FI102459B - Method for preparing water-soluble silybinine inclusion complexes - Google Patents

Description

102459

The present invention relates to a process for the preparation of water-soluble silybinine inclusion complexes from silybinine and cyclodextrin or a derivative thereof. This invention relates to a process for the preparation of water-soluble silybinine inclusion complexes from silybinine and cyclodextrin or a derivative thereof.

10 Silybinine is one of the isomers of silymarin (or a group of silymarins) - more precisely, it is the isomer most present - and has the formula: 15 ^ \ - / 0 \ ^ - ch2oh A / V0CH3

I n x OH

I II oh OH o 20

It can be obtained by extraction from the fruit of Carduus marianus,. and has interesting antihepatotoxic activity.

The preparation of silybinine is described, for example, in DE 3537656.

Silybinine itself, when formulated in appropriate proportions with suitable excipients, has interesting antihepatotoxic activity, which makes it of particular interest in a number of pathological conditions.

According to the invention, inclusion complexes can be provided by preparing a solution of silybinine and cyclodextrin in water at a pH above 7, adjusting the pH of the solution to 2 102459 below 7 by adding a suitable acid, and isolating the complex formed.

Preferably, silybinine is used with an HPLC titer of less than 90% water. Of the α-, (3- and γ-cyclodextrin and their suitable derivatives, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, eptakis-2,6-O-dimethyl-3-cyclodextrin and eptakis-2,3 , 6-0-trimethyl-p-fal-lodekstriini.

10

A preferred embodiment of the process involves suspending silybinine in water and then adding a suitable base, preferably ammonium hydroxide, sodium or potassium hydroxide, until complete dissolution of the silybinine. To the solution thus obtained, cyclodextrin or a selected derivative thereof is added in such an amount as to obtain a silybinine: cyclodextrin molar ratio of 1: 1 to 1: 4. After a period of between 15 minutes and a few hours, the solution is evaporated or, after most of the volatile base used has been removed, the pH of the solution is adjusted to a neutral or acidic pH below 7, preferably 3-4, using a suitable acid, e.g. HCl, H 3 PO 4, H 2 SO 4. The complex is then isolated by known techniques, for example by lyophilization. A solution of silyl binine and cyclodextrin can also be prepared by mixing them in water, and continuing by adding the base described above.

The inclusion complexes obtained according to the invention are water-soluble.

. This means that they have a solubility of at least 1 part to 10 parts of water (by weight) at 20 ° C.

The dissolution and release rates of the complexes obtained according to the invention are much higher compared to silybinine. An acceptable rate of release is a prerequisite for useful absorption. The good absorption of the complexes obtained according to the invention results in high levels of silybinine. The release rate (solubility) is at least 60% as determined by the USP XII paddle method in water at 37 ° C at pH 7.5, preferably at least 75%, and especially at least 85%. Most preferably it is at least 90%.

5

The novel silybinine inclusion complex with cyclodextrin can be suitably formulated into pharmaceutical compositions suitable for oral administration using excipients such as anionic surfactants such as sodium lauryl sulfate, polyalcohols having a molecular weight of 400 to 20,000, preferably 1500 to 3000, simple. attached polyvinylpyrrolidones, cellulose and derivatives such as hydroxypropylmethylcellulose, sodium carboxymethylcellulose and ethylcellulose.

15

The most preferred oral pharmaceutical forms are tablets, hard or soft gelatin capsules, and granulated products in sachets.

Figure 1 shows X-ray diagrams of silybinine, β-cyclodextrin, a physical mixture thereof and the complex of Example 1.

Figure 2 shows an X-ray diffraction pattern of the complex of silybinine, DIMED and Example 7.

Figure 3 shows an X-ray diffraction pattern of the complex of silybinine, DIMED and Example 8.

Figure 4 shows the thermal analysis curves of silybinine.

Figure 5 shows DSC curves of the complex of Example 7, and a physical mixture of silybinine and DIMED.

* • · 4 102459

Figure 6 shows DSC curves of the complex of Example 8, and a physical mixture of silybinine and HPPCD.

Figure 7 shows the UV spectrum of the complex of (1) Example 7 and (2) Example 8 complex.

The following examples are presented to illustrate the invention without limiting it.

10 Example 1

Preparation of silybinine: β-cyclodextrin inclusion complex (1: 1 molar ratio) 10 g of silybinine (titer 93%, 0.018 mol) are suspended in 200 ml of deionized water, and 75 ml of 35% ammonium hydroxide are added dropwise with stirring with a magnetic stirrer over 10 minutes. . When the addition is complete, 24.78 g of β-cyclodextrin (Fisher 13%; 0.019 mol) are added in 1 portion. Stirring is continued for a further 35 minutes and then the water is distilled off over 15 minutes in vacuo at 50 ° C. The resulting yellow solid is washed twice with 30 ml of acetone and dried to give 24.6 g of a pale yellow solid ** 'which is water-soluble and shows X-ray analysis of the silybinine: β-cyclodextrin inclusion complex (1: 1). (e.g., 20 = 18.8) characteristic peak; melting point 250 ° C.

UV (max) 287 nm (MeOH).

The inclusion complex according to the invention shows a higher dissolution compared to that seen with either silybinine or silymarin under the same experimental conditions.

• · 5 102459 This characteristic is shown in the graph of Table 1, which shows the dissolution curves for silybinine-β-cyclodextrin complex prepared according to Example 1 (148 mg of complex corresponds to 40 mg of pure silybinine), dissolved in silybinine (40 mg) and silymarin gastric acid at pH 6.8.

The evaluation has been performed according to the USP paddle method and the assays have been performed against standards known by UV.

Table 1 i

SILYBININ: DISSOLUTION TEST FOR CYCLODEXTRIN COMPLEX

Il M ... ......

'i m .......................

il Y

V I * -o- Silybinine n I - · -. silymarin I -Hi- | β-cyclode / silybinine (I / i’ll f / »» / ............................

r * ·) /

^ Jf..P - a · · · e -—---------— "O

O '2 4 il e | (| 12 H 10 uTi time (min)

The X-ray diffraction pattern of the inclusion complex of Example 1 is characterized by the presence of some significant peaks (e.g., 20 * 18.8) not included in the X-ray diffraction pattern of the powder formed by mechanically mixing the 2 components; moreover, the peaks (20 * 24.5; 14.6; 32; 22.7) characteristic of the diffraction pattern of the mechanical mixture of these 2 components are not present in the X-ray diffraction pattern of the above-mentioned inclusion complex.

«• · 6 102459 4r

Example 2 '

Preparation of silybinin: β-cyclodextrin inclusion complex (molar ratio 1: 1) 10 g of silybinine (titer 93%, 0.019 mol) are suspended in 200 ml of deionized water, and 75 ml of 35% ammonium hydroxide are added dropwise with stirring over 10 minutes with a magnetic stirrer. When the addition is complete, 24.78 g of β-cyclodextrin (Fisher 13%; 0.019 mol) are added in 1 portion. Maintain stirring for a further 35 minutes, then remove the excess gaseous ammonia in vacuo, adjust the pH to 7 with 6N hydrochloric acid, lyophilize, wash twice with 30 ml each of acetone to give 26.2 g of a yellow solid (HPLC of silybinine). titer 24.7% ta) having the spectral characteristics of the product obtained in the previous example.

Example 3

Preparation of silybinine: β-cyclodextrin inclusion complex (molar ratio 1: 2)

The operations described in the previous examples are performed using 35 g of pure silybinine and 211.7 g of β-cyclodextrin, corresponding to 183.7 g of anhydrous. 218 g of product are obtained with a silybinine titer of 15.41% and a KF · 2.5%.

Example 4

Preparation of silybinine: β-cyclodextrin inclusion complex (1: 1 molar ratio)

The operation described in Example 1 is carried out using 5.1 g of β-cyclodextrin with a titer of 98% and 2.04 g of silybinine with a titer of 91%, corresponding to 1.857 g of pure silybinine. 5.9 g of silybinine: β-cyclodextrin inclusion complex (1: 1) are obtained.

4 7 102459

Example 5 t-

Silybinine: Preparation of DIMED (eptakis-2,6-O-dimethyl-β-cyclodextrin) inclusion complex (1: 1 molar ratio)

The operation described in Example 1 is performed using 32.3 g of silybinine with a titer of 93% and 82.4 g of DIMEB. 97.2 g of silybinine: eptakis-2,6-O-dimethyl-β-cyclodextrin inclusion complex (1: 1) are obtained.

The products obtained in the above examples can be used in the preparation of pharmaceutical compositions with the addition of suitable excipients and in the ways described in the following Examples 9-13.

Example 6

Preparation of silybinine-β-cyclodextrin complex (molar ratio 1: 2.75) 109.76 g of β-cyclodextrin (96.6 mmol) was added dissolved in 600 ml of distilled water; To the solution was added 16.94 g of silybinine (35 mmol) to give a suspension. About 75 ml of 1N sodium hydroxide solution was added dropwise over 10 min with constant stirring. Silybinine readily dissolved to give a yellow solution. 2N Phosphoric acid was added to adjust the pH to between 3.2 and 3.4 (about 100 to 105 ml of phosphoric acid is required). The temperature of the solution was kept below 20 ° C during the dissolution and neutralization process using an ice bath. The color of the solution disappeared at a pH below 5. The UV spectra of the solution after a 1: 800 dilution with 50% (v / v) ethanol showed the presence of silybinine in non-io- ·; in solution in solution. The slightly cloudy beige solution was filtered through a 04 glass filter. The solid complex was isolated from the clear solution by lyophilization.

140 g of silybinine-β-cyclodextrin complex were obtained. Active ingredient content: 12.1% (w / w) measured by UV- * 8 102459 t »I · spectrometry. Weight loss on drying (105 ° C; vacuum for 2 hours): 9.5% (w / w).

Release experiments with silybinine, silymarin, and the complex of this example were performed according to USP XXII, paddle device at pH 7.5 at 37 ° C. The experiments showed 96% release (solubility) of the complex as opposed to 0% release of silybinine and 53% release of silymarin corresponding to 37.1% release of silybinine.

Example 7

Preparation of silybinine / 2,6-di-O-methyl-β-cyclodextrin complex, (molar ratio 1: 2.2) i

The procedure of Example 6 was repeated except that 110 g of 2,6-di-O-methyl-β-cyclodextrin (DIMED; 82 mmol) and 18 g of silybinine (37 mmol) were used.

140 g of silybinine / DIMED complex were obtained. Active ingredient content: 12.6% (w / w) as measured by UV spectrometry. Weight loss on drying (105 ° C under vacuum for 2 hours): 5.0% (w / w).

Example 8

Preparation of silybinine / hydroxypropyl-β-cyclodextrin complex (molar ratio 1: 2.2) 100 g of hydroxypropyl-β-cyclodextrin (HP CD) 76 mmol, PS * 3.0 (PS "average degree of substitution / cyclodextrin unit) were dissolved in 700 ml To water was added 17 g of silybinine (35 mmol) to give a suspension.

To the solution was added dropwise 70 ml of 1N sodium hydroxide over 10 min. Further work-up of the solution was identical to the preparation of the DIMED complex to give 130 g of silybinine / HPBCD complex. Content of active ingredient: 12.7% (w / w). Weight loss on drying (105 ° C ’under vacuum for 2 hours): 7.0%.

9 102459 it

X-ray analysis of the complexes of Examples 7 and 8.

X-ray powder diffraction patterns of pure silybinine, DIMED, HP-BCD, and complex samples were recorded on a Philips powder diffractometer using Cu-K radiation. Comparing the X-ray models (Figures 2 and 3), the characteristic 20 peaks of crystalline silybinine and DIMED disappeared, and the complexes showed a completely different structure in both cases.

Thermodynamic tii-hlcinmlcamt

Thermodynamic studies were performed with a Du Pont 990 Thermal Analyeer system. The drug in saunas as well as its mechanical mixtures with DIMED and HP-E CD and the complexes T6 (thermo-gravimetry), DTG (differential TG), DSC (differential scanning calorimetry) and TEA (heat generation analysis) were taken simultaneously.

DSC curves of silybinine (Figure 4) show that the degradation of the drug begins at about 130 ° C and up to 160 ° C a loss of about 4-5% is observed. The second phase of drug degradation begins above 250 ° C in the region of cyclodextrin degradation. The decomposition step at a lower temperature is suitable for differentiating complexed and uncomplexed products. The DSC curves of the complexes (Figures 5, 6) show that degradation of silybinine is observed at higher temperatures only at about 175-180 ° C.

Solubility properties of the complexes of Examples 7 and 8.

250 mg of DIMED and HPBCD can be easily dissolved in 2 ml of distilled water and pB of 1.4 hydrochloric acid to give a clear solution. The OV spectra of the aqueous solutions -: recorded after 1: 750 dilution with 50% (v / v) ethanol - unequivocally show that silybinine is present in both DIMED and HPE CD in non-ionized form (Figure 7).

10 102459

PH of aqueous solutions: DIMED complex: 3.3 HP-BCD complex: 3.4 1 day after reconstitution No precipitation was observed in the DlMED complex solution. The DIMED complex solution remained clear for more than 10 days of storage. The measured UV spectra remained unchanged, indicating good stability of silybinine complexes in solutions.

Example 9

Pharmaceutical compositions containing silybinin: β-cyclodextrin inclusion complex (molar ratio * 1: 2).

titratable silybinine 40.0 mg β-cyclodextrin 164.7 mg

Pharmacoat 606 10.0 mg sodium laurilsulfate 15.0 mg polyethylene glycol 15.0 mg lactose 103.8 mg magnesium stearate 1.5 mg

Assemble the hard gelatin capsules "1".

Silybinine and β-cyclodextrin are stirred in a water / methanol solution (7: 3) at 45 ° C for 2 hours, then the methanol is evaporated and the product is lyophilized.

The lyophilized product is kneaded with 2% w / v Pharmacoat 606 in methylene chloride / ethanol solution (9: 1). The product thus obtained is granulated with a 1.2 mm mesh mesh, dried and scraped with a 0.840 mm mesh mesh. The granulated product is sealed in capsules of size “1”.

The binder solution can be replaced with a solution of polyvinylpyrrolidone in ethanol / water or an aqueous solution of gelatin. The molecular weight of polyethylene glycol can range from 1,500 to 10,000.

* 102459 11 * * ggime £ kfe & _10

Pharmaceutical composition containing silybinine: β-cyclodextrin inclusion complex (molar ratio 1: 2.75) silybinine: 3-cyclodextrin inclusion complex 330 mg

Pharmacoat 606 10.0 mg sodium laurilsulfate 15.0 mg polyethylene glycol 15.0 mg lactose 103.8 mg magnesium stearate 1.5 mg

The inclusion complex is kneaded with 2% w / v Pharmacoat 606 in methylene chloride / ethanol solution (9: 1). The product thus obtained is granulated with a 1.2 mm mesh mesh, dried and scraped with a 0.840 mm mesh mesh. The granulated product is sealed in size "1" capsules.

The binder solution can be replaced with a solution of polyvinylpyrrolidone in ethanol / water or an aqueous solution of gelatin. The molecular weight of the polyethylene glycol can range from 1,500 to 10,000.

Example 11

Pharmaceutical composition containing silybinine β-cyclodextrin inclusion complex (molar ratio 1: 2)

The operation is performed as described in Example 9 using: titratable silybinine 40.0 mg V-cyclodextrin 188.1 mg: Example 12

Pharmaceutical composition containing silybinin: β-cyclodextrin inclusion complex (molar ratio 1: 4)

The operation is performed as described in Example 9 using: 12 102459 titratable; silybinine 40.0 mg β-cyclodextrin 328.4 mg

Pharmacoat 606 10.0 mg sodium laurilsulfate 15.0 mg polyethylene glycol 15.0 mg lactose 129.1 mg magnesium stearate 1.5 mg

Assemble the hard gelatin capsules "0".

Example 13

Pharmaceutical composition containing silybinine: DIMED inclusion complex (molar ratio 1: 2)

The operation is performed as described in Example 9 using: titratable silybinine 40.0 mg DIMED 220.77 mg

The absorption of the kmlexes of the invention has been evaluated by administering an aqueous solution of the test composition orally and measuring the bile levels of silybinin by HPLC. This test is advantageous because it is well known that silymarin-type drugs reach the highest concentrations in bile.

. . The tests were performed on Wistar rats weighing 180-200 g, to which the test compositions were administered by gastric tube.

Animals were fasted for 16 hours. The test composition was dissolved in water containing 10% gum arabic, and solutions were prepared by administering to each animal 5 ml / kg of said solution in each case.

Animals were anesthetized immediately after treatment, and excised to inspect the common bile duct. A small tube was placed there, and bile was collected over 24 hours. For the analytical assay, 1 ml of bile was treated as described in D. Lorenz et al .; 13 102459

Pharmacokinetic Studies with Silymarin in Human Serum and Bile - Meth. and Find. Exptl. Clin. Pharmacol. 6 (10) (1984) 655-661. The results obtained from the tests are listed in the following Table 2, in which: - Test A gave 20 mg / kg of silymarin according to a known commercial formulation corresponding to 8 mg / kg of pure silybinine, Test B gave 8.79 mg / kg of silybinine by HPLC titer was 91% and corresponding to 8 mg / kg of pure silybinine was used as such without excipients, in Test C 29.2 mg / kg of the sily-binine: 3-cyclodextrin inclusion complex described in Example 1 was administered. titer 27.4%, corresponding to 8 mg / kg of pure silybinine.

Table 2 product animal bile bile 0-24h% secretion volume 0-24h / μg / rat silylity pg / ml binin silybinin 12 12.2 1.0 11.9 0.6 (test B) silybinin 15 11.4 6.4 73.8 2.7 (Test A) Complex 14 of Example 1 14 14.3 17.6 256.6 11.7. (tests C)

The average amount of bile produced in 24 hours in the untreated rat is 13.4 ml.

Evaluation of the data obtained shows that administration of a composition containing the inclusion complex according to the invention as active ingredient 14 102459 allows to achieve, using the same amount of active ingredient in all tests, more than twice the silybinine concentration levels obtained in test A and almost 20 times that obtained in test B.

In a further experiment, the resorption of silymarin, silybinine and the complex of Example 6 was evaluated. To this end, the secretion of silybinin in rat bile was examined by a standard model experiment. Anesthetized male Wistar SPF rats kept under normal conditions were cannulated into the common bile duct. After administration of the test compound (5 mg silyl binin / kg), bile was collected for 5 hours. The silybinine content of the bile was determined by quantitative thin layer chromatography. The results were as follows:% secretion of silybinin in rat bile: silymarin: 11.9% ** silybinine: 0.21% a> complex: 17.3% 3> 1) Mean calculated from 5 experiments 2) Mean calculated from 10 experiments 3) Mean calculated from 7 experiments

The above data show that the complex is absorbed more and more easily than silybinine and silymarin.

Claims (6)

A process for preparing water-soluble inclusion complexes with silybinin and cyclodextrin or its derivatives, characterized in that a solution of silybinin and cyclodextrin is produced in water at a pH above 7, the pH of the solution is related to a level below 7 by adding appropriate acid, and separating the resulting complexes. A process according to claim 1, characterized in that silybinin is suspended in water, a suitable base is added to dissolve the silybinin, cyclodextrin is added, the pH is adjusted to a level below 7 by adding a suitable acid and the complex are separated, or silybinin and cyclodextrin are suspended in water, a suitable base is added to dissolve the components, the pH is adjusted to a level below 7 by adding a suitable acid, and the complex is separated. 3. A process according to claim 1 or 2, characterized in that the molar ratio of the silybinin to the cyclodextrin is 1 '. 1 - 1: 4. ... 4. A method according to any of claims 1-3, wherein the cyclodextrin is β-cyclodextrin. 30 Process according to claim 4, characterized in that the molar ratio of the silybinin to the β-cyclodextrin is 1: 2, 1: 2.75 or 1: 4. Process according to any of claims 1-5, characterized in that the cyclodextrin is selected from the group of eptakis-2,6-O-dimethyl-2-cyclodextrin and O-hydroxypropyl-β-cyclodextrin. • ·