Title of Invention

STABLE PHARMACEUTICAL COMPOSITION OF 5, 10-METHYLENE-TEROHYDROFOLATE

Abstract

The invention relates to 5, 10-methylene- (6R)-, (6S)- or (6R,S)-tetrahydrofolate (MTHF) pharmaceutical compounds whose stability is due to the adjustment thereof to a basic pH value and to a simultaneous citrate use. Said stabilization is self-producible in the absence of a reducing agent. Said invention is particularly suitable for preparing lyophilization solution or yophilisates or dry powders and dry mixtures since the stable MTHF solutions can be filled at the high concentration thereof into corresponding recipients like, :or example vials, ampoules etc. Said lyophilisates are surpassingly storable and stable. They are easily reconstituted by adding water or aqueous solutions, thereby providing diluted injected solutions ready for use with an excellent stability. Said invention also makes it possible to produce highly concentrated calcium or acid MTHF salts, even difficult-to-dissolve, in the form of isotonic physiologically compatable solutions.

Full Text

This invention relates to stable pharmacy utical compositions of 5,10-methylene-(6R)-, -(6S)- or -(6R,S)-tetrahydrofolate. In the present text, the term 5,10-methyienetetrahydrofolate (abbreviated to MTHF) relates to 5,10-methylenetetrahydrofolic acid and polyglutamates thereof in the form of the free acids, in the form of pharmaceutically acceptable salts, particularly acidic salts, as well as alkali or alkaline earth metal salts. 5,10-;nethylenetetrahydrofolic acids and polyglutamates thereof comprise both mixtures of optical isomers, particularly 1:1 mixtures of diastereoisomers, as well as optically pure diastereoisomers, particularly optically pure, natural 5,10-methylene-(6R)-tetrahydrofolic acid. Pharmaceutically acceptable salts can be acidic salts, such as sulphate or sulphonate salts, preferably sulphate salts, or can also be alkali or alkaline earth metal salts, preferably sodium, potassium, magnesium or calcium salts. MTHF is an active ingredient which is preferably used for parenteral administration in combination with fluorinated pyrimidines such as 5-fluoruracil (5-FU), which is a widely used cytostatic agent for the treatment of solid tumours [Cofactor Biokeys Pharmaceuticals. Seley, K.L. Idrugs 4 (1), 99-101 (2001 ]. MTHF is a reduced folate and achieves its chemotherapeutic effect together with the base analogue 5-FdUMP by inhibiting the enzyme thymidylate synthase (TS), which catalyses the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP), which is a corral component of DNA synthesis. Since this step constitutes the only de novo source of deox ythymidy ate in the cell, the inhibition of this key enzyme by pyrimidine bases such as 5-FU or the 5-FU prodrug capecitabine (xeloda) is one of the main starting points in cancer therapy. Deactivation of TS occurs by the formation of a covalent, inhibiting ternary complex between TS, the base analogue 5-FdUhAP, which is a metabolite of 5-FU, and MTHF. An enhaicement of the cytotoxic effect of 5-FU can be achieved by increasing the intracellular concentration of MTHF, whereupon the stability of the ternary complex is increased. This causes direct inhibition of DNA synthesis and repair, which ultimately results in cell death an 1 in the delaying of tumour growth. The pharmaceutical use of MTHF is restricted by its extremely high sensitivity to oxidation by air [Chemical Stability and Human I'lasma Pharmacokinetics of Reduced Folates. Odin, E. et al., Cancer Investigation 16 (7), 447-455 (1998). The structure of "Active Formaldehyde" (N5N10-methylene tetral ydrofolic acid), Osborn, M. J. et al., J. Am. Chem. Soc. 82, 4921-4927 (1960), Folates in 7oods: Reactivity, stability during processing, and nutritional implications. Hawkes, J., anc Villota, R. Food Sci. Nutr. 28, 439-538 (1989)]. MTHF is an addition product of tetrahydrofolic acid (THF) and formaldehyde [5,10- methylene-5,6,7,8-tetrahydrofolate]. Conformation of the Tetrahydropyrazine and Imidazolidine Rings. Poe, M. et al. Biochtmistry 18 (24), 5527-5530 (1979). Tetrahydrofolic Acid and Formaldehyde. Kallen, R. G. Methods in Enzymology 18B, 705-716 (1971)]. In aqueous solution there is an equilibrium between MTHF on the one hand and THF and formaldehyde on the other hand. The folk wing procedures have hitherto been employed for the stabilisation of MTHF solutions: Rigorous exclusion of atmospheric oxygen by the use of special technical devices for the reconstitution of solid formulations and the injection of MTHF in an air-free environment [Chemical Stability and Human Plasma Piarmacok.netics of Reduced Folates. Odin, E. et al., Cancer Investigation 16 (7), 447-455 (1998), Fluid Transfer Systems US 4,564,054]. Addition of a reducing agent such as L(+)-ascorbic acid or salts thereof, reduced y- glutathione, P-mercaptoethanol, thioglycerol, N-acetyl-L-cysteine, etc. as an antioxidant for the sensitive MTHF and for THF in particular. Stabilisation by means of cyclodextrin inclusion compounds: EP 0 579 996 (Eprova). Use of high concentrations of the active ingredient. The following methods are also known fc r the stabilisation of other tetrahydrofolic acid derivatives: Stabilisation of solutions containing 5-formyltetrahydrofolic acid by the addition of of sodium citrate, sodium acetate or sodium ihloride: EP 0 755 396 (Pharmachemie). Stabilisation of injection solutions containing a sodium or potassium salt of 5- formyltetrahydrofolic acid at a pH between 7.5 and 8.5: EP 0 677 159 (SAPEC). Stabilisation of solutions containing tbi calcium salt of 5-formyltetrahydrofolic acid by the addition of sodium citrate: US 4,931,441 (Luitpold Pharmaceutical). However, the stabilisation of 5-formyl .etrahydrofolic acid, particularly solutions thereof, cannot be compared with the stabilisation of 5,10-methylenetetrahydrofolic acid solutions. Thus the methylene group in 5,10-methylenetetra.bydrofolic acid, which is incorporated in a five-membered ring, results in properties of this substance which differ considerably from those of 5-formyltetrahydrofolic acid. This is Manifested, for example, in significantly different stability behaviour and in di fferent paths of decomposition. In contrast to 5- formyltetrahydrofolic acid, 5,10-methybnetetrat,ydrofolic acid in solution is always in equilibrium with formaldehyde and tetraf ydrofolic acid (THF), which is distinguished by its extremely high sensitivity to oxidation. In contrast, 5-formyltetrahydrofolic acid does not exhibit this dissociation behaviour and is generally very stable in pharmaceutical^ acceptable aqueous solutions, even wthout the addition of sodium citrate and sodium hydroxide. Therefore, no stable pharmaceutical comp )sitions of MTHF have hitherto been described. It has now surprisingly been found that the stability of MTHF in aqueous solutions, in suspensions and in solid forms such as powders or lyophilisates can be strikingly increased by adjusting to a basic pH, with the simultaneous use of citrate. Surprisingly, this stabilisation occurs even in the absence of a reducing agent. Thus, even without additions of reducing igents (antioxidants) and without the exclusion of atmospheric oxygen, MTHF solutions an stable for hours. This is all the more surprising since stable compositions of MTHF cannot be obtained using acetate, oxalate, maleate and salts of other acids instead of citrate. This is also in contrast to the situation for 5- formyltetrahydrofolic acid, where an effect comparable with that of citrate can be obtained with acetate (EP 0 755 396). In 5-fomyltetrahydrofolic acid solutions citrate reduces hydrolysis and oxidative cleavage of the basic skeleton and thus reduces the formation of products such as p-aminobenzoylglutamic acid and pterin- and tetrahydropterin derivatives. In contrast to this, for MTHF in the sasic pH region citrate inhibits the separation of formaldehyde (hydrolysis) from the molxule. This is a striking and surprising difference in the behaviour of these two compound;, both of which form part of the folate class of substances. Furthermore, the present invention even makes it possible to prepare difficultly soluble calcium or acidic salts [Eprova Patent: Stable salts of 5,10-methylenetetrahydrofolic acid - EP 0 537 492] of MTHF in high concentrations and in physiologically acceptable isotonic solutions. The unexpected stabilisation of MTHF with citrate at basic pH values is due to a surprising synergistic effect of the citrate buffer solution in this pH range. Complex formation between citrate and MTHF on the one hand and between citrate and the counterion (salt) of MTHF on the other hand makes a decisive contribttion to the stabilisation of the methylene group by inhibiting the separation of formaldehyde (hydrolysis) from the MTHF molecule. The formation of THF, which is extremely sensitive to oxidation, is thereby prevented, as is the decomposition of MTHF. In the compositions according to the invention, the pH is set within the range from 7.5 - 10.5, preferably 8.5 - 9.5. This effectec with the aid of aqueous sodium hydroxide and hydrochloric acid in the MTHF solution which contain citric acid, sodium dihydrogen citrate or tri-sodium citrate dihydrate as stabilisation and buffer substances. It is also possible to add reducing agents, such as L(+)-ascc rbic acid or salts thereof, reduced y-glutathione, P- mercaptoethanol, thioglycerol, N-acetyl-L -cysteine, etc. as antioxidants. The formulations according to the invention are also particularly suitable for producing lyophilisation solutions and lyophilisates or dry powders and dry mixtures, since the stable MTHF solutions can be used in high concentrations for filling corresponding vessels, e.g. phials, ampoules, etc.. The lyophilisates can be stored surprisingly well, and are surprisingly stable. They can be reconstituted withoit problems by the addition of water or aqueous solutions, and the final clear injection solutions again exhibit excellent stability properties. The claimed formulations are preferably used for parenteral administration. However, formulations are also produced for enteral (e.g. oral, sublingual or rectal) administration or for topical (e.g. transdermal) application. The formulations are preferably used directly as water-based solutions or oil-based suspensions, or as lyophilisates. Prepaiations for parenteral application comprise sterile, aqueous and nonaqueous injection solutions and suspensions of the active compounds which preferably comprise an isotonic composilion. The formulations can also be administered with a carrier, however. Suitable carriers include organic or inorganic substances which dc not react with the active ingredient, e.g. oil, benzyl alcohol, polyethylene glycol, glycerol riacetate or other fatty acid glycerides, gelatine, lecithin, cyclodextrins, carbohydrates such as lactebiose or starch, magnesium stearate, talc or cellulose. Tablets, dragees, capsules, powders, syrups, concentrates or drops are preferred for oral application, and suppositories are preferred for rectal application. Suspensions, emulsions or implants can also be used, and patches or creams can be used for topical application. The preparations can comprise stabi isers, additives for the controlled release of pharmaceutically active compounds, antioxidants, buffers, bacteriostatic agents and adjuvants for obtaining an isotonic solution. Aqueous and non-aqueous sterile suspensions can contain suspension additives and thickeners. The preparation can exist as a single-dose or as a multiple-dose container, e.g. as welded ampoules or phials with a stopper and a closure cap. They can be stored as a freez-dried product and when required can be prepared for use by adding a sterile liquid, e.g. water or a physiological salt solution. Sterile powders, granules or tablets can also be used in this manner. All preparations can additionally contair one or more separately or synergistically acting active compounds. In particular, these nclude fluorinated pyrimidine bases such as 5- fluoruracil (5-FU), capecitabine (Xelcda), tegafur, UFT, doxifluridine, 2'-deoxy-5- fluorouridine, various cytostatic agents such as gemcitabine (gemzar), docetaxel (taxotere), paclitaxel (taxol), topotecan (hycamtin), irinotecan (CPT-11), doxorubicin (rubex), mitomycin (MTC), cisplatin (CD DP), cyclophosphamide (CPM), methotrexate (amethopterin), pemetrexed (alimta), vincristine (VCR), cytarabine (Ara-C), epirubicin (ellence), oxaliplatin (eloxatin), tamoxifen (nolvadex), carboplatin (CBDCA), etoposide (etopophos), ifosfamide (ifex) or antioxidants such as vitamin C, vitamin E, glutathione, thioglycerol and acetylcysteine, as well as the two MTHF dissociation products formaldehyde and tetrahydrofolic acid. The preparation comprises between 0.001 mg and 10,000 mg MTHF per dose. Preparations which preferably contain between 1 mg and 1,000 mg MTHF per dose are used in therapy. The dosage depends on the form of theripy, on the form of application of the preparation, and on the age, weight, nutrition and condition of the patient. Treatment can commence with a smaller amount, below the optimum amount, which can be increased in order to achieve the optimum effect. The preferred dose used in therapy ranges between 1 mg and 1,000 mg per day, particularly between 100 mg and 500 mg per day. Administration can be effected either as a single dose or as a repeated dose. The preparations can be used in all fields c f application for folates. From the preceding description, one ski led in the art in this field can read the crucial elements of the invention without problems, and, without departing from the basic idea and from the scope of the invention, can make modifications and additions and can thereby adapt the invention to differing needs and condit ons. The entire disclosures of all the patent applications., patents and publications which are cited in this text are included jointly by reference. The following examples can be carried out with a similar degree of success by replacing the generic or specifically described product: and/or process conditions by those which are given in the following examples. The folbwing specific embodiments are likewise purely exemplary and should by no means be considered as having a limiting effect on the remainder of the disclosure. Examples to illustrate the invention Example 1 A lyophilisate containing 5,10-methy)ene-(6R,S)-tetrahydrofolic acid 9900 ml water were saturated with argon. 421.9 g citric acid were completely dissolved therein with stirring. 232.0 g 5,10-m ethylene-*6R,S)-tetrahydrofolic acid, calcium salt were added. The pH was adjusted to 8 0 with aiqueous sodium hydroxide, whereupon the 5,10-methylene-(6R,S)-tetrahydrotblic acid was slowly dissolved. Thereafter, the pH was adjusted to 8.5 with aqueous sodium lydroxide. The solution was filtered under sterile conditions and was introduced at 5.0 ml per phial into 10 ml glass phials. Thereafter, the solution was frozen and freeze-dried. Phials were obtained which contained 5 10-methylene-(6R,S)-tetrahydrofolic acid. Example 2 Stabilisation of 5,10-methylene-(6R,S)-1etrahydrofolic acid (lyophilisate) Phials produced as in Example 1 exhibited the following stability values (Am 1466-A): Compared with this, the untreated referer ce sample of the calcium salt of 5,10-methylene- (6R)-tetrahydrofolic acid exhibited the following stability values (Co 751): Example 3 Stabilisation of 5,10-methylene-(6R,S)- etrahydrofolic acid (solutions) Compositions prepared as in Example 1 exhibited the following stability values as a dilute solution in physiological common salt solution (AC0448): Storage at +25EC (% relative stability) w thout the exclusion of air Compositions prepared as in Example 1 exhibited the following stability values as a dilute aqueous solution (AC0447): Compositions prepared as in Example 1 exhibited the following stability values as a concentrated aqueous solution (AC0447) Storage at +25EC (% relative stability) without the exclusion of air As a comparison with the above, the foil owing stability values are given in the prior art for the calcium salt of 5,10-methylene-(6R,S)-tetrahydrofolic acid in physiological common salt solution [see Chemical Stability and Hunan Plasma Pharmacokinetics of Reduced Folates. Odin, E. et al. Cancer Investigation 16 (71, 447-455 (1998)]. Storage at +25EC (% relative stability) Example 4 Tablet containing 5,10-methylene-(6R) tetrahydrofolic acid 990 1 water were saturated with argon. 42.2 kg citric acid were completely dissolved therein with stirring. 21.4 kg 5,10-methylene-(6R,S)-tetrahydrofolic acid were added as the free acid. The pH was adjusted to 8.0 wi h aqueous sodium hydroxide, whereupon the 5,10- methylene-(6R,S)-tetrahydrofblic acid was slowly dissolved. Thereafter, the pH was adjusted to 8.5 with aqueous sodium hydroxide. The solution was filtered under sterile conditions and lyophilised. An amount of the lyophilisate containing 1,000 g 5,10- methylene-(6R)-tetrahydrofolic acid was pressed into tablets with 4 kg lactose, 1.2 kg starch, 0.2 kg talc and 0.1 kg magnesium sttarate so that each tablet contained 100 mg 5,10- methylene-(6R)-tetrahydrofoIicacid. The tablet can also be coated as a film tablet. Example 5 Suppositories containing 5-methylene-(6R,S)-tetrahydrofolic acid A lyophilisate prepared as in Example 1 and containing 500 g 5,10-methylene-(6R,S)- tetrahydrofolic acid was melted with 50 g. hydroxypropylcellulose and 2 kg of semisynthetic glycerides to form suppositories so that each suppcsitory contained 500 mg 5,10-methylene- (6R,S)-tetrahydrofolic acid. Example 6 A combination preparation containing 5,10-methylene-(6R,S)-tetrahydrofolic acid and 5-fluoruracil, amongst other ingredients A combination preparation was produced, similarly to Examples 1, 4, 5 and 7, which in addition to the usual amount of 5,10-methylene-(6R,S)-tetrahydrofolic acid for the corresponding application also contained the usual amount of 5-fluoruracil for this application. Example 7 A lyophilisate containing 5,10-methylene-(6R)-tetrahydrofolic acid 9900 ml water were saturated with argon. 316.5 g citric acid were completely dissolved therein with stirring. 212.5 g 5,10-metiylene-(6R)-tetrahydrofolic acid sulphate were added. The pH was adjusted to 8.0 with aqueous sodium hydroxide, whereupon the 5,10-methylene- (6R,S)-tetrahydrofolic acid was slowly dissolved. Thereafter, the pH was adjusted to 8.5 with aqueous sodium hydroxide. The solution was filtered under sterile conditions and 5.0 ml per phial was introduced into 10 ml glass phials. Thereafter, the solution was frozen and freeze-dried. Phials were obtained which contained f, 10-methylene-(6R)-tetrahydrofolic acid. Example 8 Stabilisation of 5,10-methylene-(6R)-ietrahydrofolic acid (solution) Compositions prepared as in Example 7 exhibited the following stability values as a concentrated aqueous solution (Am 175 3-2/a); Storage at +25EC (% relative stability), without the exclusion of air As a comparison with the above, the following stability values are given in the prior art for the calcium salt of 5,10-methylene-(6R,S)-tetrahyclrofolic acid in physiological common salt solution [see Chemical Stability and Human Plasma Pharmacokinetics of Reduced Folates. Odin, E. et al. Cancer Investigation 16 (:). 447-455 (1998)]. WE CLAIM 1. Stable pharmaceutical compositions of 5,10-methylene-(6R)-, -(6S)-or -(6R,S)- tetrahydrofolate, characterized in that the composition comprises 5,10- methylene-(6R)-, (6S)- Dr -(6R,S)-tetrahydrofolic acid or a pharmaceutical^ acceptable salt of 5,10-methylene-(6R)-, -(6S)-or -(6R,S)-tetrahydrofolic acid together with citrate, and has a pH between 7.5 and 10.5, preferably between 8.5 and 9.5. 2. Stable pharmaceutical compositions as claimed in claim 1 together with further pharmaceutically acceptable active ingredients and adjuvants. 3. A pharmaceutical compcsition as claimed in claim 2, wherein that it comprises formaldehyde as an adjuvant. 4. A pharmaceutical compcsition as claimed in claim 2, wherein that it comprises a further folate as a further active ingredient. 5. A pharmaceutical compcsition as claimed in claim 4, wherein that it comprises tetrahydrofolic acid and salts thereof as a further folate. 6. A pharmaceutical composition as claimed in claim 1, wherein that the calcium salt or an acidic salt is used as the pharmaceutically acceptable salt of 5,10- methylene- (6R)-, -(6S)- or -(6R,S)-tetrahydrofolic acid. 7. A pharmaceutical compcsition as claimed in claim 2, wherein that it comprises a cytostatic agent as a further active ingredient. 8. A pharmaceutical composition as claimed in claim 2, wherein that it comprises a fluorinated pyrimidine derivative as a further active ingredient. 9. A pharmaceutical composition as claimed in claim 8, wherein that it comprises 5-fluoruracil or a 5-fluon racil prodrug, particularly capecitabine (xeloda) as a fluorinated pyrimidine derivative. 10.A pharmaceutical composition as; claimed in any one of claims 1 to 9, additionally comprising at least one antioxidant or a radical scavenger. 11. A pharmaceutical compos tion as claimed in claim 10, wherein that it comprises vitamin C or reduced glutathione as an antioxidant or radical scavenger. 12. A pharmaceutical compos tion as claimed in any one of claims 1 to 11, wherein that the composition exist; as a lyophilisate, dry powder or dry mixture. 13. A pharmaceutical composition as claimed in any one of claims 1 to 11, wherein that the composition exist; as a lyophilisation solution. 14.A method of stabilizing compositions comprising 5,10-methylene-(6R)-, -(6S)- or - (6R,S)-tetrahydrofolate, characterized in that 5,10-methylene-(6R)-, -(6S)- or -(6R,S)-tetrahydrofolic acid is treated with citrate and is brought to a pH between 7.5. and 10.5, preferably between 8.5 and 9.5. The invention relates to 5, 10-methylene- (6R)-, (6S)- or (6R,S)-tetrahydrofolate (MTHF) pharmaceutical compounds whose stability is due to the adjustment thereof to a basic pH value and to a simultaneous citrate use. Said stabilization is self- producible in the absence of a reducing agent. Said invention is particularly suitable for preparing lyophilization solution or yophilisates or dry powders and dry mixtures since the stable MTHF solutions can be filled at the high concentration thereof into corresponding recipients like, :or example vials, ampoules etc. Said lyophilisates are surpassingly storable and stable. They are easily reconstituted by adding water or aqueous solutions, thereby providing diluted injected solutions ready for use with an excellent stability. Said invention also makes it possible to produce highly concentrated calcium or acid MTHF salts, even difficult-to-dissolve, in the form of isotonic physiologically compatable solutions.

Documents:

36-KOLNP-2006-FORM-27.pdf

36-kolnp-2006-granted-abstract.pdf

36-kolnp-2006-granted-claims.pdf

36-kolnp-2006-granted-correspondence.pdf

36-kolnp-2006-granted-description (complete).pdf

36-kolnp-2006-granted-examination report.pdf

36-kolnp-2006-granted-form 1.pdf

36-kolnp-2006-granted-form 18.pdf

36-kolnp-2006-granted-form 2.pdf

36-kolnp-2006-granted-form 26.pdf

36-kolnp-2006-granted-form 3.pdf

36-kolnp-2006-granted-form 5.pdf

36-kolnp-2006-granted-reply to examination report.pdf

36-kolnp-2006-granted-specification.pdf