.8 43.3 ± 2.3 23.5 ± 0.4 New formulation (B) 48.8 ± 1.2 57.0 ± 5.0 77.7 ± 1.8 100.8 ± 2.0 Dissolution Rate Ratio (B / A) 2.5 3.6 1.8 4.3

Experimental Example 2. Comparison of Bioavailability in White Rats

The raw material of the conventional formulation and the raw material of the silymarin formulation prepared according to Example 1 were each taken as an amount of 43 mg of total silinine, suspended in 1 mL of 1% sodium carboxymethylcellulose solution, and orally administered to rats. After anesthetizing the rats fasted 24 hours before, anesthesia was performed and a carotid cannula was inserted, and each drug was administered after sufficient rest to minimize stress. The n number of each experimental group was set to n = 9 for accurate statistical processing. Immediately before administration of the drug, approximately 0.2 mL of blood was collected at 30, 60, 90, 120, 180, 240, 360, and 480 minutes after administration, and centrifuged and 100 μl of plasma was taken as a sample. 100 μl of the sample was mixed with 100 μl of pH 4.5 and 1 M sodium acetate buffer, and 40 units of β-glucuronidase, and stirred at 60 rpm for 2 hours or more to decompose all the conjugates in the plasma into free form. Afterwards, 200 µl of a pH 8.0 and 0.5M phosphate buffer containing a standard Naringenin standard, 0.05 mg / ml, was mixed and mixed with 1.0 ml of diethyl ether, followed by shaking with diethyl ether. The layers were taken, evaporated and dried with nitrogen and dried. 1 ml of the mobile phase was added to the remaining residue to obtain a sample solution. The analysis of the sample liquid was performed by the liquid chromatograph method. The column was filled with a 5 µm octadecylated silica gel for the liquid chromatograph in a stainless steel tube having a diameter of 4.6 mm and a length of 250 mm. Was used. The flow rate was 1.0 mL / min, the detector was analyzed at 288 nm with an ultraviolet / visible light detector, the column temperature was 40 ° C., and the sample injection amount was 100 μl. The holding times of silibine, isosilbinin and internal standards were about 5.8 minutes, 6.4 minutes and 7.0 minutes, respectively. Bioavailability was evaluated by AUC (area under time-blood concentration curve), Tmax (maximum blood concentration attainment time), and Cmax (highest blood concentration), and AUC was calculated in a trapezoidal manner. The significance test for the difference in bioavailability between the two formulations was a paired-t test and one-sided test.

Bioavailability Parameters (Blood Concentration of Total Silybinin) in Rats AUC (μgmin / mL) Cmax (μg / mL) Tmax (min) Existing title 2211.8 ± 588.7 9.1 ± 3.7 93.3 ± 23.6 New Product 2912.5 ± 651.7 23.8 ± 7.7 36.7 ± 13.2

In terms of AUC, 2211.8 ± 588.7µg / min / mL of the existing formulation and 2912.5 ± 651.7µg / min / mL of the new formulation showed about 1.32 times higher bioavailability than the existing formulation (p = 0.0222, n =). 9) (Table 2). Cmax was 23.8 ± 7.7 μg / mL and 9.1 ± 3.7 μg / mL, respectively, for the new formulation and the existing formulation, indicating that the new formulation had a peak blood concentration of about 2.63 times higher than the conventional formulation (p = 0.00296, n = 9) ( Table 2). Tmax was 36.7 ± 13.2 minutes and 93.3 ± 23.6 minutes (Table 2) for the new formulation and the existing formulation, respectively, and the time to reach the peak blood concentration was about 1 hour faster than the conventional formulation (Figs.

Experimental Example 3. Acute Toxicity Test

For acute toxicity test of the silymarin preparation prepared according to the example, male rats (220-240 g) were orally administered 1% carboxymethylcellulose sodium suspension. Dosages are as follows for each experimental group. Group 1: 4,878 mg / kg (1000 mg / kg silyb, n = 5), group 2: 7,317 mg / kg (1,500 mg / kg silly, n = 5), group 3: 9,756 mg / kg (Silicin 2,000 mg / kg, n = 5), Group 4: 12,195 mg / kg (Sillibin 2,500 mg / kg, n = 5), Group 5: 14,634 mg / kg (Sillibin 3,000 mg / kg, n = 5). All groups were observed for 1 week, body weight was measured, and autopsy was observed on the last day of the experiment to observe changes in organs. In the first and second groups, no lethal or toxic effects of the experimental animals were found, but in the third, fourth, and fifth groups, which were overdose 9,756 mg / kg, the mortality rate increased. Various toxic effects such as temporary weight loss, exercise loss, and meal loss were observed. Therefore, the highest nontoxic dose is 7,317 mg / kg in group 2 and the lowest toxic expression dose is 9,756 mg / kg in group 4. LD ₅₀ was higher than 9,756 mg / kg (silinine 2,000 mg / kg), which did not increase the toxicity compared to the conventional formulation and was considered safe for oral administration.

Formulation Examples 1 to 4 Preparation of Silymarin-Containing Capsule

1) 150 mg of silymarin (content 80.0 or more, containing 41.8% of silybin)

Tromethamine 150mg

Poloxamer 407 7.5mg

Magnesium stearate

Talc appropriate amount

2) 150 mg of silymarin (content 80.0 or more, silybin 41.8%)

Tromethamine 150mg

Polysorbate 80 7.5mg

Magnesium stearate

Talc appropriate amount

3) Silymarin (content 80.0 or more, silybin 41.8%) 150 mg

Tromethamine 150mg

Sodium lauryl sulfate 0.3mg

Magnesium stearate

Talc appropriate amount

4) 150 mg of silicide (content 92.4%)

Tromethamine 150mg

Sodium lauryl sulfate 0.3mg

Magnesium stearate

Talc appropriate amount

The above components are mixed by a conventional method for producing a capsule, and then filled into a gelatin hard capsule to prepare a capsule.

As described above and as can be seen through the examples, the present invention improves water solubility and improves bioavailability than conventional formulations by properly adding and preparing tromethamine and poloxamer 407 which safely promotes absorption in the body to poorly soluble silymarin. As a result, the dissolution rate was significantly improved compared to the conventional formulation, and the bioavailability was increased in the animal experiment model using rats. In addition, the acute toxicity test in rats did not increase the toxicity compared to the conventional formulation, and LD ₅₀ is more than 9,756mg / kg and is very safe.

Claims (5)

In order to increase the bioavailability of silymarin, a poorly water-soluble substance, tromethamine is mixed in a weight ratio of 1: 0.5 to 1: 2 with respect to silymarin as a body absorption accelerator, and the bioavailability increased silymarin preparation composition. 2. The silymarin formulation composition with increased bioavailability according to claim 1, wherein the mixing ratio of silymarin and tromethamine is 1: 1 by weight. The bioavailability according to claim 1 or 2, wherein the mixture is prepared by mixing 0.05-5% by weight of floxamer 407 in a mixture of silymarin and tromethamine, dissolving with 95% ethanol and drying under reduced pressure at 40 ° C or lower. This increased silymarin formulation composition. 4. The increased bioavailability silymarin formulation composition according to claim 3, wherein the sodium lauryl sulfate is mixed in an amount of 0.01-1.0% by weight instead of floxamer 407. 3. The silymarin formulation composition with increased bioavailability according to claim 1 or 2, wherein the formulation of the silymarin formulation is a hard capsule.