Title of Invention

A MIXTURE COMPRISING A POLY-ENE MACROLIDE AND ANTIOXIDANT

Abstract

The present invention relates to a mixture comprising a poly-ene macrolide and an antioxidant. The present invention also relates to the stabilization of a pharmaceutically active ingredient sensitive to oxidation, e.g a poly-ene macrolide. The present invention provides a mixture comprising a poly-ene macrolide and an antioxidant, wherein the antioxidant is present in an amount of up to 1 % based on the poyl-ene macrolide weight. Preferably, the poly-ene macrolide is rapamycin or 40-0- (2 -hydroxy)ethyl-rapamycin and the anti oxidant is 2,6-di-tert.-butyl-4- methylphenol. The presence of the antioxidant improves the stability of the poly-ene mactolide to oxidation.

Full Text

STABILIZATION OF MACROLIDES The present invention relates to the stabilization of a pharmaceutically active ingredient sensitive to oxidation, e.g. a poly-ene macroiide, preferably a poly-ene macroiide having immunosuppressant properties, particularly rapamycins. The handling and storage particularfy in the bulk form of pharmaceutically active ingredients which are sensitive to oxidation is difficult. Special handling is necessary and often the oxidation-sensitive Ingredient is stored in air-tight packaging under protective gas, Substarnfai amounts of stabilizers are added during the formulating process of such pharmaceutically active ingredients. Poly-ene macrolrdes have satisfactory stability properties. However, it has now been found that their stability to oxygen may substantially be improved by the addition of a stabilizer, e.g. an antioxidant, during their isolation step. According to the invention, there is provided 1. A process for stabilizing a poly-ene macroiide comprising adding an antioxidant to the purified macroiide, preferably at the commencement of its isolation step. This process is particularly useful for the production of a stabilized poly-ene macroiide in bulk. The amount of antioxidant, may conveniently be up to 1%, more preferably from 0.01 to 0.5 % (based on the weight of the macroiide). Such a small amount is refen-ed to hereinafter as a catalytic amount. As alternatives to the above the present invention also provides: 2. A mixture, e.g. a bulk mixture, comprising a poly-ene macroiide and an anti-cixidant, preferably a catalytic amount thereof, preferably in solid form. 3. Use of a mixture as defined above in 2. in the manufacture of a pharmaceutical composition. Examples of poly-enes macrolides are e.g. molecules comprising double bonds, preferably conjugated double bonds, for example such having antibiotic and/or immunosuppressant properties, e.g. macrolides comprising a lactam or lactone bond and their derivatives, e.g. compounds which have a biological activity qualitatively similar to that of the natural macrolide, e.g. chemically substituted macrolides. Suitable examples include e.g. rapamycins and ascomycins. A prefen"ed poly-ene macrolide is a macrolide comprising at least 2 conjugated double bonds, e.g. 3 conjugated double bonds. Rapamycin is a Itnown lactam macrolide produceable, for example by Streptomvces hyqroscopicus. The structure of rapamycin is given in Kessler, H. et al.; 1993; Helv. Chim. Acta, 76 :117, Rapamycin has antibiotic and immunosuppressant properties. Derivatives of rapamycin are known, e.g. 16-0-substituted rapamycins, for example as disclosed In WO 94/02136 and WO 96/41807,40-O-substituted rapamycins, for example as disclosed in WO 94/09010, WO 92/05179, WO 95/14023, 94/02136, WO 94/02385 and WO 96/13273, all of which being incorporated herein by reference. Preferred rapamycin derivatives are e.g. rapamycins wherein the hydroxy in position 40 of fonnula A illustrated at page 1 of WO 94/09010 is replaced by -OR wherein R is hydroxyalkyi, hydroxyalkoxyalkyi, acylaminoalkyi or aminoalkyi, e.g. 40-O-{2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, and40-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. FK506 Is a lactam macrolide produced by Streptomyces tsukubaensis. The structure of FK606 is given in the Appendix to the Merck Index, 11th ed. (1989) as item A5. Ascomycin is described e.g. in USP 3,244,592. Ascomycin, FK506, other naturally occurring macrolides having a similar biological activity and their derivatives, e.g. synthetic analogues and derivatives are termed nollfirth/filv "Afirnmvrinc" Pvamnioe M ct/n/hatir^ QnQtna» «^ Particulariy preferred macrolides are rapamycin and 40-O-(2-hydroxy)ethyl-rapamycin. Preferred antioxidants are for example 2,6-dl-tert.-butyl-4-methylphenol (hereinafter BHT), vitamin E or C, BHT being particulariy preferred. A particulariy preferred mixture of the invention is a mixture of rapamycin or 40-O-(2-hydroxy)ethyl-rapamycin and 0.2% (based on the weight of the macrollde^ of antioxidant, preferably BHT. The antioxidant may be added to the poly-ene macrolide at the commencement of the isolation steps, preferably the final isolation step, more preferably just prior to the final precipitation step. The macrolide is preferably in a purified state. It may be dissolved in an inert solvent and the antioxidant is added to the resulting solution, followed by a precipitation step of the stabilized macrolide, e.g. in an amorphous fomi or In the fonn of crystals. Preferably the mixture of the invention is in amorphous forni. The resulting stabilized macrolide exhibits surprisingly an improved stability to oxidation and its handling and storage, e.g. In bulk fomn prior to its further processing for example into a galenic composition, become much easier. It is particulariy interesting for macrolides in amorphous fomi. The macrolide stabilized according to the invention may be used as such for the production of the desired galenic formulation. Such fonnulations may be prepared according to methods known In the art, comprising the addition of one or more phannaceutically acceptable diluent or carrier, including the addition of further stabilizer if required. Accordingly thqre is further provided: 4. A pharmaceutical composition comprising, as active ingredient, a stabilized mixture as disclosed above, together with one or more phannaceutically acceptable diluent or composition is one for oral administration, preferably a water-free compoation when the active ingredient is a lactone macroiide. The pharmaceutical compositions of the invention may comprise further excipients, e.g. a lubricant, a disintegrating agent, a surfactant, a carrier, a diluent, a flavor enhancer, etc. It may be in liquid foirn, e.g. solutions, suspensions or emulsions such as a microemulsions, e.g. as disclosed in USP 5,536,729, or in solid form, e.g. capsules, tablets, drag6es, powders (including micronized or othenwise reduced particulates), solid dispersions, granulates, etc.e.g. as disclosed in WO 97/03654, the contents of which being incorporated herein by reference, or semi-solid forms such as ointments, gels, creams and pastes. They are preferably adapted to be in a form suitable for oral administration. Preferably they are in solid form. The pharmaceutical compositions of the invention may be prepared according to known methods, by mixing the macroiide stabilized according to the invention with the additional ingredients under stirring; the ingredients may be milled or ground and if desired compressed, e.g into tablets. This invention is particuiariy interesting for rapamycin compositions in liquid or solid form. A particulariy preferred composition is a solid dispersion, e.g. comprising a stabilized rapamycin according to the invention and a carrier medium, e.g. a water-soluble polymer such as hydroxypropylmethylcellulose, e.g. as disclosed in WO 97/03654. The compositions of the invention are useful for the indications as known for the macroiide they contain at e.g. known dosages. For example, when the macroiide has immunosuppressant properties, e.g. rapamycin or a rapamycin derivative, the composition may be useful e.g. in the treatment or prevention of organ or tissue acute or chronic alio- or xeno-transplant rejection, autoimmune diseases or inflammatory conditions, asthma, proliferative disorders, e.g tumors, or hyperproliferative vascular disorders, preferably in the prevention or treatment of transplant rejection. The anrirtl int Af mar"rn\Ma anrt r\t (ho fnmw^ne-"Mnn tn ky.i A^nint^*..-^ ^-_—^ .- >. 1 1 In another aspect, this invention also provides 40-O-(2-hydroxy)ethyl-rapamycin in a crystalline form, particulariy in a substantially pure fprni. Preferably the crystal fomi is characterized by the absence or substantial absence of any solvent component; it is in non-solvate fomn. 40-O-(2-hydroxy)ethyl-rapamycin in crystalline form belongs to the monoclinic sytem. The resulting crystals have a m.p. of 146""-147""C, especially HB-S^C. To assist identification of the new crystalline form, X-ray diffraction analysis data are provided. The conditions under which ttiese data are obtained are as follows; 40-O-(2-hydroxy)ethyI-rapamycin in crystalline form may be prepared by dissolving the amorphous compound in a solvant e.g. ethyl acetate and adding an aliphatic hydrocatbon CnHsn+z (n=5, 6 or 7). After addition of the hydrocarbon, the resulting mixture may be warmed e.g. at a temperature of 25 to SCC, e.g. up to 30-35X. Storing of the resulting mixture may conveniently take place at a low temperature, e.g. below 25°C, preferably from 0 to 25°C. The crystals are filtered and dried. Heptane is preferred as an aliphatic hydrocarbon. If desired, nucleation procedures may be commenced e.g. by sonication or seeding. The present invention also provides a process for purifying 40-O-{2-hydroxy)ethyl-rapamycin comprising crystallizing 40-O-{2-hydroxy)ethyl-rapamycin from a crystal bearing medium, e.g. as disclosed above, and recovering the crystals thus obtained. The crystal bearing medium may include one or more components in addition to those recited above. A particuiariy suitable crystal bearing medium has been found to be one comprising ca. 2 parts ethyl acetate and ca. 5 parts aliphatic hydrocarbon, e.g. heptane. 40-O-(2-hydroxy)ethyl-rapamycin in crystalline form has been found to possess in vitro and in vivo immunosuppressive activity comparable to that of the amorphous fomi. In the localized GvHD, maximal inhibition (70-80%) of lymph node swelling is achieved with a dosage of 3 mg with 40-O-{2-hydroxy)ethyl-rapamycin in crystalline form. 40-O.-(2-hydroxy)ethyl-rapamycin may be useful for the same indications as known for the amorphous compound, e.g. to prevent or treat acute and chronic alio- or xeno-transplant rejection, autoimmune diseases or inflammatory conditions, asthma, proliferative disorders, e.g tumors, or hyperpn^iiferative vascular disorders, e.g as disclosed in WO 94/09010 or in WO 97/35575, the contents thereof being incorporated herein by reference. In general, satisfactory results are obtained on oral administration at dosages on the order of from 0.05 to 5 or up to 20 mg/kg/day, e.g. on the order of from 0.1 to 2 or up to 7.5 mg/kg/day administered once or, in divided doses 2 to 4x per day. Suitable daily dosages for patients dnjgs, e.g. immunosuppressive and/or immunomodulatory and/or anti-inflammatory agents, e.g. as disclosed in WO 94/09010. In accordance with the foregoing, the present invention also provides: 5. A method for preventing or treating acute or chronic alio- or xeno-transplant rejection, 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 said subject a therapeutically effective amount of 40-O-(2-fiydroxy)ethy(-rapamycin in crystalline fonn; 6. 40-O-(2-hydroxy}ethyl-rapamycin in crystalline forni for use as a phannaceutical;, e.g. in a method as disclosed above; 7. A phamiaceutical composition comprising 40-O-{2-hydroxy)ethyl-rapamycin in crystalline form together with a pharmaceutically acceptable diluent or carrier therefor; 8. A kit or package for use in immunosuppression or inflammation, including 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: Crystallisation 0.5 g amorphous 40-O-(2-hydroxy)ethyi-rapamycin is dissolved in 2.0 ml ethyl acetate at 40°C. 6.0 ml heptane is added and the solution becomes "milky". After wanning to 30°C, the solution becomes clear again. Upon cooling to CC and with scratching an oil lalls out of tiie solution. The .test tube is closed and stored at lO^C overnight. The resulting white voluminous solid is then filtered and washed with 0.5 ml of a mixture of ethyl acetate/hexane (1:2.5) and the resulting crystals are dried at 40""C under 5 mbar for 16 t a0% ^^ /^^ 250 g amorphous 40-O-(2-hydroxy)ethyl-rapamycin is dissolved in 1.01 ethyl acetate under argon with slow stirring. This solution is heated at SCC and then during 45 minutes, 1.5 I heptane is added dropwise. 0.25 g of seed crystals prepared as disclosed above are added under the same conditions in portions. The mixture is further stin-ed at SCC over a period of 2 hours and the crystailisation mixture is cooled to 25°C over 1 hour and then to 10"C for 30 minutes and filtered. The crystals are washed with 100 ml of a mixture eth^^ acetate/hexane (2:3). Subsequent drying is performed at SOX and ca 5 mbar. m.p. 146.5""C IRinKBr: 3452,2931,1746,1717,1617, 1453,1376,1241,1191,1163,1094,1072, 1010,985,896 cm"" Single X-ray structure with coordinates are indicated in Figures 1 to 3 beiow. Example 2: Production of stabilized 40-O-(2-hydToxy)ethyI-rapamycin 100g 40-O-(2-hydroxy)ethy!-rapamycin are dissolved in 6001 abs. ethanol. After addition of 0.2g BHT, the resulting solution is added dropwise with stining to 3.01 water within 1 hour. The resulting suspension is stirred for an additional 30 minutes. Filtration with subsequent washing (3x200 ml water/ethanol at a yN 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 dried product contains 0.2% (w/w) BHT. The resulting product shows improved stability on storage. The sum of by-products and degradation products In % after 1 week storage are as follows: Compound 50°C in open flask Ex. 2 (0.2% BHT} 1.49 Without BHT >10 The procedure of above Example may be repeated but using, as active ingredient, rapamycin. CLAIMS 1. A mixture comprising a poly-ene macrolide and an antioxidant. 2. A mixture according to claim 1, wherein the antioxidant is present in an amount of up to 1 % based on the macrolide weight. 3. A mixture according to claim 1, wherein the antioxidant is present in an amount of 0.2% based on the macrolide weight, 4. A mixture according to claim 1, wherein the antioxidant is 2,6-di-tert,-butyl-4-methylphenol, 5. A mixture according to claim 1, wherein the poly-ene macrolide is rapamycin or 40-O-(2-hydroxy)ethyl-rapamycin. 6. A mixture according to claim 1, in solide form. 7. A pharmaceutical composition comprising, as active ingredient, a mixture according to claim 1 together with one or more pharmaceutically acceptable carrier or diluent. 8. A process for stabilizing a poiy-ene macrolide comprising adding an antioxidant to the purified macrolide. 9. 40-O-{2-hydroxy)ethyl-rapamycin in crystalline form. 10. The compound according to claim 9, in crystaliine non-solvate form. 11. The compound according to claim 9, having a crystal lattice a= 14.37 A, b= 11.24A, c= 18.31 A. the volume being 2805 A^ 12. A phamiaceutical composition comprising a compound according to claim 11 or 12, together with one or more phamnaceutically acceptable diluents or carriers therefor. 13. A process for purifying 40-O-(2-hydroxy)ethyl-rapamycin, comprising crystallizing 40-0-(2-hydroxy)ethyl-rapamycin from a crystal bearing medium and recovering the crystals thus obtained. ■ 14. A mixture substantially as herein described with reference to the accompanying drawings. 15. A pharmaceutical composition substantially as herein described with reference to the accompanying drawings. 16. A process for stabilizing a poly-ene macrolide substantially as herein described with reference to the accompanying drawings. t

Documents:

in-pct-2001-0783-che abstract duplicate.pdf

in-pct-2001-0783-che abstract.pdf

in-pct-2001-0783-che claims duplicate.pdf

in-pct-2001-0783-che claims.pdf

in-pct-2001-0783-che correspondence others.pdf

in-pct-2001-0783-che correspondence po.pdf

in-pct-2001-0783-che description (complete)-duplicate.pdf

in-pct-2001-0783-che description (complete).pdf

in-pct-2001-0783-che drawings.pdf

in-pct-2001-0783-che form-1.pdf

in-pct-2001-0783-che form-19.pdf

in-pct-2001-0783-che form-26.pdf

in-pct-2001-0783-che form-3.pdf

in-pct-2001-0783-che form-4.pdf

in-pct-2001-0783-che form-5.pdf

in-pct-2001-0783-che others.pdf

in-pct-2001-0783-che pct.pdf

in-pct-2001-0783-che petition.pdf