Inclusion complexes with silybinin, their preparation and pharmaceutical compositions containing them

Abstract: The invention relates to novel inclusion complexes of Silybinin with a cyclodextrin selected from the group consisting of α-, β- and γ-cyclodextrin and a suitable derivative thereof and to the method for the preparation thereof. The novel inclusion complexes of Silybinin showed a highly improved bioavailability in comparison to Silybinin which makes them particularly interesting in the treatment of various hepatotoxic conditions.

Description: The object of the present invention relates to inclusion complexes of Silybinin with a cyclodextrin or with a derivative thereof, to a process for their preparation as well as to pharmaceutical compositions containing said inclusion complexes as active ingredients. Silybinin is one of the isomers -more precisely it is the isomer therein present in the highest amount- which constitute Silymarin (or Silymarin group) and has the formula: It may be obtained by extraction from the fruit of Carduus marianus, and is endowed with an interesting antihepatotoxic activity. The preparation of Silybinin is for example disclosed in DE-A-35 37 656. Silybinin itself, formulated with suitable excipients in suitable ratios, shows an interesting antihepatotoxic activity which renders it of a particular interest in several pathological conditions. It is know in the literature that cyclodextrin is able to form inclusion complexes with pharmaceutically active compounds and that these complexes show an improved solubility and consequently an improved bioavailability in respect to the pharmaceutical compounds considered that studies carried out on the cyclodextrin behaviour in the formation of inclusion complexes (Jozsef Szejtli, Cyclodextrin Technology, Kluwer Academic Publishers, page 84), stated that the presence of hydroxyl groups in the guest molecule hinders the complex formation with the cyclodextrin molecule and also that the stability of the complex formation with the cyclodextrin molecule with a guest molecule is proportional to the hydrophobic character of the substituents present. These results would have suggested that, in the case of Silybinin as guest molecule, the presence in its molecule of four hydroxyl groups would have hindered the inclusion complex formation and that, because of the relationship between the hydrophobic character of the substituents and the stability of the possible inclusion complex, this latter would have been unstable. Unexpectedly according to the invention the silybinin/cyclodextrin inclusion complex was obtained and proved to be stable. According to present invention, Silybinin, suitably treated with a cyclodextrin selected from the group consisting of α-,β-, γ- cyclodextrin and the O-C₁-C₄-alkyl and hydroxy-C₁-C₄-alkyl derivatives forms a novel inclusion complex which compared to the Silybinin isomer shows an improved bioavailability. A further object of the present invention is constituted by the process for the preparation of an inclusion complex of Silybinin with a cyclodextrin selected from the group consisting of α-,β- and γ-cyclodextrin and the above-mentioned derivatives thereof. A further object of the present invention is represented by the pharmaceutical compositions suitable for the oral administration which contain as active principle an inclusion complex of Silybinin with a cyclodextrin selected from the group consisting of α-, β- and γ-cyclodextrin and the above-mentioned derivatives thereof optionally together with pharmaceutically acceptable carriers and/or adjuvants. Preferably Silybinin having a HPLC titre on anhydrous higher than 90%, is used. Among α-,β-and γ-cyclodextrin and the suitable derivatives thereof, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, heptakis-2,6-0-dimethyl-β-cyclodextrin and heptakis-2,3,6-0-trimethyl-β-cyclodextrin are particularly preferred. Complexes of β-cyclodextrin and Silybinin wherein the molar ratio of silybinin to β-cyclodextrin is 1:1 to 1:4 and 1:2, 1:2.75 or 1:4 are especially preferred. More particularly, for the preparation of the inclusion complex of the present invention, Silybinin and cyclodextrin or its above-mentioned derivatives, are mixed, optionally in warm conditions, at the Silybinin: cyclodextrin molar ratio of from 1:1 to 1:4, in an aqueous solution of a suitable organic solvent such as a di-C₁-C₄-alkyl or C₅-C₇ -cycloalkyl ketone or an C₁-C₄-alkyl alcohol and therefrom the desired inclusion complex is obtained. Suitable solvents among the ketones may be acetone, cyclohexanone, methylisobutylketone and diethylketone. Among the alcohols are suitable methyl, ethyl and isopropyl alcohol. Particularly preferred are methyl and ethyl alcohols. More particularly an aqueous solution of an alcohol, such as for example methyl or ethyl alcohol, containing the desired components is made to react and then the product which forms is precipitated by cooling. In an alternative, the components are dissolved in the aqueous solution of the selected alcohol, the solution is concentrated by evaporation and submitted to lyophilization or dried by evaporating or spray drying. According to the process, which is encompassed within the scope of the present invention, the inclusion complex of the invention may be easily obtained by preparing a solution of Silybinin and cyclodextrin at a pH above 7, adjusting the pH to below 7 and isolating the complex formed. This process comprises suspending Silybinin in water and then adding to it a suitable base, preferably ammonium hydroxide, sodium or potassium hydroxide, until complete dissolution of Silybinin. To the so obtained solution, cyclodextrin is added, or its above-mentioned derivatives, to such an extent to have the molar ratio Silybinin: cyclodextrin from 1:1 to 1:4. After a period of time, varying from 15 minutes to a few hours, the solution is evaporated or after having removed the majority of the base used, it is neutralized or acidified to a pH below 7, preferably 3 to 4, using a suitable acid, for example HCl, H₃PO₄,H₂SO₄. The complex is then isolated according to known techniques, for example by lyophilization. The solution of Silybinin and cyclodextrin can also be prepared by admixing them in water and continuing by adding a base as stated above. The solubilization and the release rate of the complexes according to the invention are much higher as compared to Silybinin. An acceptable release rate is a premise for an useful absorption. The good absorption of the present complexes results in high Silybinin levels. The inclusion complex of Silybinin with a cyclodextrin according to the present invention can be conveniently formulated in pharmaceutical compositions suitable for the oral administration, using excipients like anionic surfactants, such as sodium laurylsulphate, polyalcohols having molecular weight from 400 to 20,000, preferably from 1,500 to 3,000, simple and reticulate polyvinylpyrrolidones, cellulose and derivatives, such as hydroxypropylmethylcellulose, sodium carboxymethylcellulose and ethylcellulose. The most preferred oral pharmaceutical forms are tablets, hard or soft gelatin capsules and granulated product in bags. Brief description of the drawings: Fig.1/a-1/d shows X-ray diagrams of Silybinin, β-cyclodextrin, a physical mixture thereof and of the complex of example 1 Fig.2 shows the X-ray diffraction patterns of Silybinin, DIMEB and the complex of Example 6 Fig.3 shows the X-ray diffraction patterns of Silybinin, HPβCD and the complex of Example 7 Fig.4 shows thermoanalytical curves of Silybinin Fig.5 shows DSC curves of the complex of Example 6 and of a physical admixture of Silybinin and DIMEB Fig.6 shows DSC curves of the complex of Example 7 and of physical admixture of Silybinin and HPβCD Fig.7 is the UV-spectrum of the complex of Example 6 (1) and Example 7 (2) The following Examples are given with the purpose of illustrating the invention without limiting it. Example 1 Preparation of the Silybinin: β-cyclodextrin inclusion complex (molar ratio 1:1) Grams 10 of Silybinin (titre 93%, 0.019 moles) are suspended in 200 ml deionized water and under magnetical stirring 75 ml 35% ammonium hydroxyde are dropped in 10 minutes therein. When the addition is completed 24.78 g β-cyclodextrin (Fischer 13%; 0.019 moles) are added thereto all at once. Stirring is maintained for a further 35 minutes then the gaseous ammonia excess is removed under vacuo, pH is adjusted to 7 adding 6N hydrochloric acid, it is lyophilized, washed twice with 30 ml each of acetone to give 26.2 g of a yellow solid (HPLC titre in Silybinin 24.7% t.a.) soluble in water, which on X-ray analysis shows the characteristic peaks due to the Silybinin: β-cyclodextrin inclusion complex (1:1) (e.g. 20=18.8) and melting point 250°C. UV (max) 287 nm (MeOH) The inclusion complex according to the present invention shows a higher dissolution in comparison to the dissolution demonstrated, in the same experimental conditions, by either Silybinin and Silymarin. This characteristic is made evident by the diagram of Table 1 where the dissolution curves of the complex Silybinin: β-cyclodextrin, prepared according to Example 1 (148 mg of complex corresponding to 40 mg of pure Silybinin), of Silybinin (40 mg) and of Silymarin dissolved in gastric juice at pH 6,8 are reported. The evaluation has been carried out according to the USP paddle method and the determinations have been performed by UV in comparison to known standards. The X-ray diffraction diagram of the inclusion complex according to Example 1 is characterized by the presence of some significant peaks (e.g. 20=18.8) which cannot be revealed in the X-ray diffraction diagram of the powder formed by the mechanical mixture of the two components; furthermore in the X-ray diffraction diagram of the above mentioned inclusion complex are not present those peaks (20=24.5; 14.6; 32; 22.7) which are characteristics of the diffraction diagram of the mechanical mixture of the two components. Example 2 Preparation of the Silybinin: β-ciclodextrin inclusion complex (molar ratio 1:2) Operation is carried out as described in the previous Example using 35 g of pure Silybinin and 211.7 g β-cyclodextrin corresponding to 183.7 g on anhydrous. Grams 218 of the product having HPLC titre in Silybinin 15.41% and KF=2.5%, are obtained. Example 3 Preparation of the Silybinin γ-cyclodextrin inclusion complex (molar ratio 1:1) Operation is carried out as described in Example 1 using 5.1 g γ-cyclodextrin having titre 98% and 2.04 g Silybinin having titre 91% corresponding to 1.857 g pure Silybinin. Grams 5.9 of Silybinin: γ-cyclodextrin inclusion complex (1:1) are obtained. Example 4 Preparation of the Silybinin: DIMEB (heptakis-2,6-0-dimethyl-β-cyclodextrin) inclusion complex (molar ratio 1:1) Operation is carried out as described in Example 1 using 32.3 g of Silybinin having titre 93% and 82.4 g DIMEB. Grams 97.2 of Silybinin: heptakis-2,6-0-dimethyl-β-cyclodextrin inclusion complex (1:1) are obtained. The products obtained as described in the previous Examples may be used for preparing pharmaceutical compositions by addition of suitable excipients and according to the operation described in the following Examples. Example 5 Preparation of Silybinin-β-cyclodextrin-complex (molar ratio 1:2.75) Grams 109.76 β-cyclodextrin (96,6 mmol) were dissolved in 600 ml of distilled water; 16.94 g of Silybinin (35 mmol) were added to the solution giving a suspension. About 75 ml of 1N sodium hydroxide solution was added dropwise over a period of 10 min with continuous stirring. Silybinin easily dissolved resulting in a yellow solution. 2N phosphoric acid solution was added to adjust the pH between 3.2-3.4 (about 100-105 ml of phosphoric acid is needed). The temperature of the solution during the dissolution and the neutralization process was kept below 20°C by using an ice bath. The solution lost Its colour at a pH below 5. The UV-spectra of the solution after 1:800 dilution with 50% (v/v) ethanol showed that Silybinin was present in unionized form in the solution. The slightly opalescent beige tone solution was filtered across a 04 glass filter. The solid complex was isolated from the clear solution by freeze drying. Grams 140 of Silybinin-β-cyclodextrin complex was obtained. Active ingredient content: 12.1 % (w/w) measured by UV-spectrophotometry. Loss on drying (at 105°C under vacuo 2h): 9.5 % (w/w). Release esperiments with Silybinin, Silymarin and the complex of this Example were carried out according to USP XXII, paddle apparatus at pH 7.5 and 37°C. The experiments showed a 96 % release (solubility) of the complex in contrast to 0 % release of Silybinin and 53 % total release of Silymarin corresponding to 37.1 % Silybinin release. Example 6 Preparation of Silybinin:heptakis-2,6-di-O-methyl-β-cyclodextrin-complex (molar ratio 1:2.2) The method of Example 5 was repeated except that 110 g heptakis-2,6-di-O-methyl-β-cyclodextrin (DIMEB; 82 mmol) and 18 g Silybinin (37 mmol) were used. Grams 140 of Silybinin-DIMEB complex was obtained. Active ingredient content: 12.6 % (w/w) measured by UV-spectrophotometry. Loss on drying (at 105°C under vacuo 2h): 5.0 % (w/w). Example 7 Preparation of Silybinin-hydroxypropyl-β-cyclodextrin- complex (molar ratio 1:2.2) Grams 100 hydroxypropyl-β-cyclodextrin (HP CD) 76 mmol. PS = 3.0 (PS = average degree of substitution per cyclodextrin unit) was dissolved in 700 ml of water. Grams 17 silybinin (35 mmol) was added, resulting in a suspension, approx. 70 ml of 1N sodium hydroxide was added dropwise to the solution over a period of 10 min. Further processing of the solution is identical with the preparation of DIMEB complex and 130 g of Silybinin-HPβCD complex was obtained. Active ingredient content: 12.7 % (w/w). Loss on drying (at 105°C under vacuo 2h): 7.0 %. X-ray analysis of the complexes of Examples 6 and 7. X-ray power diffraction diagrams of pure Silybinin, DIMEB, HP-βCD and the complex samples were recorded on Philips powder diffractometer using Cu-K radiation. Comparing the X-ray patterns (Fig.2,3), the characteristic 20 peaks of the crystalline Silybinin and DIMEB disappeared, the complexes showed a completely different structure in both cases. Thermoanalytical studies Thermoanalytical investigations were carried out on a Du Pont 990 Thermal Analyser System. TG (Thermogravimetry) DTG (Differential TG) DSC (Differential Scanning Calorimetry) and TEA (Thermal Evolution Analysis) curves of the drug, as well as those of its mechanical mixtures with DIMEB and HP-βCD and the complexes were taken simultanously. DSC curves of Silybinin (Fig.4) indicates that the decomposition of the drug begins at about 130°C and up to 160°C about 4-5 % loss of weight is observed. The second decomposition step of the drug starts above 250°C in the region of the cyclodextrin decomposition. The decomposition step at the lower temperature is suitable to differentiate the complexed and the non-complexed products. DSC curves of the complexes (Fig.5,6) show that the decomposition of Silybinin is observed at higher temperature only at about 175-180°C. Solubility properties of the complexes of Examples 6 and 7. Milligrams 250 of DIMEB and HPβCD complex can easily be dissolved in 2 ml of distilled water and in pH 1.4 hydrochloric acid resulting in a clear solution. The UV-spectra of the aqueous solutions - recorded after 1: 750 dilution with 50 % (v/v) ethanol - show undoubtly that Silybinin is included both in DIMEB and HPβCD in unionized form (Fig.7). The pH of the aqueous solutions: DIMEB complex: 3.3 HP-βCD complex: 3.4 After one day following the dissolution no precipitation was observed in the DIMEB-complex solution. DIMEB complex solution remained clear more than 10 days of storage. UV-spectra recorded remained unaltered indicating the good stability of Silybinin complexes in solutions. Example 8 Pharmaceutical composition containig the Silybinin: β-cyclodextrin inclusion complex (molar ratio = 1:2). Silybinin to titre 40.0 mg β-cyclodextrin 164.7 mg Pharmacoat 606 10.0 mg Sodium laurylsulphate 15.0 mg Polyethyleneglycol 15.0 mg Lactose 103.8 mg Magnesium stearate 1.5 mg Hard gelatin capsules of size "1". Silybinin: and β-cyclodextrin are mixed in a water/methyl alcohol solution (7:3) at 45°C for two hours, then methyl alcohol is evaporated and the product lyophilized. It is analyzed by X-ray (PW1800 Philips diffractometer for powder; CUKα radiation; Ni filter; Kv40mA20) and it shows a diffractogram which does not correspond to the simple addition of the β-cyclodextrin and Silybinin spectra, but where are present some novel signals (such as for example 20=43.3) while there are missing some peaks which on the are characteristic of the mechanical mixture of the two components (20=24.5; 14.6; 32; 22.7). That confirms the formation of the inclusion complex. The lyophilized product is kneaded with a 2% w/v Pharmacoat 606 in methylene chloride/ethyl alcohol (9:1) solution.The so obtained mixture is granulated on a 1.2 mm mesh sieve, dried and scraped on a 0.840 mm mesh sieve. The granulated product is operculated in capsules of size "1". The binding solution may be replaced by a polyvinylpyrrolidone solution in ethyl alcohol/water or by an aqueous gelatin solution. The polyethyleneglycol molecular weight may vary from 1,500 to 10,000. Example 9 Pharmaceutical composition containig the Silybinin: β-cyclodextrin inclusion complex (molar ratio = 1:2.75) The inclusion complex is kneaded with a 2% w/v Pharmacoat 606 in methylene chloride/ethyl alcohol solution.The so obtained mixture is granulated on a 1.2 mm mesh sieve, dried and scraped on a 0.840 mm mesh sieve. The granulated product is operculated in capsules of size "1". The binding solution may be replaced by a polyvinylpyrrolidone solution in ethyl alcohol/water or by an aqueous gelatin solution. The polyethyleneglycol molecular weight may vary from 1,500 to 10,000. Example 10 Pharmaceutical composition containing the Silybinin: γ-cyclodextrin inclusion complex (molar ratio 1:2). Operation is carried out as described in Example 9 using: Silybinin to titre 40.0 mg γ-cyclodextrin 188.1 mg Example 11 Pharmaceutical composition containing the Silybinin: β-cyclodextrin inclusion complex (molar ratio 1:4). Operation is carried out as described in Example 8 using: Hard gelatin capsules of size "0". Example 12 Pharmaceutical composition containing the Silybinin: DIMEB inclusion complex (molar ratio 1:2). Operation is carried out as described in Example 8 using: Silybinin to titre 40.0 mg DIMEB 220.77 mg The absorption of the complexes of the invention has been evaluated by orally administering an aqueous solution of the composition under examination and measuring by HPLC the Silybinin bile levels. This test has been preferred because it is well known that drugs of the Silymarin kind reach in the bile the highest concentration. The experiments have been carried out on Wistar rats weighing 180-200 g to which the compositions under examination were administered by gastric intubation. The animals were kept on an empty stomach for 16 hours. The composition to be tested was dissolved in water containing 10% of gum Arabic and were prepared solutions so that each animal was administered, each time with an amount of 5 ml/kg of said solution. Immediately after the treatment the animals were anaesthetized and operated to check the coledocous duct. A small tube was inserted therein and the bile was collected over a period of 24 hours. For the analytical determination 1 ml bile was treated 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 from the tests are listed in the following Table 2 where: in test A, there were administered 20 mg/kg of Silymarin, according to a known commercial formulation which corresponds to 8 mg/ kg of pure Silybinin, in test B, there were administered 8.79 mg/kg of Silybinin having a HPLC titre of 91%, which corresponds to 8 mg/kg of pure Silybinin, used such as without excipients, in test C, there were administered 29.2 mg/kg of the Silybin: β-cyclodextrin inclusion complex, described in Example 1, which possess a HPLC, titre in Silybinin 27.4% corresponding to 8 mg/kg of pure Silybinin. Product Number of animals Bile Volume Bile 0-24hr mcg/ml 0-24hr mcg/rat % excreted Silybinin Silybinin (Test B) 12 12.2 1.0 11.9 0.6 Silybinin (Test A) 15 11.4 6.4 73.8 2.7 Complex of Ex.1 (Test C) 14 14.3 17.6 256.6 11.7 The average volume of bile produced in 24 hours by a non-treated rat is 13.4 ml. The evaluation of the data obtained shows that the administration of the composition containing as active ingredient the inclusion complex according to the invention allows to obtain, using the same amount of active ingredient in all the tests,concentration levels in Silybinin more than doubled when compared to those obtained in test A and nearly twenty times those obtained in test B. In a further experiment the resorption of Silymarin, Silybinin and of the complex of Example 5 was evaluated. For this purpose the excretion of Silybinin in the rat bile was determined in a conventional model experiment. Anesthetized male Wistar SPF rats kept under conventional conditions, were cannulated at the ductus choledochus. After intragastric administration of the test compound (5 mg Silybinin/kg) the bile fluid was collected for 5hr. The Silybinin content in the bile fluid was determined by quantitative thin layer chromatography. The results were as follows: % excretion of Silybinin in rat bile: The above data show that the complex is more and easier absorbed than Silybinin and Silymarin.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE An inclusion complex of Silybinin with a cyclodextrin selected from α-, β-, γ-cyclodextrin, and the O-C₁-C₄-alkyl and hydroxy-C₁-C₄-alkyl derivatives thereof. A complex of claim 1, wherein the molar ratio of Silybinin to cyclodextrin is 1:1 to 1:4. A complex of claim 1 or 2, wherein the cyclodextrin is β-cyclodextrin. The complexes of claim 3, wherein the molar ratio of Silybinin to β-cyclodextrin is 1:2, 1:2.75 or 1:4. The complexes of claim 1 or 2, wherein the O-C₁-C₄-alkyl and hydroxy-C₁-C₄-alkyl cyclodextrin derivative is selected from the group consisting of heptakis-2,6-0-dimethyl-β-cyclodextrin, heptakis-2,3,6-0-trimethyl-β-cyclodextrin and O-hydroxypropyl-β-cyclodextrin. A process for preparing the inclusion complexes of claim 1 which comprises preparing a solution of Silybinin and cyclodextrin in water or in an aqueous alkyl alcohol at a pH above 7, adjusting the pH of the solution below 7 adding a suitable acid and isolating the formed complex. The process of claim 6, which comprises suspending Silybinin in water, adding a suitable base to dissolve Silybinin, adding the cyclodextrin, lowering the pH below 7 by adding a suitable acid and isolating the complex, or suspending Silybinin and the cyclodextrin in water, adding a suitable base to dissolve the components, adjusting the pH to below 7 by adding a suitable acid and isolating the complex. The process of claim 6, wherein the alkyl alcohol is methyl alcohol or ethyl alcohol. A pharmaceutical composition comprising a therapeutically effective amount of one or more complexes according to anyone of claims 1 to 5, optionally together with pharmaceutically acceptable carriers and/or adjuvants. A process for preparing the pharmaceutical composition of claim 9 which comprises providing an effective therapeutical amount of at least one complex of anyone of claims 1 to 5 in a form suitable for administration, optionally incorporated into a pharmaceutically acceptable carrier and/or adjuvant. The use of at least a complex according to anyone of claims 1 to 5 for preparing a pharmaceutical composition for hepatotherapy. Claims for the following Contracting States : ES, GR A process for preparing an inclusion complex of Silybinin with a cyclodextrin selected from α-, β-, γ-cyclodextrin and the O-C₁-C₄-alkyl and hydroxy-C₁-C₄ -alkyl derivatives thereof which comprises preparing a solution of Silybinin and cyclodextrin in water or in an aqueous alkyl alcohol at a pH above 7, adjusting the pH of the solution below 7 and isolating the formed complex. The process of claim 1, wherein the O-C₁-C₄-alkyl and hydroxy-C₁-C₄-alkyl derivative is selected from heptakis-2,6-0-dimethyl-β-cyclodextrin, heptakis-2,3,6-0-trimethyl-β-cyclodextrin and O-hydroxypropyl-β-cyclodextrin. The process of claims 1 and 2, which comprises suspending Silybinin in water, adding a suitable base to dissolve Silybinin, adding the cyclodextrin, lowering the pH below 7 by adding a suitable acid and isolating the complex, or suspending Silybinin and the cyclodextrin in water, adding a suitable base to dissolve the components, adjusting the pH to below 7 by adding a suitable acid and isolating the complex. A process according to claim 1, wherein the alkyl alcohol is methyl alcohol or ethyl alcohol. A process according to claims 1-4, wherein the molar ratio of Silybinin to cyclodextrin is 1:1 to 1:4. A process according to any one of claims 1-5, wherein the cyclodextrin is β-cyclodextrin. A process according to claim 6, wherein the molar ratio of Silybinin to β-cyclodextrin is 1:2, 1:2.75 or 1:4. A process according to any one of the previous claims wherein the obtained inclusion complex is used, optionally incorporated into a pharmaceutically acceptable carrier or adjuvant, as an active principle for pharmaceutical compositions with antihepatotoxic activity.

Inventors: BOSONE ENRICO DR, CAPONI ROBERTO DR, MONTEROSSO VINCENZO DR, SZEJTLI JOZSEF PROF DR, VALCAVI UMBERTO PROF, WACHTER WILFRIED PETER DR RER, BOSONE, ENRICO, DR., CAPONI, ROBERTO, DR., MONTEROSSO, VINCENZO, DR., SZEJTLI, JOZSEF, PROF. DR., VALCAVI, UMBERTO, PROF., WACHTER, WILFRIED PETER, DR. RER. NAT.
Applicants: ISTITUTO BIOCHIMICO ITALIANO, MADAUS AG, IBI- ISTITUTO BIOCHIMICO ITALIANO GIOVANNI LORENZINI S.P.A., MADAUS AKTIENGESELLSCHAFT
Application year: 1990
Main classification: C08B37
IPC codes: C07D407, A61P1, A61K31, C08B37, A61K47