HU212940B - Process for producing silybinin inclusion complexes and pharmaceutical compositions comprising same - Google Patents

Description

The present invention relates to inclusion complexes of silibinin with β-cyclodextrin or a derivative thereof and to a process for the preparation of such compositions.

Silibinin is one of the isomers that make up silymarin (the silymarin group), more specifically, the most abundant isomer represented by formula (A).

The fruit of the Carduus marianus plant is extractive and has interesting antihepatotoxic activity. The preparation of silibinin is described, for example, in DE-A-3 537 656.

Silibinin itself, with the appropriate binders, formulated in the proper proportions, exhibits an interesting antihepatotoxic activity, which plays a particularly important role in certain pathological conditions.

It has now been found that silibinin forms a novel inclusion complex with β-cyclodextrin or O- (C 1 -C 4 alkyl) and hydroxy (C 1 -C 4 alkyl) derivatives which exhibits better bioavailability compared to the silibinin isomer.

The invention thus relates to a process for the preparation of an inclusion complex of silibinin with β-cyclodextrin or a derivative thereof as defined above.

It is a further object of the present invention to provide pharmaceutical compositions for oral use which contain as an active ingredient a inclusion complex of silibin with β-cyclodextrin or a derivative thereof. Preferably, a product with a silibinin content of greater than 90% by weight on a dry basis is used for HPLC. Particularly preferred β-cyclodextrin and its derivatives are β-cyclodextrin, hydroxypropyl 4-cyclodextrin, heptakis (2,60-dimethyl) -cyclodextrin and heptakis (2,3,9-trimethyl) 4-cyclodextrin.

The inclusion complex of the present invention can be prepared by preparing a solution of silibinin and β-cyclodextrin at a pH above 7, then adjusting the pH to below 7 and isolating the resulting complex. The process is carried out by suspending the silibinin in water and adding a suitable base, preferably ammonium hydroxide, sodium or potassium hydroxide, until the silibinin is completely dissolved. The resulting solution should have a cyclodextrin or selected cyclodextrin molar ratio of 1: 1 to 1: 4. After a waiting time of 15 minutes to several hours, the solution is evaporated or, after removal of most of the volatile base used, the solution is neutralized or acidified to a pH below 7, preferably to a pH of 3 to 4, with a suitable acid such as hydrochloric using. The complex is then isolated by known techniques such as lyophilization. Alternatively, a solution of silibinin and cyclodextrin can be prepared by mixing them in water and proceeding with the addition of a base as described above.

The inclusion complexes of the present invention are water soluble. This means that their solubility at 20 'Con is at least one part in 10 parts water (parts by weight).

The solubilization and efficiency dissolution rates of the complexes of the present invention are much higher than those of silibinin. Proper dissolution rate is a prerequisite for proper absorption. Good absorption of the present complexes results in high silibinin levels. The dissolution rate (solubility) of the active ingredient in water at 37 ° C, pH 7.5, as determined by the USP ΧΧΠ rotor blade method is at least 60%, preferably at least 75%, most preferably at least 85%, most preferably at least 90%.

An inclusion complex of silibinin with a cyclodextrin can be formulated according to the present invention into a pharmaceutical composition for oral use such as binding agents, such as anionic surfactants such as lauryl sulfate sodium salt; Polyalcohols having a molecular weight of 400 to 2000, preferably 1500 to 3000; simple and cross-linked polyvinylpyrrolidones; cellulose and its derivatives such as hydroxypropylmethylcellulose, sodium salt of carboxymethylcellulose and ethylcellulose.

Most preferred oral dosage forms are tablets, hard and soft gelatine capsules and sachet granules.

Brief Description of the Drawings: The

Figure 1 shows an X-ray diagram of the physical mixture of silibinin, β-cyclodextrin, and the complex of Example 1. THE

Figure 2 shows an X-ray diffraction diagram of the complex of silibinin, DIMEB (heptakis (2,6-O-dimethyl) -? - cyclodextrin) and Example 5. THE

Figure 3 shows an X-ray diffraction diagram of the complex of silibinin, β-CD (hydroxypropyl-β-cyclodextrin) and Example 6. THE

Figure 4 shows thermoanalytical curves of silibinin.

The

Figure 5 illustrates the complex of Example 5 and silibinin and

Displays DSC (differential scanning calorimetry) curves of a physical mixture of DIMEB. THE

Figure 6 is a graph of the complex of Example 6 and silibinin and

Shows the DSC curves of the physical mixture of ΗΡ-β-CD. THE

Figure 7 shows the complex of Example 5 (1) and Figure 6.

The complex (2) has an ultraviolet (UV) spectrum.

The following examples illustrate the invention without limiting it in any way.

Example 7

Preparation of a 7: 7 molar inclusion complex of silibinin: $ - cyclodextrin A 93% silibinin-containing product (0.019 mol) was suspended in 200 ml of deionized water and stirred with magnetic stirrer (75 ml) of 35% ammonium hydroxide solution over 10 minutes. When the addition of ammonium hydroxide was complete, 24.78 g of β-cyclodextrin (Fischer 13%; 0.019 mol) was added suddenly. Stirring is continued for another 35 minutes,

The excess ammonia gas was removed in vacuo and the pH adjusted to slightly below 7 with 6N hydrochloric acid. After lyophilization, it is washed with 2 x 30 ml of acetone and dried. 26.2 g of a yellow, water-soluble solid are obtained (HPLC analysis contains 24.7% silibinin, which by X-ray analysis shows the typical peaks of the silibinin: p-cyclodextrin 1: 1 inclusion complex (e.g. 20 = 18.8). 250 DEG C., UV max at 287 nm (methanol).

The inclusion complex of the present invention shows a greater dissolution of the active ingredient than either silibinin or silymarin under the same experimental conditions.

This characteristic is illustrated by the diagram in Figure 8, which shows the active ingredient of the silibinin-cyclodextrin inclusion complex (148 mg equivalent to 40 mg pure silibinin), silibinin (40 mg) and silymarin prepared in Example 1 in a pH of 6.8 dissolution curves.

The experiment was performed using the USP rotary vane method and determined by measuring ultraviolet absorption against known drug-containing standards.

The X-ray diffraction pattern of the inclusion complex of Example 1 is characterized by some significant peaks (such as 20 = 18.8) which are not included in the X-ray diffraction pattern of the powder obtained by mechanical mixing of the two components; furthermore, the peaks (20 = 24.5; 14.6; 32; 22.7) characteristic of the diffraction diagram of the mechanical mixture of the two components are absent from the X-ray diffraction pattern of the above inclusion complex.

Example 2

Preparation of a 1: 2 molar inclusion complex of silybinin: $ - cyclodextrin

The procedure was carried out as in the previous example, using 35 g of pure silibinin and 181.7 g of anhydrous 211.7 g of β-cyclodextrin. 218 g of product are obtained having a silibinin titer of 15.41% by HPLC and a water content of 2.5% by the Kari Fischer method.

Example 3

Preparation of silibinin: DIMEB [heptakis (2,6-O-dimethyl) - $ - cyclodextrin] 1: 1 molar inclusion complex

The procedure was carried out as described in Example 1, with 32.3 g of 93% silibinin product

82.4 g DfMEB. 97.2 g of a 1: 1 molar inclusion complex of silibinin-heptakis (2,6-O-dimethyl) - cyclodextrin are obtained.

The products obtained as described in the previous examples can be used for the preparation of pharmaceutical compositions with the addition of a suitable excipient, as described in Examples 9-13. using the operations described in the examples.

Example 4

Preparation of silibinin: $ - cyclodextrin in a 1: 2.75 molar complex

109.76 g (96.6 mmol) of β-cyclodextrin are suspended in 600 ml of distilled water and 16.94 g (35 mmol) of silibinin are added. About 75 ml of 1M sodium hydroxide solution are added dropwise over 10 minutes with constant stirring. Silibinin dissolves readily to give a yellow solution. A solution of 0.66 M phosphoric acid is then added to adjust the pH to a value between 3.2 and 3.4 (this requires about 100-105 mL of phosphoric acid). The temperature of the mixture was maintained below 20 ° C during the dissolution and neutralization process using an ice bath. The solution becomes colorless under pH 5; The UV spectrum after dilution with 50: 1 v / v ethanol of 1: 800 indicates that silibinin is present in the solution in its non-ionized form. The slightly opalescent solution of the crude wool is filtered through a 0.4 glass filter. The solid complex is isolated from the crude solution by lyophilization.

140 g of silibinin-β-cyclodextrin complex are thus obtained. It contains 12.1% w / w of active ingredient (as measured by UV spectrophotometry). Loss on drying (105 ° C, vacuum dried for 2 hours): 9.5% w / w.

Dissolution experiments for silibinin, silymarin and the complex in this example were performed using the USP XXII rotary vane dissolution experiments at USP XXII rotary vane at pH 7.5 at 37 ° C. The experiments show 96% solubility (solubility) for the complex as opposed to 0% for silibinin and 53% for total silymarin, which corresponds to 37.1% of silibinin.

Example 5

Preparation of Silibinin: [heptakisil 2,6-O-dimethyl) - [beta] -cyclodextrin] 1: 2.2 Mole Complex The procedure of Example 4 is repeated, except that 110 g (82 mmol) of DIMEB [heptakis (2 6-O-dimethyl) -cyclodextrin] and 18 g (37 mmol) of silibinin were used.

140 g of silibinin: DIMEB are thus obtained. The content of active ingredient as measured by UV spectrophotometry was 12.6% w / w. Loss on drying (105 DEG C., vacuum dried for 2 hours): 5% w / w.

Example 6

Preparation of Silibinin: (Hydroxypropyl - $ - Cyclodextrin) 1: 2.2 Mole Complex

100 g (76 mmol) PS = 3.0 (PS = average degree of substitution per unit of cyclodextrin) is dissolved in 700 ml of water, 17 g (35 mmol) of silibinin is added to hydroxypropyl-β-cyclodextrin (ΗΡ-β-CD), resulting in a suspension, 70 ml of 1 M sodium hydroxide solution are added dropwise over 10 minutes. Further processing of the solution is the same as used for the preparation of the DIMEB complex. 130 g of silibinin: HP ^ CD complex are thus obtained. Active ingredient content: 12.7% w / w. Loss on drying (105 DEG C., vacuum dried for 2 hours): 7.0%.

X-ray analysis of the complexes of Examples 5 and 6.

X-ray diffraction diagram of pure silibinin, DMEB, ΗΡ-β-CD and complex samples Philips por-diff3

HU 212 940 Β using a CU-K radiation. Comparison of the X-ray diffraction diagrams (Figures 2 and 3) shows that the characteristic 20 peaks of crystalline silibinin and DIMEB have disappeared, the complexes showing a completely different structure in both cases.

Thermoanalytical studies

Thermal analysis was performed on a Du Pont 990 Thermal Analyzer System. TG (thermogravimetric), DTG (differential thermogravimetric), DSC (differential scanning calorimetric), and TEA (thermal analysis; Thermal Evolution) are simultaneously recorded for the active ingredient and its mechanical mixtures and complexes with DIMEB and ΗΡ-β-CD. Analysis).

The DSC curves of silibinin (Fig. 4) show that the decomposition of the active substance starts at about 130 'C and at a weight loss of about 4-5% up to 160' C. The second degree of degradation of the drug starts above 250 ° C, within the degradation range of cyclodextrin. The lower temperature decomposition step is suitable for distinguishing between complexed and non-complexed product. The DSC curves of the complexes (Figures 5 and 6) show that the degradation of silibinin occurs only at higher temperatures of about 175-180 ° C.

Solubility properties of the complexes of Examples 5 and 6.

250 mg of DIMEB and ΗΡ-β-CD are readily soluble in 2 ml of distilled water and pH 1.4 hydrochloric acid to give a clear solution. The UV spectra of the aqueous solutions, after dilution with 1: 750 v / v 50% v / v ethanol, undoubtedly show that silibinin is enclosed in both DIMEB and ΗΡ-β-CD in non-ionised form ( Figure 7).

PH of aqueous solutions:

DIMEB Complex: 3.3.

ΗΡ-β-CD complex: 3.4.

One day after dissolution, no precipitate formed in the DIMEB complex solution. DIMEB Complex solution remains sharp for more than 10 days. The UV spectra remain unchanged, indicating good stability of the silibinin complexes in solution.

Example 7

Silibinin. ? -cyclodextrin 1: 2 molar inclusion complex Silibinin (active ingredient; titration) 40.0 mg β-cyclodextrin 164.7 mg

Pharmacoat 606 10.0 mg

Sodium lauryl sulfate 15.0 mg

Polyethylene glycol 15.0 mg

Lactose 103.8 mg

Magnesium stearate 1.5 mg "7" hard gelatin capsules.

Silibinin and β-cyclodextrin were mixed in water: methanol (7: 3) at 45 ° C for 2 hours, then methanol was evaporated and the product was lyophilized.

The lyophilized product is kneaded in a 2% w / w solution of Pharmacoat 606 in methylene chloride: ethanol (9: 1). The mixture thus obtained is one

It is granulated through a sieve with a mesh size of 1.2 mm, dried and passed through a sieve with a mesh size of 0.840 mm. The granulated product is filled into "1" capsules.

The binding solution used may be replaced by an aqueous solution of polyvinylpyrrolidone in ethanol or an aqueous solution of gelatin. The molecular weight of the polyethylene glycol can range from 1500 to 10,000.

Example 8

Silibinin. ? -cyclodextrin 1: 2.75 molar inclusion complex

Silibinin-β-cyclodextrin complex 330.0 mg

Pharmacoat 606 10.0 mg

Sodium lauryl sulfate 15.0 mg

Polyethylene glycol 15.0 mg

Lactose 103.8 mg of magnesium stearate 1.5 mg

The inclusion complex was kneaded with a 2% w / v solution of Pharmacoat 606 in methylene chloride: ethanol. The mixture thus obtained is one

It is granulated through a sieve with a mesh size of 1.2 mm, dried and passed through a sieve with a mesh size of 0.840 mm. The granulated product is filled into size 1 capsules.

The binding solution used may be replaced by an aqueous solution of polyvinylpyrrolidone in ethanol or an aqueous solution of gelatin. The molecular weight of the polyethylene glycol can range from 1500 to 10,000.

Example 9

Pharmaceutical composition containing a 1: 4 molar inclusion complex of silibinin: β-cyclodextrin The procedure was carried out as described in Example 7, using the following ingredients:

Silibinin active ingredient content 40.0 mg β-cyclodextrin 328.4 mg Pharmacoat 606 10.0 mg Sodium laureth sulfate 15.0 mg Polyethylene glycol 15.0 mg Lactose 129.1 mg Magnesium stearate 1.5 mg

Hard gelatine capsules of size Ο ''.

Example 70

Silibinin.DIMEB 1: 2 Molecular Inclusion Complex Preparation The procedure was carried out as described in Example 7, using the following ingredients:

Silibinin (active ingredient; by titration) 40.0 mg DIMEB 220.77 mg

Absorption of the complexes of the present invention is evaluated by administering an aqueous solution of the test composition orally and measuring the level of bile silibinin by high performance liquid chromatography (HPLC). This is exam4

EN 212 940 Β gal is advantageous because it is well known that silymarin-like drugs reach the highest concentrations in the bile.

The experiments were carried out on Wistar rats weighing 180-200 g, and the compositions to be tested were administered by gastric intubation (gastric tube). The animals were fasted for 16 hours. Dissolve the preparation to be examined in water containing 10% gum arabic to obtain solutions in which each animal receives 5 ml / kg each time.

Immediately after treatment, the animals are anesthetized and operated to check for common bile duct (choledochus). A small tube was introduced and the bile was collected for 24 hours. For analytical testing, 1 ml of bile is used as described in D. Lorenz et al., Pharmacokinetic Studies with Silymarin in Human Serum and Bile - Meth. and Find. Exp't'l. Clin. Pharmacol. 6 (10), 655-661 (1984).

The results of the assays are shown in Table 1, wherein assay A uses 20 mg / kg of silymarin as a known commercial preparation, corresponding to 8 mg / kg of silibinin;

for test B, 91%, 8.79 mg / kg silibinin corresponding to 8 mg / kg pure silibinin according to HPLC assay, without binder;

Test C uses 27.4% of the 29.2 mg / kg silibinin-β-cyclodextrin inclusion complex corresponding to 8 mg / kg pure silibinin by HPLC measurement.

Table 1

Product Number of experimental animals Bile volume. Bile 024 hours pg / ml 0.24 hours pg / rat % selected silibinin Silibinin (Test B) 12 12.2 1.0 11.9 0.6 Silibinin (Test A) 15 11.4 6.4 73.8 2.7 Complex Example 1 (Test C) 14 14.3 17.6 256.6 11.7

The average amount of bile produced by an untreated rat per 24 hours is 13.4 ml.

An evaluation of the data obtained shows that the use of the inclusion complex composition of the invention as active ingredient, using the same amount of active ingredient in each assay, allows for concentrations of silibinin to be more than twice as high as in Assay A and almost 20 times higher than Assay B.

In a further experiment, the absorption of silymarin, silibinin and the complex described in Example 4 was investigated. To this end, the excretion of silibinin in the rat bile is measured by a conventional model experiment. Under normal conditions, anesthetized male Wistart SPF rats were cannulated into the common bile duct (ductus choleduchus). Following intragastric administration of the test compound (5 mg silibinin / kg), the bile is collected for 5 hours. Its silibinin content is determined by quantitative thin layer chromatography.

The following results are obtained:

% Silibinin excretion in rat bile silymarin 11.9% ^υ silibinin 0.21% ² > Complex 17.3% ³ '

Average of ¹ '5 experiments Average of ² ' 10 experiments

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

Claims (7)

PATENT CLAIMS CLAIMS 1. A process for the preparation of a water-soluble inclusion complex of silibinin with β-cyclodextrin or a 0- (1-4C) alkyl or hydroxy (1-4C) alkyl derivative thereof, characterized in that the silibinin and β-cyclodextrin, An aqueous solution is prepared at pH, the pH of the solution is adjusted to below 7 with acid, and the resulting complex is isolated. 2. A process according to claim 1, wherein the silibinin is suspended in water, added to a solution by addition of a base, and then the β-cyclodextrin or said derivative is added to the solution, the pH of the solution is adjusted to below 7 or the silibinin and The β-cyclodextrin or said derivative is suspended in water, added to the solution by addition of a base, the pH of the solution is adjusted to below 7 with acid and the resulting complex is isolated. 3. The process according to claim 1 or 2, wherein the silibinin and β-cyclodextrin are used in a molar ratio of 1: 1 to 1: 4. The process according to claim 3, wherein the silibinin and β-cyclodextrin are used in a molar ratio of 1: 1, 1: 2.75 or 1: 4. 5. Process according to any one of claims 1 to 3, characterized in that the derivative is heptakis (2,6O-dimethyl) -β-cyclodextrin, heptakis (2,3,6-O-trimethyl) -? - cyclodextrin or O- (hydroxypropyl) -? - cyclodextrin. . 6. A process for the preparation of a pharmaceutical composition comprising a water-soluble inclusion complex of silibinin with β-cyclodextrin or a 0- (C 1-4 alkyl) or hydroxy (C 1-4 alkyl) derivative thereof, characterized in that HU 212 940 Β 1-5. The at least one complex prepared according to any one of claims 1 to 6, together with the usual excipients, is processed into a pharmaceutical composition. 7. The method of claim 6, wherein the medicament is for treating the liver.