MXPA01005720A - Macrolides - Google Patents

Abstract

The invention relates to the stabilization of poly-ene macrolides and to a particular macrolide obtained in crystalline form.

Description

STABILIZATION OF MACRO IDOS The present invention relates to the stabilization of a pharmaceutically active ingredient sensitive to oxidation, for example a polyene macrolide, preferably a polyene macrolide having immunosuppressive properties, in particular rapamycins. The handling and storage, particularly in the bulk form, of pharmaceutically active ingredients that are sensitive to oxidation is difficult. Special handling is necessary, and often the oxidation sensitive ingredient is stored in an air-tight package under a protective gas. Substantial amounts of stabilizer are added during the process of formulating these pharmaceutically active ingredients. Polyene macrolides have satisfactory stability properties. However, it has now been found that its stability to oxygen can be substantially improved by the addition of a stabilizer, for example an antioxidant, during its isolation step. According to the invention, there is provided: 1. A process for stabilizing a polyene macrolide, which comprises adding an antioxidant to the purified macrolide, preferably at the beginning of its isolation step.

This process is particularly useful for the production of a stabilized polyene macrolide in bulk. The amount of antioxidant can conveniently be up to 1 percent, more preferably 0.01 to 0.5 percent (based on the weight of the macrolide). This small amount is subsequently referred to herein as a catalytic amount. As alternatives to the above, the present invention also provides: 2. A mixture, for example a bulk mixture, comprising a polyene macrolide and an antioxidant, preferably a catalytic amount thereof, preferably in solid form. The mixture may be in the form of particles, for example in crystallized or amorphous form. It can be in a sterile or substantially sterile condition, for example in a condition suitable for pharmaceutical use. 3. The use of a mixture as defined above in 2, in the manufacture of a pharmaceutical composition. Examples of polyene macrolides are, for example, molecules comprising double bonds, preferably conjugated double bonds, for example those having antibiotic and / or immunosuppressive properties, for example macrolides comprising a lactam or lactone bond and their derivatives, for example compounds having a biological activity qualitatively similar to that of the natural macrolide, for example chemically substituted macrolides. Suitable examples include, for example, rapamycins and ascomycines. A preferred polyene compound is a macrolide comprising at least 2 conjugated double bonds, for example, 3 conjugated double bonds. Rapamycin is a known lactam macrolide that can be produced, for example, by Streptomyces hyqroscopicus. The structure of rapamycin is given in Kessier, H. and collaborators; 1993; Helv. Chim. Acta, 76: 117. Rapamycin has antibiotic and immunosuppressive properties. The rapamycin derivatives are known, for example, 16-O-substituted rapamycins, for example as disclosed in International Publications Nos. WO 94/02136 and WO 96/41807, 40-O-substituted rapamycins, for example as disclose in International Publications Numbers WO 94/09010, WO 92/05179, WO 95/14023, 94/02136, WO 94/02385, and WO 96/13273, all of which are incorporated herein by reference. Preferred rapamycin derivatives are, for example, rapamycins wherein the hydroxyl at position 40 of formula A illustrated on page 1 of International Publication Number WO 94/09010, is replaced by -OR, where R is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, for example 40-O- (2-hydroxy) ethyl-rapamycin, 40-O- (3-hydroxy) propyl-rapamycin, and 40-O- [2- (2-hydroxy) ethoxy] ethyl-rapamycin. Ascomycins, of which FK-506 and ascomycin are the best known, form another class of lactam macrolides, many of which have potent immunosuppressive and anti-inflammatory activity. FK-506 is a lactam macrolide produced by Streptomyces tsukubaensis. The structure of FK-506 is given in the Appendix to the Merck Index, n to Edition (1989) as point A5. Ascomycin is described, for example, in U.S. Patent No. USP 3,244,592. Ascomycin, FK-506, other naturally occurring macrolides, which have a similar biological activity, and their derivatives, for example synthetic analogues and derivatives, are collectively referred to as "Ascomycins". Examples of the synthetic analogues or derivatives are, for example, halogenated ascomycins, for example 33-epi-chloro-33-deoxy-ascomycin, as disclosed in European Patent Number EP-A-427, 680, derivatives of tetrahydropyran, for example as disclosed in European Patent Number EP-A-626,385. Particularly preferred macrolides are rapamycin and 40-O- (2-hydroxy) ethyl-rapamycin. Preferred antioxidants are, for example, 2,6-diterbutyl-4-methylphenol (later in the present BHT), vitamin E or C, with BHT being particularly preferred. A particularly preferred mixture of the ition is a mixture of rapamycin or 40-O- (2-hydroxy) ethyl-rapamycin, and 0.2 percent (based on the weight of the macrolide) of antioxidant, preferably BHT. The antioxidant can be added to the polyene macrolide at the beginning of the isolation steps, preferably the final isolation step, more preferably just before the final precipitation step. The macrolide is preferably in a purified state. It can be dissolved in an inert solvent, and the antioxidant is added to the resulting solution, followed by a precipitation step of the stabilized macrolide, for example in an amorphous form, or in the form of crystals. Preferably the mixture of the ition is in an amorphous form. The resulting stabilized macrolide surprisingly exhibits improved oxidation stability, and its handling and storage, for example in bulk form, before further processing, for example, in a galenic composition, becomes much easier. It is particularly interesting for macrolides in an amorphous form. The stabilized macrolide according to the ition can be used as such for the production of the desired galenic formulation. These formulations can be prepared according to methods known in the art, comprising the addition of one or more pharmaceutically acceptable diluents or carriers, including the addition of additional stabilizer if required.

Accordingly, there is further provided: 4. A pharmaceutical composition comprising, as an active ingredient, a stabilized mixture as disclosed above, together with one or more pharmaceutically acceptable diluents or vehicles. The composition of the invention can be adapted for oral, parenteral, topical (e.g., skin), ocular, nasal, or inhalation (e.g., pulmonary) administration. A preferred composition is one for oral administration, preferably a water-free composition when the active ingredient is a lactone macrolide. The pharmaceutical compositions of the invention may comprise additional excipients, for example a lubricant, a disintegrating agent, a surfactant, a carrier, a diluent, a flavor improver, and the like. They may be in liquid form, for example solutions, suspensions, or emulsions, such as microemulsions, for example as disclosed in U.S. Patent No. USP 5,536,729, or in solid form, e.g., capsules, tablets, dragees, powders (including micronized or otherwise reduced particles), solid dispersions, granules, etc., for example as disclosed in International Publication Number WO 97/03654, the content of which is incorporated herein by reference, or forms semi-solid, such as ointments, gels, creams, and pastes. Preferably they are adapted to be in a form suitable for oral administration. Preferably they are in a solid form. The pharmaceutical compositions of the invention can be prepared according to the known methods, by mixing the stabilized macrolide according to the invention, with the additional ingredients, under agitation; the ingredients can be ground or crushed, and if desired can be compressed, for example in tablets. This invention is particularly interesting for rapamycin compositions in liquid or solid form. A particularly preferred composition is a solid dispersion, for example comprising a rapamycin stabilized according to the invention, and a carrier medium, for example a water soluble polymer, such as hydroxypropylmethylcellulose, for example as disclosed in International Publication. Number WO 97/03654. The compositions of the invention are useful for the known indications for the macrolide they contain, for example in known dosages. For example, when the macrolide has immunosuppressive properties, for example rapamycin or a rapamycin derivative, the composition may be useful, for example in the treatment or prevention of rejection of allo- or xeno-acute or chronic organ or tissue transplantation. , autoimmune diseases or inflammatory conditions, asthma, proliferative disorders, for example tumors, or hyperproliferative vascular disorders, preferably in the prevention or treatment of transplant rejection. The amount of macrolide and the composition to be administered depend on a number of factors, for example the active ingredient used, the conditions to be treated, the duration of the treatment, and so on. For example, for rapamycin or 40-O- (2-hydroxy) ethyl-rapamycin, a daily dosage form suitable for oral administration comprises 0.1 to 10 milligrams, to be administered once or in divided form. In another aspect, this invention also provides 40-O- (2-hydroxy) ethyl-rapamycin in a crystalline form, particularly in a substantially pure form. Preferably, the crystal form is characterized by the absence, or by the substantial absence, of any solvent component; it is in a form that is not solvate. 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form belongs to the monoclinic system. The resulting crystals have a melting point of 146-147 ° C, especially 146.5 ° C. To assist in the identification of the new crystalline form, X-ray diffraction analysis data are provided. The conditions under which these data are obtained are as follows: Temperature 293 (2) K Wavelength 1.54178 Á Space group P2? Unitary cell dimensions at 14.378 (2) A b 11.244 (1) Á c 18.310 (2) Á ß 108.58 (1) ° Volume 2805.8 (6) Á3 Z 2 Density (calculated) 1,134 g / cm3 Absorption coefficient 0.659 mm "1 F (000) 1040 Crystal size 0.59 x 0.11 x 0.03 mm Range of? For data collection 2.55 to 57.20. Reflections collected 4182 Independent reflections 4037 [R (int) = 0.0341] Intensity decay 32% Refinement method Least squares of full matrix on F2 Data / restrictions / parameters 3134/1/613 Good fit on F2 1,055 indices R 'end R? = 0.0574, wR2 = 0.1456 [1 >; 2 sigma (1)] Largest peak difference and 0.340 and -0.184 e / Á3 hole. 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form can be prepared by dissolving the amorphous compound in a solvent, for example ethyl acetate, and adding an aliphatic hydrocarbon CnH2n + 2 (n = 5, 6 or 7). After the addition of the hydrocarbon, the resulting mixture can be heated, for example, at a temperature of 25 ° C to 50 ° C, for example up to 30 ° C-35 ° C. The storage of the resulting mixture can conveniently take place at a low temperature, for example less than 25 ° C, preferably from 0 ° C to 25 ° C. The crystals are filtered and dried. Heptane is preferred as an aliphatic hydrocarbon. If desired, nucleation procedures can be started, for example by sonication or seeding. The present invention also provides a process for purifying 40-O- (2-hydroxy) ethyl-rapamycin, which comprises crystallizing 40-O- (2-hydroxy) ethyl-rapamycin from a medium containing crystal, for example as disclosed above, and recover the crystals thus obtained. The crystal carrying medium may include one or more components in addition to those mentioned above. It has been found that a particularly suitable glass containing medium is one which comprises approximately two parts of ethyl acetate and about 5 parts of aliphatic hydrocarbon, for example heptane. It has been found that 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form possesses immunosuppressive activity in vi tro and in vivo comparable to that of the amorphous form. In localized GvHD, the maximum inhibition (70-80 percent) of swelling of the lymph node is achieved with a dosage of 3 milligrams with 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form. 40-O- (2-hydroxy) ethyl-rapamycin can be useful for the same known indications for the amorphous compound, for example to prevent or treat acute or chronic allo-or xeno-transplantation, autoimmune diseases or inflmatory conditions, asthma, proliferative disorders, for example tumors, or hyperproliferative vascular disorders, for example as disclosed in International Publication Number WO 94/09010, or in International Publication Number WO 97/35575, the content of which is incorporated herein as reference. In general, satisfactory results are obtained after oral administration in dosages of the order of 0.05 to 5 or up to 20 milligrams / kilogram / day, for example in the order of 0.1 to 2 or up to 7.5 milligrams / kilogram / day, administered once, or in divided doses 2 to 4 times a day. The daily dosages suitable for the patients, therefore, are of the order of up to 10 milligrams, for example from 0.1 to 10 milligrams. 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form can be administered by any conventional route, for example orally, for example tablets or capsules, or nasally or pulmonarily (by inhalation). It can be administered as the sole active ingredient, or together with other drugs, for example immunosuppressive and / or immunomodulatory and / or anti-inflammatory agents, for example, as disclosed in International Publication Number WO 94/09010. In accordance with the foregoing, the present invention also provides: A method for preventing or treating rejection of allo-or xeno-acute or chronic transplantation, autoimmune diseases or inflammatory conditions, asthma, proliferative disorders, or hyperproliferative vascular disorders, in a subject in need of such treatment, which method comprises administering to this subject a therapeutically effective amount of 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form; 6. 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form, for use as a pharmaceutical product; for example, in a method as disclosed above; 7. A pharmaceutical composition comprising 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form, together with a pharmaceutically acceptable diluent or carrier therefor, - 8. A kit or pack for use in immunosuppression or inflammation. , which includes a pharmaceutical composition as disclosed above, and a pharmaceutical composition comprising an immunosuppressant or immunomodulatory drug, or an anti-inflammatory agent. The following examples illustrate the invention without limiting it.

Example 1; Crystallization 0.5 grams of amorphous 40-O- (2-hydroxy) ethyl-rapamycin are dissolved in 2.0 milliliters of ethyl acetate at 40 ° C. 5.0 milliliters of heptane are added, and the solution becomes "milky". After heating to 30 ° C, the solution becomes clear again. Upon cooling to 0 ° C, and with scraping, an oil drops from the solution. The test tube is closed and stored at 10 ° C overnight. The resulting white bulky solid is then filtered and washed with 0.5 milliliters of a mixture of ethyl acetate / hexane (1: 2.5), and the resulting crystals are dried at 40 ° C under 5 mbar for 16 hours. In this way 40-O- (2-hydroxy) ethyl-rapamycin is obtained in a crystalline form, which has a melting point of 146.5 ° C. Crystallization can be performed on a larger scale as follows: 250 grams of amorphous 40-O- (2-hydroxy) ethyl-rapamycin are dissolved in 1.0 liter of ethyl acetate under argon with slow stirring. This solution is heated to 30 ° C, and then for 45 minutes, 1.5 liters of heptane are added per drop. 0.25 grams of seed crystals prepared as disclosed above under the same portion conditions are added. The mixture is further stirred at 30 ° C for a period of 2 hours, and the crystallization mixture is cooled to 25 ° C for 1 hour, and then at 10 ° C for 30 minutes, and filtered. The crystals are washed with 100 milliliters of a mixture of ethyl acetate / hexane (2: 3). The subsequent drying is carried out at 50 ° C and at about 5 mbar. P.f. 146.5 ° C. IR in KBr: 3452, 2931, 1746, 1717, 1617, 1453, 1376, 1241, 1191, 1163, 1094, 1072, 1010, 985, 896 was "1 The simple X-ray structure with coordinates is indicated in Figures 1 to 3 later.

Example 2 Production of stabilized 40-O- (2-hydroxy) ethyl-rapamycin Dissolve 100 grams of 40-O- (2-hydroxy) ethyl-rapamycin in 600 liters of absolute ethanol. After the addition of 0.2 grams of BHT, the resulting solution is added dropwise with stirring to 3.0 liters of water within 1 hour. The resulting suspension is stirred for an additional 30 minutes. Filtration with subsequent washing (200 milliliters of water / ethanol 3 times, in a volume / volume ratio of 5: 1) results in a moist white product, which is further dried under vacuum (1 mbar) at 30 ° C. for 48 hours. The resulting dry product contains 0.2 percent BHT (w / w).

The resulting product shows better storage stability. The sum of the by-products and degradation products in percentage after one week of storage is as follows: Compound 50 ° C in an open flask Example 2 (0.2% BHT) 1.49 Without BHT > The procedure of the previous example can be repeated, but using, as an active ingredient, rapamycin.

Claims (13)

1. CLAIMS 1. A mixture comprising a polyene macrolide and an antioxidant.
2. 2. A mixture according to claim 1, wherein the antioxidant is present in an amount up to 1 percent, based on the weight of the macrolide.
3. 3. A mixture according to claim 1, wherein the antioxidant is present in an amount of 0.2 percent, based on the weight of the macrolide.
4. 4. A mixture according to claim 1, wherein the antioxidant is 2,6-diterbutyl-4-methylphenol.
5. 5. A mixture according to claim 1, wherein the polyene macrolide is rapamycin or 40-0- (2-hydroxy) ethyl-rapamycin.
6. 6. A mixture according to claim 1, in solid form.
7. 7. A pharmaceutical composition comprising, as an active ingredient, a mixture according to claim 1, together with one or more pharmaceutically acceptable carriers or diluents.
8. 8. A process for stabilizing a polyene macrolide, which comprises adding an antioxidant to the purified macrolide.
9. 9. 40-O- (2-hydroxy) ethyl-rapamycin in crystalline form.
10. 10. The compound according to claim 9, in a crystalline form which is not solvate.
11. 11. The compound according to claim 9, having a glass lattice of: a = 14.37 A; b = 11.24 A; c = 18.31 Á, with volume 2805 Á3.
12. 12. A pharmaceutical composition comprising a compound according to claim 9 or 10, together with one or more pharmaceutically acceptable diluents or carriers therefor.
13. 13. A process for purifying 40-0- (2-hydroxy) ethyl-rapamycin, which comprises crystallizing 40-O- (2-hydroxy) ethyl-rapamycin from a medium containing crystal, and recovering the crystal thus obtained .