SK278519B6 - Water soluble inclusive complex of silybinine together with cyclodextrine, preparation method thereof and pharmaceutical composition contaning that complex - Google Patents

Abstract

This patent describes water soluble inclusive complexes of silybinine together with cyclodextrine, chosen of the group containing cyclodextrine alpha, cyclodextrine beta, cyclodextrine gamma and the suitable derivatives of them. These new inclusive complexes improve a biological availability in a significant measure, when comparing it with silybinine alone and these complexes are suitable for healing of different hepatotoxic states, as a result of that.

Description

Technical field

The invention relates to silybinin inclusion complexes with cyclodextrin or derivatives thereof, to a process for the preparation of these inclusion complexes and to pharmaceutical compositions containing the inclusion complexes as active ingredient.

BACKGROUND OF THE INVENTION

Silybinine is one of the isomers forming silymarin (or a silymarin group); more specifically, it is the isomer that is contained therein in the largest amount. It has the following general formula:

Silybinin can be obtained by extraction from Carduus marianus fruit and has interesting antihepatotoxic properties. For example, the preparation of silybinin is described in DE-A-3537656.

Silybinin itself formulated with suitable excipients in suitable proportions exhibits an interesting antihepatotoxic efficacy, predisposing it to therapeutic use in the field of some pathological conditions.

It has now been found that silybinin can advantageously be used in the form of inclusion complexes with cyclodextrin. This also constitutes the essence of the invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a silybinin suitably treated with a cyclodextrin selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin, and a suitable derivative thereof, especially an O-C1-C4-alkyl and hydroxy-C1-C4-alkyl derivative. an inclusion complex that exhibits improved bioavailability compared to the silybinin isomer.

The present invention also provides a process for preparing a silybinin inclusion complex with a cyclodextrin selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin, and a suitable derivative thereof.

The present invention also provides a pharmaceutical composition suitable for oral administration comprising as active ingredient a silybinin inclusion complex with a cyclodextrin selected from the group consisting of alpha-cyclodextrin, betacyclodextrin and gamma-cyclodextrin, and a suitable derivative thereof.

Preferably, silybinine is used whose purity in the dry state is greater than 90% (high performance liquid chromatography HPLC).

Among cyclodextrin derivatives including alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin and a suitable derivative, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, eptakis-2,6-O-dimethyl-beta-cyclodextrin and eptakis-2,3 are particularly preferred. , 6-0-trimethyl-BCTA-cyclodextrin.

The inclusion complexes of the invention can be obtained by preparing a solution of silybinin and cyclodextrin at a pH greater than 7, then adjusting the pH to less than 7 and isolating the complex formed. The process comprises suspending the silybinin in water and then adding a suitable base, preferably ammonium hydroxide, sodium hydroxide or potassium hydroxide, until complete dissolution of the silybinin. To the solution thus obtained is added cyclodextrin or a derivative thereof in an amount such that a molar ratio of silybinin / cyclodextrin of 1: 1 to 1: 4 is achieved. After a period of time ranging from 15 minutes to a few hours, the solution is evaporated or, after a substantial amount of the volatile base used has been removed, neutralized or acidified to a pH of less than 7, preferably pH 3-4, using an acid, hydrochloric acid, phosphoric acid or sulfuric acid. The complex formed is then isolated by known methods, for example by lyophilization.

A solution of silybinin and cyclodextrin can also be prepared by mixing these substances in water and adding a base as previously described.

The inclusion complexes of the invention are water soluble. That is, at least one part by weight of the complex of the invention is dissolved in 10 parts by weight of water at 20 ° C.

The solubilization and release rate of the complexes of the invention is much higher than the solubilization and release rate of silybinin alone.

Acceptable release rates are a prerequisite for useful absorption. The good absorption of the complexes according to the invention is due to the high level of silybinin achieved in this case. The release rate (solubility) determined according to USP XII (in water 37 ° C at pH 7.5 and paddle mixing) in this case is at least 60%, preferably at least 75% and especially at least 85%. The most preferred release rate is at least 90%.

The silybinin-cyclodextrin inclusion complex of the invention may preferably be formulated in a pharmaceutical composition suitable for oral administration using excipients such as anionic surfactants such as lauryl sulfate, polyalcohols having a molecular weight of from 400 to 20,000, preferably from 1,500 to 3,000 , simple and cross-linked polyvinylpyrrolidones and cellulose and derivatives thereof such as hydroxypropylmethylcellulose and sodium carboxymethylcellulose and ethylcellulose.

The most preferred oral pharmaceutical forms are tablets, hard or soft gelatin capsules, and a granular product in the sachets.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated in more detail by the accompanying drawings, in which:

Fig. 1 shows a diffraction X-ray diffraction pattern of silybinin, beta-cyclodextrin, their physical mixture and the complex of Example 1;

Fig. 2 shows a diffraction X-ray diffraction pattern of silybinin, DIMEB and the complex of Example 7;

Fig. 3 is a diffraction X-ray diffraction pattern of silybinin, HPbetaCD and the complex of Example 8; FIG. 4 shows the thermoanalytic curves of silybinin,

SK 278519 Β6

Fig. Figure 5 shows DSC curves of the complex of Example 7 and the physical mixture of silybinin and DIMEB. Figure 6 shows DSC curves of the complex of Example 8 and the physical mixture of silybinin and HPbetaCD. 7 shows the UV spectrum of the complex of Example 7 (1) and the complex of Example 8 (2).

In the following, the invention will be explained in more detail by means of specific embodiments thereof, which, however, are illustrative only and are not intended to limit the scope of the invention as defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of the silybinin-betacyclodextrin inclusion complex (1: 1 molar ratio) g of silybinine (93% purity, 0.019 moles) is suspended in 200 ml of deionized water and 75 ml of 35% aqueous solution are added dropwise with magnetic stirring over 10 minutes. ammonium hydroxide solution. When the addition of ammonium hydroxide is complete, 24.78 g of beta-cyclodextrin (Fisher 13%, 0.019 mol) is added in one portion. Stirring was continued for a further 35 minutes, after which water was distilled off under vacuum at 50 ° C for 15 minutes. A yellow solid is obtained, which is washed twice with 30 ml of acetone and dried to give 24.6 g of a light yellow, water-soluble solid.

The X-ray diffraction pattern of this product shows characteristic peaks of the silybinin-beta-cyclodextrin inclusion complex (1: 1) (e.g., 20 = 18.8). Melting point of the complex: 250 ° C UV (max.) = 287 nm (MeOH).

This silybinin-beta-cyclodextrin inclusion complex has higher solubility under the same experimental conditions as silybinin and silymarin. This is evident from the following Table 1, which shows the solubility values of the silybinin-beta-cyclodextrin inclusion complex prepared according to Example 1 (148 mg silybinin-beta-cyclodextrin complex corresponds to 40 mg pure silybinin), silybinin alone (40 mg) and silymarin in gastric juice at pH 6.8.

This evaluation was carried out according to the above USP method and the determination of the contents of the individual substances was performed in UV light by comparison with known standards.

Table 1 Solubility of the silybinin-beta cyclodextrin, silybinin and silymarin inclusion complex

Test substance Solubility (%) after: 1 min. 4 min. 5 min. 10 min 20 min. Silybinín 6 6,5 7 8 9 silymarin 34 47 49 50 55 Inclusion complex beta-silybinin 74 90 95 97 100 cyclodextrin

The X-ray diffraction pattern of the silybinin-beta-cyclodextrin inclusion complex prepared according to Example 1 has several characteristic significant peaks (e.g., 20 = 18.8) that are not present in the powder X-ray diffraction pattern of a mechanical mixture of both components, i. silybinin and beta-cyclodextrin; furthermore, no peaks (20 = 24.5; 14.6; 32; 22.7) are characteristic of the diffraction X-ray diffraction pattern of the silybinin-beta-cyclodextrin inclusion complex characteristic of the diffraction X-ray diffraction pattern of said mechanical mixture of said individual components.

Example 2

Preparation of the silybinin-betacyclodextrin inclusion complex (1: 1 molar ratio) g of silybinine (93% purity, 0.010 mol) was suspended in 200 ml deionized water and 75 ml of 35% aqueous solution was added dropwise with magnetic stirring over 10 minutes. ammonium hydroxide solution. After the addition of ammonium hydroxide was complete, 24.78 g of beta-cyclodextrin (Fisher 13%, 0.010 mol) was added in one portion. Stirring was continued for a further 35 minutes, after which excess ammonia gas was removed in vacuo, the pH was adjusted to 7 with 6 N hydrochloric acid and the mixture was lyophilized. After washing twice with 30 ml of acetone, a yellow solid is obtained.

Product yield: 26.2 grams, silybinin content (determined by HPLC): 24.7%.

The complex obtained has the same spectral characteristics as the product of the previous example.

Example 3

Preparation of silybinin-beta-cyclodextrin inclusion complex (molar ratio 1: 2)

Proceed as in the previous examples, using 35 g of pure silybinine and 211.7 g of beta-cyclodextrin (corresponding to 183.7 g of dry matter). 218 g of a product containing (HPLC) 15.41% silybinin and 2.5% KF are obtained.

Example 4

Preparation of silybinin-gamma-cyclodextrin inclusion complex (molar ratio 1: 1)

The procedure was as in Example 1 using 5.1% gamma-cyclodextrin with a purity of 98% and 2.04 g of a silybinin with a purity of 91% (corresponding to 1.857 g of pure silybinin). 5.9 g of silybinin-gamma-cyclodextrin inclusion complex (1: 1) are obtained.

Example 5

Preparation of the silybinin-DIMEB inclusion complex (eptakis-2,6-0-dimethyl-beta-cyclodextrin) (molar ratio 1: 1)

The procedure was as in Example 1 using 32.3 g of silybinine with a purity of 93% and 82.4 eptakis-2,6-O-dimethyl-beta-cyclodextrin. 97.2 g of silybinin-eptakis-2,6-O-dimethyl-beta-cyclodextrin inclusion complex (1: 1) are obtained.

The products obtained in the previous examples can be used to prepare pharmaceutical compositions by adding suitable excipients according to the procedures described in Examples 9 to 13 below.

Example 6

Preparation of silybinin-beta-cyclodextrin complex (molar ratio 1: 2.75)

109.76 g of beta-cyclodextrin (96.6 mmol) are dissolved in 600 ml of distilled water. 16.94 g of silybinin (35 mmol) are then suspended in the solution thus obtained. About 75 ml of N aqueous sodium hydroxide solution are then added dropwise to the suspension with stirring over 10 minutes. Silybinin dissolves slightly to form a yellow solution. To adjust the pH of the solution to between 3.2 and 3.4, a 2 N aqueous phosphoric acid solution is added to the solution (about 100-105 ml of said aqueous phosphoric acid solution must be added thereto). During said dissolution and said neutralization, it is kept below 20 ° C using an ice bath. The solution loses color at a pH below 5.

The ultraviolet spectrum of this solution, when diluted 1: 800 with 50% ethanol, shows that silybinin is present in the solution in an unionized form. The slightly opalescent beige-colored solution is then filtered through a sintered glass 04 filter. The solid complex is isolated from the solution by freeze-drying.

140 g of silybinin-beta-cyclodextrin complex are obtained. Active substance content (measured by UV spectrophotometry): 12.1% by weight. The loss on drying (105 ° C under vacuum for 2 hours) is 9.5% by weight.

Tests were performed to determine the release rate of silybinin alone, silymarin and silybinin-beta-cyclodextrin complex using USP XXII (paddle stirring at pH 7.5 and 37 ° C). These assays showed 96% solubility of the complex compared to 0% silybinin solubility and 53% total silymarin solubility, corresponding to 37% silybinin solubility.

Example 7

Preparation of silybinin-2,6-di-O-methyl-beta-cyclodextrin complex (molar ratio 1: 2,2)

The procedure is as in Example 6 except that 110 g of 2,6-di-O-methyl-beta-cyclodextrin (DIMEB, 82 mmol) and 18 g of silybinin (37 mmol) are used. 140 g of silybinin DIMEB complex are obtained. Active ingredient content (measured by UV spectrophotometry): 12.6% by weight. Loss on drying (at 105 ° C under vacuum for two hours): 5.0% by weight.

Example 8

Preparation of silybinin-hydroxypropyl-beta-cyclodextrin complex (molar ratio 1: 2.2)

100 g of hydroxypropyl-beta-cyclodextrin (HPbetaCD, 76 mmol; mean degree of substitution per cyclodextrin unit [PS] = 3.0) is dissolved in 700 ml of water. 17 g of silybinin (35 mmol) are then added to the resulting solution, and about 70 ml of 1N aqueous sodium hydroxide solution are added dropwise to the resulting suspension over a period of 10 minutes. Further treatment of the solution is the same as for the preparation of the DIMEB complex and 130 g of silybinin-HPbetaCD complex is obtained. Active ingredient content: 12.7% by weight.

Loss on drying (at 195 ° C under vacuum for 2 hours): 7.0% by weight.

X-ray diffraction analysis of the complexes of Examples 7 and 8 Diffraction X-ray diffraction patterns of pure silybinin, DIMEB and HPbetaCD and the corresponding complexes were recorded on a Philips powder diffractometer using Cu-K-radiation. A comparison of the X-ray diffraction matrices (Figs. 2, 3) shows that 20 complex maxima of crystalline silybinin and DIMEB have disappeared in the complexes, which have a completely different structure in both cases.

Thermoanalytical studies

Thermoanalytical studies were performed on the Du Pont thermal analyzer system 990. TG- (thermogravimetry), DTG- (differential thermography), DSC- (differential scanning calorimetry) and TDA (thermal evolution analysis) curves of the active ingredients alone were obtained, mechanical mixtures with DIMEB and HPbetaCD and their complexes with DIMEB and HPbetaCD.

The DSC curves of silybinin (Fig. 4) show that the decomposition of this substance starts at about 130 ° C and up to 160 ° C, with a weight loss of about 4-5% determined. The second decomposition step of silybinin begins at a temperature greater than 250 ° C in the region of decomposition of the cyclodextrin. The decomposition step taking place at a lower temperature is suitable for distinguishing between complex and uncomplexed product. The DSC curves of the complexes (Figs. 5, 6) show that silybinin degradation occurs only at a higher temperature of about 175-180 ° C.

Solubility of the complexes of Examples 7 and 8

250 mg of the DIMEB- and HPbetaCD-complex were readily dissolved in 2 ml of distilled water, and at pH 1.4 adjusted with hydrochloric acid the solution became clear. The UV-spectrum of the aqueous solutions recorded after dilution of 1: 750 with 50% ethanol indicates beyond doubt that silybinin is complexed with DIMEB and HPbetaDC in unionized form (Fig. 7). pH of aqueous solutions: DIMEB complex: 3.3 HPbetaCD complex: 3.4

In the case of the DIMEB complex solution, there is no solid phase excretion one day after the preparation of this solution. The DIMEB complex solution remains clear during storage for more than 10 days. Also, the UV-spectra remain unchanged during this time, indicating good stability of said silybinin complexes in solutions.

Example 9

Pharmaceutical composition comprising the silybinin-beta-cyclodextrin inclusion complex (molar ratio 1: 2)

Silybinin 40.0mg

Beta-cyclodextrin 164.7mg

Pharmacoat 606 10.0mg

Sodium lauryl sulphate 15.0mg

Polyethylene glycol 15.0mg

Lactose 103.8mg

Magnesium stearate 1.5mg

Size 1 hard gelatine capsules.

SK 278519 Β6

The silybinin and beta-cyclodextrin were stirred in a 7: 3 v / v mixture of water and methanol at 45 ° C for two hours, then the methanol was evaporated and the remaining solution was lyophilized. The lyophilized product thus obtained is mixed with a 2% solution of Pharmacoat 606 in a 9: 1 by volume mixture of methylene chloride and ethanol. The resulting mixture is granulated on a 1.2 mm mesh screen, dried and separated from the waste on a 0.840 mm mesh screen. The granular product is then encapsulated in size 1 gelatin capsules.

Said binder solution may be replaced by a solution of polyvinylpyrrolidone in a mixture of ethanol and water or an aqueous solution of gelatin. The molecular weight of the polyethylene glycol may range from 1,500 to 10,000.

Example 10

Pharmaceutical composition comprising silybinin-beta-cyclodextrin inclusion complex (molar ratio 1: 2.75) Silybinin-beta-cyclodextrin inclusion complex 330.0 mg

Pharmacoat 606 10.0mg

Sodium lauryl sulphate 15.0mg

Polyethylene glycol 15.0mg

Lactose 103.8mg

Magnesium stearate 1.5mg

The inclusion complex was triturated with a 2% solution of Pharmacoat 606 in a mixture of methylene chloride and ethanol. The mixture thus obtained is then granulated on a 1.2 mm mesh screen, dried and the granules obtained are separated from waste on a 0.840 mm mesh screen. The granular product is then encapsulated in size 1 capsules.

Said binder solution may be replaced by a solution of polyvinylpyrrolidone in a mixture of ethanol and water or an aqueous solution of gelatin. The molecular weight of the polyethylene glycol may range from 1,500 to 10,000.

Example 11

Pharmaceutical composition comprising the silybinin-gamma-cyclodextrin inclusion complex (molar ratio 1: 2)

The procedure was as in Example 9, using: Silybinin 40.0 mg a

Gamma-cyclodextrin 188.1 mg.

Example 12

Pharmaceutical composition comprising the silybinin-beta-cyclodextrin inclusion complex (molar ratio 1: 4)

The procedure is as in Example 9, using:

Silybinin 40.0 mg

Beta-cyclodextrin 328.4 g

Pharmacoat 606 10.0 mg

Sodium lauryl sulfate 15.0 mg

Polyethylene glycol 15.0mg

Lactose 129.1mg

Magnesium stearate 1.5mg.

Size 0 hard gelatine capsules

Example 13

Pharmaceutical composition comprising silybinin-DIMEB inclusion complex (1: 2 molar ratio)

The procedure is as in Example 9, using:

Silybinin 40.0 mg a

DIMEB 220.77 mg.

The absorption of the complexes of the invention was evaluated by oral administration, and the level of silybinin in the bile was examined and measured by high performance liquid chromatography (HPLC). This test was chosen because it is known that the active ingredients of the silymarin group are most concentrated in the bile.

These experiments were performed with Wistar rats having a body weight of 180-200 g. The test compositions were administered to these experimental animals by gastric intubation. The animals were not fed for 16 hours. The test composition was dissolved in water containing 10% arabic gum, each animal being treated with 5 ml of each solution per kilogram of body weight.

Immediately after administration of the test compound, the test animals were anesthetized and operated to block the common bile duct and insert the catheter into the bile duct to collect the bile for 24 hours. For analytical determination, 1 ml of bile is always treated as described by D. Lorenz et al., Pharmacokinetic Studies with Silymarin in Human Serum and Bile - Meth. and Find. Exptl. Clin. Pharmacol. 6 (10), 655-661 (1984).

The results obtained by this test are shown in Table 2 below, where:

- in Test A, 20 mg / kg of silymarin was administered as a known commercial formulation, corresponding to 8 mg / kg of pure silybinine,

Test B gave 8.79 mg / kg silybinin having a purity of 91% (determined by high performance liquid chromatography - HPLC), corresponding to 8 mg / kg pure silybinin; silybinin is administered as such, without excipients,

in Test C, 29.2 mg / kg of the silybinin-beta-cyclodextrin inclusive complex described in Example 1 was administered having a silybinin content of 27.4%, corresponding to 8 mg / kg of pure silybinin.

Table 2

Product Number of animals Bile volume Bile 0-24 hrs. pg / ml 0-24 hrs. gg / rat % silybinin excreted Silybinin (Test B) 12 12.2 1.0 11.9 0.6 Silybinin (Test A) 15 11.4 6.4 73.8 Complex of Examples 1 14 14.3 17.6 256.6 11.7 (test C)

The mean volume of bile produced over 24 hours by control rats (no drug) was 13.4 ml.

The analysis of the results obtained shows that by administering a composition containing the inclusion complex as an active ingredient, otherwise otherwise equal amounts of active ingredient in all tests, the silybinin concentration level is more than twice that of Test A and nearly 20-fold compared to the composition used in Step B.

In another assay, the resorption of silymarin, silybinin and the complex of Example 6 was evaluated. To this end, the excretion of silybinin in rat bile was determined under conventional model conditions. Anesthetized Wistar SPF male rats held under conventional conditions were subjected to a procedure in which

A cannula was inserted into the bile duct. Following intragastric administration of the test compound (5 mg silybinin / kg), bile fluid was collected for 5 hours. The silybinin content of the bile liquid was determined by quantitative thin layer chromatography.

The following results were obtained:

Use of at least one complex according to any one of claims 1 to 6 for the preparation of a pharmaceutical composition for hepatotherapy.

drawings

Percentage excretion of silybinin in rat bile: silymarin: 11.90 g ¹ ' Silybinín: 0.21 g ² ' complex: 17.30 g ³⁾

⁰ average calculated from 5 experiments ²⁾ average calculated from 10 experiments average calculated from 7 experiments

These results show that the complex of the invention is more and more readily absorbed than silybinin and silymarin.

Claims (7)

PATENT CLAIMS A water-soluble inclusion complex of silybinin with a cyclodextrin having a water solubility of at least 60% as determined by USP XXII, wherein the cycolodextrin is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and a derivative thereof). The molar ratio of silybinin to cyclodextrin is 1: 1 to 1: 4 The water-soluble inclusion complex according to claim 1, characterized in that it contains as cyclodextrin beta-cyclodextrin. A water-soluble complex according to claim 1, characterized in that the molar ratio of silybinin to beta-cyclodextrin is 1: 2, 1: 2.75 or 1: 4. The water-soluble inclusion complex according to claim 1, wherein the cyclodextrin is selected from the group consisting of eptakis-2,6-O-dimethyl-beta-cyclodextrin, eptakis-2,3,6-O-trimethyl-beta- cyclodextrin; and O-hydroxypropyl-beta-cyclodextrin. A process for preparing an inclusion complex according to claim 1, comprising preparing a solution of silybinin and cyclodextrin in water at a pH of greater than 7, adjusting the pH of the obtained solution to a pH of less than 7 by adding an acid, and isolating the formed complex. The method of claim 7, comprising suspending silybinin in water, adding a base to dissolve silybinin, adding cyclodextrin, lowering the pH to less than 7 by adding an acid, and isolating the complex or suspending silybinin and cyclodextrin in water, adding a base to dissolve these compounds, adjust the pH to less than 7 by adding an acid, and isolate the complex. A pharmaceutical composition comprising a therapeutically effective amount of at least one inclusion complex according to any one of claims 1 to 6, optionally in combination with pharmaceutically acceptable carriers and / or excipients.