PT99146B - METHOD FOR THE PREPARATION OF INCLUSIVE COMPLEXES WITH SILIBININ AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM - Google Patents

Description

description

The object of the present invention relates to silibinine inclusion complexes with a cyclodextrin or derivative thereof, a process for its preparation as well as pharmaceutical compositions containing said inclusion complexes as active ingredients.

Silybin is one of the isomers, more precisely it is the isomer that is present in greater proportion, which make up Silymarin (or the Silymarin group) that has the formula

J.M. ’

OH Ο

It can be obtained by extracting the fruit of Cardus marianus, and is considered to have an interesting anti-hepatotoxic activity. The preparation of Silibiine is for example referred to in DE-A-35 37 656.

Silibinin itself, formulated with suitable excipients in suitable proportions, reveals an interesting anti-hepatotoxic activity that makes it of particular interest in various pathological conditions.

It has been demonstrated, and is the object of the present invention, that Silibinin, suitably treated with a cyclodextrin chosen from the group consisting of <? (-, and ^ -cyclodextrin and a suitable derivative thereof, in particular those derived from 0-alkyl (1 -4C) and hydroxy (1-4C) alkyl forms a new inclusion complex which, compared to the silibinine somer, reveals superior bioavailability.

Another object of the present invention is a process for the preparation of a Silibinin inclusion complex with a cyclodextrin chosen from the group consisting of O (-, β - and y-cyclodextrin and a suitable derivative thereof.

An additional object of the present invention is represented by pharmaceutical compositions suitable for oral administration that contain a silibinin inclusion complex with a chosen cyclodextrin as an active ingredient2

in the group consisting of <-, 73 - and γ-cyclodextrin and a suitable derivative thereof.

Silibinin is preferably used, having a purity determined by HPLC (High Performance Liquid Chromatography) greater than 90%. Among Ύ -, J ³ - θ Ϊ -cyclodextrin and their suitable derivatives, / 3-cyclodextrin, hydroxypropyl- / 3-cyclodextrin, eptaquis-2,6-0-dimethyl-β-cyclodextrin and eptaquis- are particularly preferred 2,3,6-0-trimethyl- / 3-cyclodextrin.

Inclusion complexes of the invention can be obtained by preparing a solution of Silybin and cyclo dextrin at a pH greater than 7, adjusting the pH to values less than 7 and isolating the complex formed. This process comprises suspending Silibinine in water and then adding it to a suitable base, preferably ammonium hydroxide, sodium or potassium hydroxide until Silibinine is completely dissolved. To the solution thus obtained, the cyclodextrin or its chosen derivative is added to an extent so as to have a 1: 1 to 1: 4 ratio of silibinin: cyclodextrin. After a period of time, ranging from 15 minutes to a few hours, the solution is evaporated or after most of the volatiles have been removed from the base used, it is neutralized or acidified to a pH value below 7, preferably 3 to 4, using a suitable acid, for example HCl, H2 PO4, HgSO4.

The complex is then isolated according to known techniques, for example by lyophilization. The solution of Silibinin and cyclodextrin can also be prepared by mixing it in water and continuing to add the base as mentioned above.

The inclusion complexes of the present invention are soluble in water. This means that they have a solubility of at least one part in 10 parts of water with O_hase in weight at 20 C.

The solubilization and release rate of the complexes according to the invention are much higher when compared to Silibinin. An adequate release rate = 3 =

it is a condition for useful absorption. The good absorption of the present complexes results in high levels of Silibinin. The release rate (solubility) is at least 60% determined according to the USP XII method (Pharmacopeia Nortre-Ame. \ 'O ricana) in water at 37 Ca and a pH of 7.5, preferably at least 75 # and in particular at least 85 More preferably it is a Silibinin inclusion complex with a cyclodextrin according to the present invention can be conveniently formulated in pharmaceutical compositions suitable for oral administration, using excipients such as anionic surfactants, such as sodium lauryl sulfate, polyalcohols having molecular weights of 400 to 20,000, preferably from 1,500 to 3,000, simple and cross-linked polyvinylpyrrolidones, cellulose and derivatives, such as, for example, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and ethylcellulose.

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

Brief description of the drawings;

Figure 1 shows an X-ray diagram of Silibinin, -cyclodextrin, a physical mixture and the complex of Example 1.

Fig. 2 shows X-ray diffraction patterns of Silibinin, DIMEB (eptaquis-2,6-0-dimethyl - /? - cyclodextrin) and the complex of Example 7.

Fig. 3 shows the X-ray diffraction patterns of silybin, HB # CD (hydroxypropyl - / - cyclodextrin) and the Complex of Example 8.

Fig. 4 shows silibinin thermoanalytic curves.

Fig. 5 shows DSC curves (Callus = 4 =

Differential Scanning Rheometry) of the Example 7 complex and a physical mixture of Silybin and DIMEB.

Example 8 and a

Fig. 6 shows DSC curves of the complex physical mixture of Silybin and HPjSCD.

Fig. 7 is the UV (ultraviolet) spectrum of the complex of Example 7 (1) and Example 8 (2).

The following Examples are presented for the purpose of illustrating the invention without, however, limiting it.

Example 1

Preparation of the SilibininsA-oiolodextrin inclusion complex (molar ratio 1: 1)

10 g of Silibinine (purity 93 0.019 moles) are suspended in 200 ml of deionized water and with magnetic stirring 75 ml of 35% ammonium hydroxide are added dropwise over 10 minutes. When the addition is complete, 24.78 g of -cyclodextrin (Fisher 13%; 0.019 moles) are added at once. Stirring is continued for a further 35 minutes and then the water is distilled off in 15 minutes under vacuum at 50 ° C. The yellow solid that forms is washed twice with 30 ml of acetone and dried to obtain 24.6 g of a light yellow solid, soluble in water, which by X-ray analysis reveals the characteristic peaks due to the inclusion of Silybin: J5-cyclodextrin (1: 1) (for example 20 = = 18.8); melting point of 250 ° C. UV (max) 287 nm (MeOH).

inclusion complex according to the present invention reveals a superior dissolution compared to the dissolution demonstrated, under the same experimental conditions by Silybin and Silymarin.

This characteristic is made evident by the diagram in Table 1 showing the dissolution curves of the Silibinin complex: -β-cyclodextrin prepared according to Example 1 (148 g of the complex corresponding to

mg of pure Silybin); Silybin (40 mg) and Silymarin dissolved in gastric juice at pH 6.8 are also mentioned.

The evaluation was done according to the paddle method (USE) and the determinations were made in UV light in comparison with known standards.

Table 1

SILIBININE COMPLEX DISSOLUTION TEST:

: j3-CYCLODEXTRIN llX)

O 'i Ί 0

-o-Silybinin • “Silymarin

-CD / Silybin, - ₍ —j

IO 12 H 10 18 20

Iknepuln} X-ray diagram of the inclusion complex prepared according to Example 1 is characterized by the presence of some significant peaks (for example 20 = 18.8) that do not exist in the X-ray diagram of the powder formed by the mechanical mixture of the two components; furthermore, in the X-ray diffraction diagram of the inclusion complex mentioned above, these peaks are not present (20 = 24.5; 14.6;

32; 22,7) that are characteristic of the diffraction diagram of the mechanical mixture of the two components.

Example 2

Preparation of the Silibinin inclusion complex: jS-cyclodextrin (molar ratio 1: 1)

10 g of Silybin (93 l of purity, 0.019 moles) are suspended in 200 ml of deionized water with magnetic stirring and 75 ml of 35% ammonium hydroxide are added dropwise over 10 minutes. When the addition is complete, 24.78 g of β-cyclodextrin (Fisher 13 $ 0.019 moles) are added in one go. Stirring is continued for a further 35 minutes and then the excess ammonia gas is removed in vacuo, the pH is adjusted to 7 by adding 6N hydrochloric acid, freeze-drying is carried out, washing twice with 30 ml of each instead of acetone to obtain 26.2 g of a yellow solid (the purity determined by HPLC in Silybin is 24.7% ta) having the spectrum characteristics as those of the product of the previous Example.

Example 3

Preparation of the Silibinin inclusion complex; β -cyclodextrin (molar ratio 1: 2)

The operation is carried out as described in the previous Examples using 35 g of pure Silybin and 211.7 g of -cyclodextrin corresponding to 183.7 g of anhydrous. 218 g of the product are obtained which have a HPLC-determined purity in Silibinin of 15.41% and KF = 2.5%.

Example 4

Preparation of the Silibinin inclusion complex: y-cyclodestrin (molar ratio 1; 1)

The operation is carried out as described in Example 1 using 5.1 g of 98% purity y -cyclodextrin and 2.04 g of 91% purity silibinine corresponding to 1.857 g of pure silibinin. 5.9 g of the

inclusion of Silibinin: -cyclodextrin (1: 1).

Example 5

Preparation of the Silibinin inclusion complex: DIMEB (eptaquis-2,6-0-dimethyl- Λ-cyclodextrin) (1: 1 molar ratio)

The operation is carried out as described in Example 1 using 32.3 g of Silybin having a purity of 93% θ 82.4 g of DIMEB. 97.2 g of Silibinin inclusion complex: eptaquis-2,6-O-dimethyl - „/ 3-cyclodextrin (1: 1) are obtained.

The products obtained in the manner described in the previous Examples can be used for the preparation of pharmaceutical compositions by adding suitable excipients according to the operations described in Examples 9a 13Example 6

Preparation of the Silibinin- / 3-cyclodextrin inclusion complex (molar ratio 1: 2.75)

79.76 g of / (- cyclodextrin (96.6 mmol)) were dissolved in 600 ml of distilled water: 19.94 g of Silybin (35 mmol) were added to the solution resulting in a suspension. 75 ml of 1N sodium hydroxide solution over a period of 10 minutes with continuous agitation.Silibinine was rapidly dissolved resulting in a yellow solution.

A 2N phosphoric acid solution was added to adjust the pH value between 3.2 and 3.4 (about 100 to 105 ml of phosphoric acid are needed). The temperature of the solution during dissolution and the neutralization process was kept below 20 ° C by using an ice bath. The solution lost its color at a pH value below 5. The UV spectra of the solution after a dilution of 1: 800 with ethanol at 50? (v / v) revealed that Silibinin was pre = 8 =

- feels in an ionized form in the solution. The slightly beige and translucent solution was filtered through a 0.4 glass filter. The solid complex was isolated from the clear solution by lyophilization.

140 g of Silibinin-β-cyclodextrin complex were obtained. The active ingredient content was

12.1 $ (by weight) measured by UV spectrophotometry. Drying time (at 105 ° C under vacuum in 2h): 9.5% (by weight). Release experiments were carried out with Silibinine, Silimari na and the complex of this Example according to USP XXII, with the paddle device at pH 7.5 and 37 ° C. The experiments revealed a 96% release (solubility) of the complex in | in contrast to a 0% release of Silybin and a 53% total release of Silymarin corresponding to a 37.1% release of Silybin.

Example 7

Preparation of the Silibinin-2,6-di- / 3-cyclodextrin inclusion complex (2: 2.2 molar ratio)

The procedure of Example 6 was repeated with the exception that 110 g of 2,6-di-0-methyl- / 3-cyclodextrin (DIMEB; 82 mmol) and 18 g of Silibinine (37 mmol) were used.

140 g of the Silibi | nina: DIMEB. The active ingredient content was 12.6% (by weight) as measured by UV spectrophotometry. Loss on drying (loss of weight at 105 ° C in a 2h vacuum): 5.0% (by weight).

Example 8

Preparation of the Silibinin-hydroxypropyl-P-cyclodextrin complex) (molar ratio 1: 2.2)

100 g of hydroxypropyl-β-cyclodextrin (HPCD) 76 mmol were dissolved. PS = 3.0 (PS = average degree of substitution per unit of cyclodextrin) in 700 ml of water, 17 g of Silibinine (35 mmol) were added, resulting in

in a suspension, and approximately 70 ml of 1N sodium hydroxide was added to the solution within 10 minutes. The further processing of the solution is identical to the preparation of the DIMEB complex and 130 g of the Silibinin-HPACD complex were obtained.

active ingredient content was 12.7% (by weight). Loss on drying (weight loss at 105 ° C ^and empty in 2h): 7.0%.

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

The X-ray powder diffraction diagrams of pure Silibinin, DIMEB, HP-jã »CD and of the complex samples were recorded on a Philips powder diffractometer using Cu-Kj3 radiation. Comparing the X-ray patterns (Fig. 2, 3) · It appears that 20 crystalline Silibinine and DIMEB peaks disappeared, and the complexes revealed a completely different structure in both cases.

Thermoanalytical studies

Thermoanalytical investigations were carried out in a DuPont 990 Thermal Analysis System device. TG (thermogravimetry), DTG (Differential Thermogravimetry), DSC (Differential Scanning Calorimetry) and TEA (Thermal Evolution Analysis) curves were performed simultaneously for the curves. the medicine, as well as for its mechanical mixtures with DIMED and ΗΡ-βίϊΟ and complexes.

The DSC curves of Silibinine (Fig. 4) indicate that the decomposition of the drug starts at about 130 ° C and up to 16 ° C a weight loss of 4 to 5 was observed. The second phase of drug breakdown begins above 250 ° C in the cyclodextrin decomposition region. The decomposition phase at a lower temperature is adequate to differentiate complexed and non-complexed products. The DSC curves of the complexes (Fig. 5, 6) reveal that the decomposition of Silybin is observed at a temperature higher than just

about 175-180 C.

Solubility properties of the complexes of Examples 7 and 8,

250 g of DIMEB and HPjSCD complex can be easily dissolved in 2 ml of distilled water and at a pH of 1.4 hydrochloric acid resulting in a clear solution. The UV spectra of the aqueous solutions recorded after a 1: 750 dilution with 50% ethanol (by volume) undoubtedly reveal that Silibinine is included in DIMEB and in HP / 5BC in an unionized form (Fig. 7).

PH value of aqueous solutions:

DIMEB complex: 3.3 HP - complex, / 3 CD: 3.4

One day after dissolution, no precipitation in the DIMEB complex solution was observed. The DIMEB complex solution remained transparent more than 10 days after storage. The recorded UV spectra remained unchanged indicating the good stability of the Silibinin complexes in solutions.

Example 9

Pharmaceutical compositions containing the silibinin inclusion complex: y ^ -cyclodextrin (molar ratio = 1: 2).

Silibinine for holder 40.0 mg -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

Size 1 "hard gelatin capsules.

Silibinin and J3-cyclodextrin are mixed in a water / methyl alcohol solution (7: 3) at 45 ° C for 2 hours, and then the methyl alcohol is evaporated.

lyophilized product is kneaded with a mixture of 2? p / v of Pharmacoat 606 in methylene chloride / ethyl alcohol solution (9: 1). The mixture thus obtained is granulated on a 1.2 mm sieve, dried and passed through a 0.840 mm sieve. The granulated product is filled into ”1” size capsules.

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

Example 10

Pharmaceutical composition containing the inclusion complex of

Silibinin-J ⁵ -cyclodextrin (molar ratio = 1: 2.75)

Silibinin Complex: 7 ^ -cyclodextrin 330 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

inclusion complex is kneaded with a 2% w / v solution of Pharmacoat 606 in methylene chloride / ethyl alcohol solution. The mixture thus obtained is granulated on a 1.2 mm sieve, dried and passed through a 0.840 mm sieve. The granulated product is filled into "1" size capsules.

The binder solution can be replaced

by a solution of polyvinylpyrrolidone in ethyl alcohol / water or by an aqueous aqueous solution of gelatin. The molecular weight of the polyethylene glycol can vary from 1,500 to 10,000.

Example 11

Pharmaceutical composition containing the inclusion complex

Silibinin: y -cyclodextrin (1: 2 molar ratio).

The operation was carried out in the manner described in Example 1 using:

Silibinin for 40.0 mg γ-cyclodextrin 188.1 mg

Example 12

Pharmaceutical composition containing the inclusion complex

Silibinine: / 6 -cyclodextrin (molar ratio 1: 4).

The operation was performed as described in Example 9 using:

Silibinine for holder 40.0 mg y5_cyclodextrin 328.4 mg Pharmacoat 606 10.0 mg Sodium lauryl sulfate 15.0 mg Polyethylene glycol 15.0 mg Lactose 129.1 mg Magnesium stearate 1.5 mg

Size O ”hard gelatin capsules.

Example 13

Pharmaceutical composition containing the inclusion complex

Silybin: DIMEB (molar ratio 1: 2) '.

The operation is carried out as described13

in Example 9 using:

Silibinin for 40.0 mg titrator

DIMEB 220.77 mg

The absorption of the complexes of the invention was evaluated by oral administration of an aqueous solution of the composition under examination by measuring the levels of Silibinin in the Bile by HPLC. This trial was preferred because it is well known that Silymarin-type drugs reach bile at maximum concentration.

The experiments were carried out on Wistar rats weighing 180-200 g to which the study compositions were administered by gastric incubation. The animals were kept on an empty stomach for 16 hours. The composition to be tested was dissolved in water containing 10% gum arabic and solutions were prepared so that each animal was administered, each time, with an amount of 5 ml / kg of the said solution.

Immediately after treatment, the animals were anesthetized and operated to check the choledochosus tube. A small tube was inserted in this place and bile was collected over a period of 24 hours.

For analytical determination was treats | of 1 ml of bile as described in the following publication:

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 data obtained in the tests are reported in Table 2 below where:

in test A, 20 mg / kg of Silymarin was administered according to a known commercial formulation corresponding to 8 mg / kg of pure Silybinin,

- in test B, 8.79 mg / kg of silibinin were administered. having a purity determined by HPLC of 91 1 °, which color = 14 =

responds to 8 mg / kg of pure silibinine, used as it is without excipients, in test C, 29.2 mg / kg of the silibinin inclusion complex was administered; 7 -cyclodextrin, described in Example 1, which has a purity in Silybinin of 17.4% determined by HPLC, corresponding to 8 mg / kg of pure Silybinin.

Table 2

Product Number of animals Volume in Bile Bile 0-24hr mcg / ml % 0-24hr mcg / mouse Silybin segregated Silibi- nina (Test B) 12 12.2 1.0 11.9 0.6 Silibi- nina (Test A) 15 11, 4 6.4 73.8 2.7 Complex Ex. 1 14 14.3 17.6 256.6 11.7

(Test C) average volume of bile produced in 24 hours by an untreated rat is 13.4 ml.

The evaluation of the data obtained reveals that the administration of the composition containing the inclusion complex according to the invention as an active ingredient allows obtaining, using the same amount of active ingredient in all

the tests, values of concentration in Silibinina more than doubled when compared with those obtained in test A and practically twenty times those obtained in test B.

In another experiment, the reabsorption of Silymarin, Silybin and the complex of Example 6 was evaluated. For this purpose, the secretion of Silybin in bile of the rat was determined in a conventional experimental model. Anesthetized male Wistar SPF rats were cannulated and maintained under conventional conditions in the choledochosus tube. After intragastric administration of the test compound (5 mg Silibinin / kg), the bile fluid was collected for 5 hours. The value of Silibinin in the bile fluid was determined by quantitative thin layer chromatography.

The results obtained were as follows:% of Silibinin secretion in the bile of the rat:

Silymarin:

Silybin:

Complex:

1) Average calculated 2) Average calculated 3) Average calculated

11.9%

2}

0.21% ¹ 3)

17.3% ^{J out} of 5 experiments out of 10 experiments out of 7 experiments

The aforementioned data show that the complex is more easily absorbed than Silibinine and Silamarine.

¢ - 16

Claims (1)

= CLAIMS = _ 1st _ Process for the preparation of a water-soluble inclusion complex of Silibinin with a cyclodextrin chosen from icx, J3, Y -cyclodextrin and a derivative of the appropriate one characterized by preparing a solution of Silibinin and cyclodextrin in water at a value of pH greater than 7, adjust the pH of the solution to values below '7 by adding a suitable acid and isolate the complex formed. - Process according to claim 1, characterized in that the Silybin is suspended in water, a suitable base is added to dissolve the Silybin, added to the cyclodextrin, the pH value is lowered to values below 7 per addition of a suitable acid and isolate the complex, or suspend Silibinine and cyclodextrin in water, add a suitable base to dissolve the components, adjust the pH to values below 7 by adding a suitable acid and isolate the complex. Process according to claim 1, characterized in that a complex is obtained which has a molar ratio of silibinin to cyclodextrin from 1: 1 to 1: 4. - 4th Process according to claim 1 or 3) characterized in that the cyclodextrin is -cyclodextrin. Process according to claim 4, characterized in that the molar ratio of Silibinine to _t -3-cyclodextrin is 1: 2, 1: 2.75 or 1: 4. 6. A process according to claim 1 or 3, characterized in that the cyclodextrin is chosen from the group consisting of eptaquis-2,6-0-dimethyl-J ³ -cyclodextrin, eptaquis-2,3,6-0-trimethyl- J ³ -cyclodextrin and O-hydroxypropyl-J ³ -cyclodextrin. _ 7§ _ Process according to any of the preceding claims, characterized in that complexes are obtained having a solubility (release) in water of at least 60 determined according to the USP XXII (Far macopoeia North American) process. 8. A process for the preparation of a pharmaceutical composition characterized in that a therapeutically effective amount of one or more complexes is incorporated as an active ingredient when prepared according to any of claims 1 to 7 optionally in association with pharmaceutically acceptable vehicles and / or adjuvants. The applicant also claims the priority of European patent applications filed on October 4, 1990, under serial number 90118964.7. Lisbon, October 4, 1991 19 = RESUME PROCESS FOR PREPARING INCLUSION COMPLEXES WITH SILIBININ AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM The invention relates to a process for the preparation of a water-soluble inclusion complex of Silybin with a cyclodextrin chosen from ΰζ, J3, T -cyclodextrin and a suitable derivative comprising preparing a solution of Silybin and cyclodextrin in water at a pH value above 7, adjust the pH of the solution to values below 7 by adding a suitable acid and isolate the complex formed.