Stabilized tacrolimus composition

[0001] The present invention relates to stabilized pharmaceutical compositions comprising tacrolimus. The tacrolimus compositions of the invention contain a stabilizing agent useful for preventing or reducing the formation of degradation products of tacrolimus during storage.

The present invention also relates to a process for the preparation of a stable pharmaceutical composition of tacrolimus.

BACKGROUND OF THE INVENTION [0002] Tacrolimus is a macrolide lactone also known as FK506, fugimycin or tsucubaenolide, which is a pharmaceutically active compound, i.e. a drug substance. Many tacrolimus compositions are marketed under the trade names of e.g. Prograf®, Advagraf® and Protopic® and are used as immunosuppressive agents to prevent graft rejection, i.e. rejection of transplanted organs. Tacrolimus forms can also be used topically in a wide range of conditions.

[0003] Tacrolimus is produced by the bacterium Streptomyces tsukubaensis, and the name is an abbreviation that is derived from the "immunosuppressive tsukuba macrolide". Tacrolimus belongs to the class of macrolides derived from L-pipecolic acid produced by the Streptomyces strain also including, for example, rapamycin (sirolimus), ascomycin and meridamycin, which have valuable pharmacological properties.

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Tacrolimus (numbering of tricosine according to USP) [0004] In 2009, the USP issued a monograph of tacrolimus monohydrate (Pharmacopeial Forum 35 (2) 20 [March-April 2009], pp. 310-314, USP Pharmacopeial Convention Inc.) using a systematic name for tacrolimus based not on the IUPAC / octacose convention - structure, but on the convention of natural products, it is assumed that the basic structure is tricosine (tricosene) (a three-membered macrocycle).

This differs from the IUPAC-style nomenclature used in US Pat. No. 4,894,366 for a tacrolimus structure based on a 28-membered ring (octacose) numbered clockwise from the carbon in the tetrahydropyran ring.

[0005] Tacrolimus exerts its activity by binding to the immunophilin FKBP12 to give a complex that inhibits calcineurin, a calcium dependent phosphatase involved in signal transduction that leads to the transcription of the lymphokine gene.

[0006] The presence of degradation products in a pharmaceutical formulation, including a pharmaceutical composition containing tacrolimus as an active pharmaceutical ingredient, is highly undesirable because it imposes an increased risk to patients.

[0007] In addition, there are very strict regulatory restrictions with respect to impurities present in the pharmaceutical formulation both in the freshly prepared formulation and the pharmaceutical formulations after storage, i.e. during their storage life. Accordingly, it is necessary to monitor and document the formation of any possible degradation product resulting from the pharmaceutical formulation itself, especially any degradation product generated from the active ingredient, or to control the amount of degradation product present in the formulation during storage or to prevent or reduce it creating a degradation product during manufacture or during the storage period of the formulation, depending on the nature of each degradation product that may be present,

[0008] Tacrolimus is a poorly soluble substance that when administered in a crystalline form can have very low bioavailability due to the relatively small amount of fluid available for solubilisation in the gastrointestinal tract, particularly in the lower part of the intestine, including the large intestine. Accordingly, several attempts have been made to prepare a solution in the solid phase, preferably in the form of solid dispersions, as well as previously disclosed in the formulation of tacrolimus by Hone et al, Transplantation Proceedings, vol. XIX, No. 5, Suppl 6 (October), 1987: pp. 17-22, which discloses solid dispersions of various formulations. In the "Establishment of the new preparation method for solid dispersion formulation of tacrolimus", Yamashita Kazunari et al, International Journal of Pharmaceutics 2003, vol. 267 No. 1-2, pp.

[0009] Solvents are generally undesirable in the manufacture of pharmaceuticals, potential traces need to be closely monitored, and solvent-containing production is expensive. Thus, it is highly desirable for many pharmaceutical companies to be able to form solid dispersions without using solvents, so that approved products having sufficient absorption capacity may be introduced into the drug market, despite the low solubility of the active ingredient. However, a disadvantage of such solid dispersions, especially of molecular dispersions, is motility and increased exposure of molecules in the formulation, which increases the risk of chemical degradation compared to conventional crystalline formulations.

WO2005 / 020993, WO2005 / 020994, WO2008 / 0145143 and WO2010 / 005980 disclose tacrolimus pharmaceutical compositions with improved bioavailability and reduced peak to lowest level, compared to commercially available tacrolimus products, in particular tacrolimus compositions containing a solid dispersion. Tacrolimus in polyethylene glycol (PEG).

[0011] For formulations containing tacrolimus, especially formulations containing components that may, as starting materials in the production process, contain traces of metals or metal compounds,

For the oxidation and other undesirable but unavoidable impurities, there is a need to prevent the formation of degradation products from tacrolimus or, at least, maintain an acceptable, low concentration of such degradation products during the entire storage period of the formulation, which is typically formulated in utility forms, such as capsules (soft and hard), tablets or granules in a sachet, or as a liquid for injection, or as a topical product.

BRIEF SUMMARY OF THE INVENTION [0012] The present invention relates to stabilized pharmaceutical compositions. In accordance with one embodiment of the invention, the pharmaceutical composition comprises tacrolimus or a tacrolimus analog dissolved or dispersed in a carrier and a stabilizing agent. The stabilizing agent may be an auxiliary substance that regulates the pH. Preferably, the stabilizing agent is capable of providing a pH below 7 in the composition as measured after redispersion of the composition in water, more preferably a pH in the range of 2.5 to 5 or from 2.5 to 4 or 3 to 3.6 or 3 to 3.5. Suitable stabilizing agents include, but are not limited to, inorganic acids, inorganic bases, inorganic salts, organic acids, organic bases, and pharmaceutically acceptable salts thereof. The stabilizing agent may be a chelating agent. E.g, the stabilizing compound may be an organic acid selected from mono-, di-, oligo- and polycarboxylic acids, e.g., succinic acid, citric acid, tartaric acid, acrylic acid, benzoic acid, malic acid, maleic acid, oxalic acid, sorbic acid and their mixtures. In one preferred embodiment, the stabilizing agent is oxalic acid, tartaric acid and / or citric acid. One preferred stabilizing agent is tartaric acid. the stabilizing agent is oxalic acid, tartaric acid and / or citric acid. One preferred stabilizing agent is tartaric acid. the stabilizing agent is oxalic acid, tartaric acid and / or citric acid. One preferred stabilizing agent is tartaric acid.

The composition preferably comprises a stabilizing effective amount of a stabilizing agent (e.g. an amount effective to prevent or reduce the formation rate of tacrolimus degradation products).

[0014] In one embodiment, the amount of stabilizer is in the range of about 0.05% wt / wt. up to about 5% w / w relative to the total weight of tacrolimus, carrier and stabilizing agent. The composition may contain at least 0.05% wt / wt, at least 0.1% wt / wt or at least 0.2% wt / wt. and less than 3% w / w, less than 2% w / w, less than 1% w / w, less than 0.8% w / w, or no more than 0.6% w / w. / wt. stabilizing agent.

[0015] Odpowiednie nośniki obejmują polimery hydrofilowe lub amfifilowe mieszające się z wodą. Korzystnym nośnikiem jest mieszanina glikolu polietylenowego i poloksameru.

[0016] The composition may contain less than 0.5% w / w. 8-epitacrolimus, the main degradation product of tacrolimus after storage. In addition, the composition may be substantially free (e.g., contain less than 1, 0.5, 0.2, 0.1 and 0.05% wt / wt) from the organic solvent or residual organic solvents.

The invention further relates to any oral dosage form, including but not limited to tablets, capsules and sachets, wherein in the presence of a stabilizing agent or excipient such as an organic acid, the formation of tacrolimus degradation products is reduced (8 epitacrolimus, dien, epimerC4 , regioisomer A) disclosed herein. The preferred organic acid is tartaric acid.

[0018] By means of the present invention, it is possible to prevent or reduce the formation of a possible degradation product in a pharmaceutical formulation, in particular any product

The use of tacrolimus degrades and thus controls or controls the maximum tolerable amount of degradation product present in the formulation during the storage period, or to prevent or reduce the formation of the degradation product during manufacture or shelf life of the formulation, depending on the nature of the degradation product that may be present in either the pharmaceutical formulation product.

DETAILED DESCRIPTION OF THE INVENTION [0019] The present invention is based on the finding that a very important, i.e. the major product of degradation of tacrolimus is the previously-undetected C8-epimer of tacrolimus, also defined as 8-epitacrolimus, having the formula:

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The structure of 8-epitacrolimus was determined by single crystal X-ray diffraction using graphite monochrome radiation Mo Κα (λ = 0.71073A) on a Kappa CCD diffractometer, and data collection using COLLECT and data reduction using EvalCCD (Skytte DM et al. .: Synthesis and characterization of an epimer of tacrolimus, an immunosuppresive drug in J. Nat Prod, 23 April 2010; 73 (4): 776-9., Which is hereby incorporated by reference).

[0021] This compound - epimer-8S (isomer) has been fully characterized by spectroscopic techniques:

epitactrolimus (3S, 4R, 5S, 8S, 9E, 12S, 14S, 15R, 16S, 18R, 19R, 26aS) -5,19-dihydroxy-14,1620 dimethoxy-15,19-epoxy-5,6,8, 11,12,13,14,15,16,17,18,19,24,25,26,26a-Hexadecahydro-3 - [(1E) -2- [(1R, 3R, 4R) -4-hydroxy-3 methoxycyclohexyl] -1-methylethenyl] -8- (2-propen-1-yl) -4,10,12,18tetrametylo-3H-pyrido [2,1-c] [1,4] oksaazacyklotrikozyno-1,7, 20.21 (4H, 23H) -tetone: Colorless prisms, mp 179-182 ° C [CH3CN-H2O (60:40)]; [Α]²⁵ο -1.2 (c 0.725, CHCl 3); UV (CH 3 CN) (ε) 202 nm (9,500); CD (CH 3 CN) λ max ([θ]) 231 (-18 300), 297 (+23 400); IR (KBr ^ max 3580, 3431, 2934, 1753, 1724, 1704, 1633, 1452, 1193, 1170, 1091, 1050 cm)^-1.

[0022] It has been found that the formation of 8-epitak rolimus for each patient takes place under relatively mild conditions, usually under mild basic conditions. However, it is believed that the production can also take place under mild acid conditions. Accordingly, this epimer-8S (tacrolimus isomer) can be formed during tacrolimus manipulation, e.g. during its isolation from fermentation broths, purification or preparation of pharmaceutical compositions and probably also in vivo as a metabolite of tacrolimus. Since it is also known that even very small structural modifications of tacrolimus result in significant changes in pharmacological profiles (as in the case of ascomycin (Sierra-Paredes et al., CNS Neurosci. Ther., 2008, vol. 14, pp. 36-46), the tacrolimus analogue .

[0023] Other tacrolimus degradation products not yet disclosed have also been identified by HPLC in a mixture containing tacrolimus

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EP 2 575 769 B1

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[0024] As used herein, the term "active ingredient" or "pharmaceutically active ingredient" means any ingredient that is intended to provide pharmacological or other direct activity in the diagnosis, cure, alleviation, treatment or prevention of disease, or affect the structure or any function of the organism human or other animals. The term includes those components that may undergo chemical change during the manufacture of the medicinal product and are present in the modified form of the medicinal product intended to deliver specific activity or activity.

[0025] In the present context, the term "stabilizing compound", "stabilizer" or "stabilizer" are used interchangeably.

[0026] In the present context, the term "hydrophilic" describes that something "resembles water", i.e. a hydrophilic molecule or part of a molecule is one that is usually electrically polarized and is capable of hydrogen bonding to water molecules, allowing faster dissolution in the water. water than in oil or other "non-polar" solvents.

[0027] In the present context, the term "amphiphilic" describes a molecule (as a surfactant) having a polar, water-soluble group attached to a water-insoluble hydrocarbon chain. Therefore, one end of the molecule is hydrophilic (polar) and the other is hydrophobic (non-polar).

[0028] In the present context, the term "hydrophobic" means a compound with a tendency to be electrically neutral and non-polar, and thus prefers other neutral and non-polar solvents or molecular environments.

[0029] As used herein, the term "vehicle" includes, but is not limited to, any solvent or carrier (e.g., carrier fluid) in a pharmaceutical product that does not have a pharmacological role. For example, water is a carrier for xylocaine, and propylene glycol is a carrier for many antibiotics.

[0030] As used herein, the term "solid dispersion" means a drug substance or active ingredient dispersed or dissolved in an inert support, carrier, diluent or matrix in the solid state. The drug substance or active ingredient may be in the form of particles, often very well

The particulate material or individual molecules. Accordingly, the term "solid dispersion" includes what is sometimes referred to as a solid solution.

[0031] As used herein, the term "analog" means a chemical compound that is structurally similar to another.

[0032] The term "drug" means a compound intended for use in diagnosing, healing, alleviating, treating or preventing a disease in a human or other animal.

[0033] In this context, the term "dosage form" means the form in which the drug is administered to a patient. It could be a parenteral, topical, tablet, oral (liquid or dissolved powder), suppository, inhalation, percutaneous, etc.

[0034] As used herein, the term "bioavailability" means the degree to which a drug or other substance becomes available to the target tissue after administration.

[0035] As used herein, the term "bioequivalence" means a scientific basis on which generic drugs and generic names are compared with one another. For example, drugs are bioequivalent if they go into circulation at the same rate when administered at similar doses under similar conditions. The parameters commonly used in bioequivalence studies are t max, cm max, AUC 0 - infinity, AUC 0 - t. Other important parameters may be W50, W75 and / or MRT. Therefore, at least one of these parameters can be used to determine if there is bioequivalence. Furthermore, in the present context, two compositions are considered bioequivalent if the value of the parameter used is in the range of 80-125% of the value for Prograf® (e.g., New Drug Application No. 050708 in the US

[0036] In one embodiment, a pharmaceutical composition of the invention comprises a solid dispersion of tacrolimus in a dispersion medium comprising a carrier and a stabilizing compound (also referred to as a stabilizing agent).

[0037] Preferably, the pH of the composition is less than 7 (as measured after redispersion of the composition in water), more preferably the pH is in the range of 2.5-5.0, more preferably 2.5 to 4.5, even more preferably from 2.5 to 4, even more preferably from 3 to 4, especially from 3.0 to 3.6. The pH can be provided by a stabilizing agent and / or can be adjusted with an inorganic or organic acid or mixtures thereof.

[0038] Suitable stabilizing and stabilizing compounds for use in a composition of the invention include, but are not limited to, inorganic acids, inorganic bases, inorganic salts, organic acids, organic bases, and pharmaceutically acceptable salts thereof.

[0039] The organic acid is preferably a mono-, di-, oligo- and polycarboxylic acid. Non-limiting examples of suitable organic acids are acetic acid, succinic acid, citric acid, tartaric acid, acrylic acid, benzoic acid, malic acid, maleic acid, oxalic acid and sorbic acid, and mixtures thereof. Preferred organic acids are selected from the group consisting of oxalic acid, tartaric acid and citric acid.

[0040] The pharmaceutically acceptable salt with an organic acid or inorganic acid is preferably an alkali metal salt or alkaline earth metal salt. Preferred examples of such salts are sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate, calcium hydrogen phosphate, dicalcium phosphate, sodium sulfate, potassium sulfate, calcium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydrogen carbonate, calcium carbonate, carbonate

Magnesium acetate, sodium acetate, potassium acetate, calcium acetate, sodium succinate, potassium succinate, calcium succinate, sodium citrate, potassium citrate, calcium citrate, sodium tartrate, potassium tartrate, calcium tartrate, zinc gluconate and zinc sulfate.

[0041] Suitable inorganic salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride and magnesium chloride.

[0042] When the composition of the present invention comprises as an organic acid stabilizing agent, the organic acid is usually present in a concentration of less than 5% w / w. or less than 3% w / w. or less than 2% w / w. or less than 1% w / w, or less than 0.8% w / w. or less than 0.6% w / w, or at least 0.05% w / w, or at least 0.1% w / w, or at least 0.2% w / w . with respect to the total amount of tacrolimus, carrier and stabilizing agent.

[0043] It has been found that the formation of the main degradation product of 8-epitacrolimus is reduced in the presence of a metal chelating agent in the composition. Accordingly, the stabilizing agent used in the composition of the present invention is preferably a metal chelating agent, i.e. capable of binding a metal ion. The metal chelating agent may be an organic acid, preferably citric acid, tartaric acid, oxalic acid or a mixture of any of these acids. For example, it has been shown that the use of 0.5% w / w. citric acid instead of 0.05% w / w. improved the stability of tacrolimus.

[0044] Furthermore, as shown in Example 3, it has been found that the composition of the present invention, using as stabilizing agent an organic acid, such as tartaric acid in a concentration ranging from about 0.05% wt / wt. up to 0.60% wt / wt it will balance the formation of the main degradation product of 8-epitacrolimus to form three other degradation products that can be formed in the composition during storage. The use of tartaric acid in the concentration range from 0.10% wt / wt. up to 0.50% wt / wt. is even more preferred, such as in the range of from 0.10 to 0.30% w / w. or from 0.10% to 0.20% w / w, such as about 0.15% w / w. relative to the total weight of tacrolimus, vehicle and stabilizing agent.

[0045] Thus, in another embodiment of the invention, the stabilizing agent in the tacrolimus composition is tartaric acid. For example, the composition may contain from about 0.01% to about 5% w / w. tartaric acid based on the total weight of tacrolimus, vehicle and stabilizing agent. The compositions may contain at least 0.01% w / w. tartaric acid, at least 0.05% wt / wt. tartaric acid, at least 0.1% w / w. tartaric acid, at least 0.15% wt / wt. tartaric acid, at least 0.2% w / w. tartaric acid, at least 0.4% w / w. tartaric acid, at least 0.5% w / w. tartaric acid, at least 0.6% w / w. tartaric acid, at most 0.75% w / w. tartaric acid, at most 0.8% w / w. tartaric acid, at most 1% w / w. tartaric acid, at most 2% w / w. tartaric acid, at most 3% w / w. tartaric acid or at most 5% w / w. tartaric acid. In a preferred embodiment, the tacrolimus composition contains 0.15% w / w. tartaric acid based on the total weight of tacrolimus, vehicle and stabilizing agent. [0046] As shown in the examples herein, also tartaric acid and oxalic acid had an improved stabilizing effect compared to citric acid when present in the tacrolimus composition according to the invention in an amount of 0.5% w / w.

[0047] Stabilizing compounds that prevent the degradation of the active substance are desirable, as well as specific excipients less susceptible to interaction with tacrolimus or its analog or degradation products. However, the prevention of degradation products is further complicated by the fact that

One additive added to prevent the first degradation may itself lead to a second degradation product, which may require the addition of a stabilizing excipient. In addition, preventing degradation does not always give a simple linear effect, but there may be windows for optimal effect.

[0048] In yet another embodiment of the present invention, the composition contains 8-epitacrolimus in an amount of less than 0.5% wt / wt, such as less than 0.2% wt / wt, based on the total weight of the composition ( tacrolimus, carrier, stabilizing agent).

[0049] As demonstrated in the examples, the composition of the present invention is stable and contains less than 0.5% 8-epitacrolimus after 12 weeks as well as 10 storage months at 25 ° C and 60% relative humidity.

[0050] In yet another embodiment, the pharmaceutical composition of the present invention comprises a solid dispersion of tacrolimus in a carrier mixture and a stabilizing agent, wherein the composition comprises (a) no more than 0.5% more 8-epitacrolimus after storage at 40 ° C at relative humidity. 75% for 5 weeks compared to pharmaceutical compositions before storage, or (b) no more than 0.2% more 8-epitacrolimus after storage at 25 ° C and 60% relative humidity for 5 weeks compared to the pharmaceutical composition before storage, or no more than 0.5% more 8-epitacrolimus after storage at 25 ° C and 60% relative humidity for 1 year compared to pharmaceutical compositions before storage.

[0051] Limit of the degradation product defined in the guidelines (ICH Theme 3 QB (R2) of the International Conference on Harmonization (ICH)): Note for Guidance on Impurities in New Drug Products, CPMP / ICH / 2738/99 June 2006; <a href="http://www.ema">www.ema</a>. eu.int) depends on the amount of drug to be administered daily. The daily dose range of tacrolimus is generally 1 mg to 20 mg. The recommended daily dose for a commercially available tacrolimus product - Prograf® for adult patients after kidney transplantation (in combination with nitropioprine) 0.2 mg / kg / day (Prograf® tacrolimus capsules, Astellas Pharma US Inc.; product label, 09H011-PRG WPI revised August 2009). Assuming an average adult weighs 70 kg, the initial daily dose of Prograf® would be 14 mg. For a maximum daily dose in the range of 10 to 100 mg, the limit of ICH is 0.5% for a single degradation product.

[0052] The amount of tacrolimus in the composition of the invention may be any amount useful as a medicine, or for the preparation of a solid dosage form (e.g., a pharmaceutically effective amount). Typically, the composition comprises from about 0.01% wt / wt. up to about 10% w / w tacrolimus based on the total weight of the composition, such as from about 0.1% w / w. up to about 10% w / w tacrolimus or from about 0.5% w / w up to about 5% tacrolimus, or from about 1% w / w up to about 4% w / w tacrolimus.

[0053] In yet another embodiment of the invention, the carrier is a hydrophilic, amphiphilic or water-miscible carrier, preferably having a melting point (freezing or solidification temperature) of at least 20 ° C, more preferably at least 30 ° C, more preferably at least 40 ° C, more preferably at least 50 ° C, even more preferably at least 52 ° C, even more preferably at least 55 ° C, even more preferably at least 59 ° C, especially at least 61 ° C, in particular at least 65 ° C. Preferably, the carrier is a polymer.

[0054] Examples of suitable hydrophilic or water miscible carriers are those selected from the group consisting of polyethylene glycols, polyoxyethylene oxides, poloxamers, polyoxyethylene stearates, poly-epsilon caprolactone, polyglycolyzed glycerides such as Gelucire® and mixtures thereof.

EP 2 575 769 B1

However, also certain amphiphilic carriers may be useful, including those carriers disclosed herein, which may be amphiphilic in addition to being miscible with water.

[0055] A preferred carrier is polyethylene glycol (PEG), in particular PEG with an average molecular weight of at least 1500, preferably at least 3000, more preferably at least 4000, especially at least 6000. For example, PEG can have an average molecular weight in the range of 1500 to 35000, from 3000 to 35000, from 3000 to 20,000, from 4000 to 20,000, from 3000 to 10000, or from 4000 to 10000. The polyethylene glycol may preferably be mixed with one or more other hydrophilic or water miscible carriers, for example a poloxamer, preferably in a ratio (by weight) between 1: 3 and 10: 1, preferably between 1: 1 and 5: 1, more preferably between 3: 2 and 4: 1, especially between 2: 1 and 3: 1, and especially around 7: 3. A specific example of a suitable mixture is a mixture of PEG6000 and poloxamer 188 in a ratio of 7: 3.

[0056] For polyethylene glycols (PEG), the melting point (freezing point or pour point) increases as the average molecular weight increases. For example, for PEG 400 it is in the range of 4-8 ° C, for PEG 600 it is in the range of 20-25 ° C, for PEG1500 it is in the range of 44-48 ° C, for PEG2000 it is about 52 ° C, for PEG 4000 it is higher about 59 ° C, for PEG 6000 is about 65 ° C, and for PEG 8000 is about 61 ° C.

Suitable poloxamers (also referred to as polyoxypropylene polyoxyethylene block copolymers) are, for example, poloxamer 188 (having an average molecular weight of about 8,400 and a melting point of about 50-54 ° C), poloxamer 237, poloxamer 338 or poloxamer 407 or other block copolymers of oxide ethylene and propylene oxide, such as the Pluronic® and / or Tetronic® series. Suitable block copolymers of the Pluronic® series include polymers having a molecular weight of about

000 or above, such as from about 4,000 to about 20,000 and / or in a (Brookfield) viscosity of from about 200 to about 4,000 cP, such as from about 250 to about 3,000 cP. Suitable examples include Pluronic® F38, P65, P68LF, P75, F77, P84, P85, F87, F88, F98, P103, P104, P105, F108, P123, F123, F127, 10R8, 17R8, 25R5, 25R8, etc. Suitable copolymers Tetronic® blocks include polymers having a molecular weight of about 8,000 or more, such as from about 9,000 to about 35,000 and / or a viscosity (Brookfield) of about 500 to about 45,000cp, such as from about 600 to about 40,000. Viscosities given above are determined at 60 ° C for substances that are pastes at room temperature and 77 ° C for substances that are solid at room temperature.

[0058] Other suitable hydrophilic or miscible carriers may be polyvinylpyrrolidones, polyvinyl-polyvinyl acetate copolymers (PVP-PVA), polyvinyl alcohol (PVA), polymatacrylic polymers (Eudragit RS, Eudragit RL, Eudragit NE, Eudragit® E), cellulose derivatives, including hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), methylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, pectins, cyclodextrins, galactomannans, alginates, xanthan, carrageenans, xanthan gums and mixtures thereof.

[0059] "Polyglycolized glycerides" means a mixture of mono-, di- and triglycerides and mono- and polyethylene glycol diesters (PEG), preferably with a molecular weight of from 200 to 600, where it is suitable free glycerol and free PEG, which HLB value is regulated by PEG chain length and whose melting point is regulated by the length of fatty acid chains, PEG and by the degree of saturation of the fatty chains and thus of the starting oil; examples of such mixtures are Gelucire®. Gelucire® compositions are inert semi-solid waxy materials that are amphiphilic and are available with different physical properties. They are inherently surface active and

The microscopic beads or vesicles are dispersed or dissolved in aqueous micelle-forming media. They are identified by the value of their melting point / HLB. The melting point is expressed in degrees Celsius and the HLB (hydrophilic-lipophilic balance) is a numerical scale extending from 0 to about 20. Lower HLB values ​​mean more lipophilic and hydrophobic substances, and higher values ​​mean more hydrophilic and lipophobic substances. The affinity of the compound for water or oily substances is determined and its HLB value is determined experimentally. One or a mixture of different classes of Gelucire® excipient can be chosen to obtain the desired melting temperature characteristics and / or HLB values. They are a mixture of monoesters, diesters and / or triesters of long-chain glycerides (C12 to C18) of fatty acids, and PEG (mono- and / or di-) esters of long-chain (C12 to C18) fatty acids and may include free PEG. Gelucire® compositions are generally described as fatty acid esters with glycerol and PEG esters or as polyglycolized glycerides. Gelucire® compositions have a wide melting temperature range of from about 33 ° C to about 64 ° C, most commonly from about 35 ° C to about 55 ° C, and different HLB values ​​from about 1 to about 14, most often from about 7 to about 14. For example, Gelucire® 50/13 means a melting temperature of approximately 50 ° C and an HLB value from about 13 for this Gelucire® class. Gelucire® compositions are generally described as fatty acid esters with glycerol and PEG esters or as polyglycolized glycerides. Gelucire® compositions have a wide melting temperature range of from about 33 ° C to about 64 ° C, most commonly from about 35 ° C to about 55 ° C, and different HLB values ​​from about 1 to about 14, most often from about 7 to about 14. For example, Gelucire® 50/13 means a melting temperature of approximately 50 ° C and an HLB value from about 13 for this Gelucire® class. Gelucire® compositions are generally described as fatty acid esters with glycerol and PEG esters or as polyglycolized glycerides. Gelucire® compositions have a wide melting temperature range of from about 33 ° C to about 64 ° C, most commonly from about 35 ° C to about 55 ° C, and different HLB values ​​from about 1 to about 14, most often from about 7 to about 14. For example, Gelucire® 50/13 means a melting temperature of approximately 50 ° C and an HLB value from about 13 for this Gelucire® class.

[0060] Further examples of substances useful as carriers are:

i) polyethoxylated fatty acids, e.g., polyethylene glycol mono- or diesters, or mixtures thereof, such as e.g. polyethylene glycol mono- or diesters with lauric acid, oleic acid, stearic acid, myristic acid, ricinoleic acid, the polyethylene glycol may be selected from PEG 4, PEG 5, PEG 6, PEG 7, PEG 8, PEG 9, PEG 10, PEG 12, PEG 15, PEG 20, PEG 25, PEG 30, PEG 32, PEG 40, PEG 45, PEG 50, PEG 55, PEG 100, PEG 200, PEG 400, PEG 600, PEG 800, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 5000, PEG 6000, PEG 7000, PEG 8000, PEG 9000, PEG 1000, PEG 10,000, PEG 15,000, PEG 20,000, PEG 35,000, ii) fatty acid esters with glycerol and polyethylene glycol, i.e. esters as mentioned above but in the form of individual fatty acid glyceryl esters;

iii) esters of glycerol, propylene glycol, ethylene glycol, PEG or sorbitol from e.g. vegetable oils, such as hydrogenated castor oil, almond oil, palm kernel oil, castor oil, apricot seed oil, olive oil, peanut oil, hydrogenated palm kernel oil and the like, iv) polyglycerylated fatty acids, such as e.g. polyglycerol stearate, polyglycerol oleate, polyglycerol ricinoleate, polyglycerol linolate,

v) propylene glycol fatty acid esters such as, for example, propylene glycol monolaurate, propylene glycol ricinoleate and the like, vi) mono- and diglycerides, e.g. glyceryl monooleate, glyceryl dioleate, glyceryl monooleate and / or dioleate, glyceryl caprylate, glycerin caprate etc .;

vii) sterol and sterol derivatives;

viii) fatty acid esters of sorbitan and polyethylene glycol (PEG-sorbitan fatty acid esters), such as PEG esters of various molecular weights indicated above and various Tween® series;

ix) polyethylene glycol alkyl ethers, such as e.g. PEG-oleyl ether and PEG-lauryl ether;

x) sugar esters, such as, e.g., sucrose monopalmitate and sucrose monolaurate;

Xi) polyethylene glycol alkyl phenols, e.g. the Triton® X or N series;

xii) polyoxyethylene-polyoxypropylene block copolymers, such as the Pluronic® series, the Synperonic® series, Emkalyx®, Lutrol®, Supronic® etc. The general term for these polymers is "poloxamers" and the relevant examples in this context are Poloxamer 105, 108 , 122, 123, 124, 181, 182, 183, 184, 185, 188, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338 , 401, 402, 403 and 407;

xiii) sorbitan fatty acid esters, such as the Span® series or Ariacel® series, such as, for example, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monooleate, sorbitan monostearate, and the like;

xiv) esters of fatty acids with lower alcohols, such as e.g. oleate, isopropyl myristate, isopropyl palmitate and the like;

xv) ionic surfactants including cationic, anionic and amphoteric surfactants, such as e.g. fatty acid salts, bile salts, phospholipids, phosphoric acid esters, carboxylates, sulfates and sulfonates, etc.

[0061] In a further aspect, the present invention relates to a process for the preparation of a stable pharmaceutical composition comprising a solid dispersion of tacrolimus in a carrier mixture and a stabilizing agent, wherein the pH in the composition is from 2.5 to 7, as measured after re-dispersing in water, which method comprises the steps of: i) dissolving the stabilizing agent in the carrier; and ii) adding or dissolving tacrolimus in the carrier mixture, and iii) optionally adjusting the pH of the composition.

[0062] WO 2005/020993 discloses tacrolimus formulations that may be useful in combination with the stabilizing agent (s) disclosed herein for the stabilized tacrolimus composition. Pharmaceutical compositions and dosage forms that can be optimized to provide a stable composition of the invention are also illustrated in Examples 1-16 in WO 2005/020993, which is incorporated herein by reference. These exemplary formulations and compositions can be further optimized by the addition of stabilizing excipients of the present invention, and as disclosed herein, to prevent the formation of degradation products as described.

[0063] Furthermore, the invention relates to an oral dosage form of tacrolimus, where the formation of the 8-epitacrolimus degradation product is reduced in the presence of a stabilizing agent. The stabilizing agent is preferably an organic acid. The preferred organic acid is tartaric acid. The oral dosage form is preferably a solid dosage form such as tablets, capsules, sachets and other forms of use customarily used to administer medications to patients in need thereof.

[0064] Any combination of the above-described elements in all its possible variations is covered by the invention, unless otherwise indicated herein or clearly contradictory to the context.

List of performances:

[0065]

A pharmaceutical composition comprising a solid dispersion of tacrolimus in a carrier mixture and a stabilizing agent capable of providing a pH in the composition below 7.

2. The composition according to embodiment 1, wherein the pH is in the range 2.5-4.0.

3. The composition according to embodiment 1, wherein the pH is in the range 3.0-3.5.

EP 2 575 769 B1

4. The composition according to embodiment 1, wherein the stabilizing agent is selected from the group consisting of inorganic acids, inorganic bases, inorganic salts, organic acids, organic bases and their pharmaceutically acceptable salts.

5. The composition according to embodiment 1, wherein the stabilizing agent is a chelating compound.

6. The composition according to embodiment 1, wherein the stabilizing agent is an organic acid selected from mono-, di-, oligo and polycarboxylic acids.

7. The composition according to embodiment 3, wherein the organic acid is selected from the group consisting of succinic acid, citric acid, tartaric acid, acrylic acid, benzoic acid, malic acid, maleic acid and sorbic acid.

8. The composition of embodiment 7, wherein the organic acid is selected from the group consisting of oxalic acid, tartaric acid and citric acid.

9. A composition according to embodiment 8 wherein the tartaric acid is present in a concentration of less than 5% w / w, or less than 3% w / w, or less than 2% w / w, or less than 1% w / w, or less than 0.8% w / w, or less than 0.6% w / w, or at least 0.05% w / w, or at least 0.1% w / w, or at least 0.2% w / w, based on the total amount of tacrolimus and carrier and stabilizing agent.

10. The composition of embodiment 1, wherein the carrier comprises a hydrophilic, amphiphilic or water miscible polymer.

11. The composition according to embodiment 1, wherein the carrier is a mixture of polyethylene glycol and poloxamer.

12. An oral dosage form comprising a pharmaceutical composition according to any of the previous embodiments.

13. A dosage form according to embodiment 1 which contains 8-epithacrolimus in an amount of less than 0.5% w / w.

14. A use embodiment according to embodiment 1 which contains an anhydrous tacrolimus degradation product in an amount of less than 0.5% w / w.

15. A method for reducing the concentration of tacrolimus degradation products in a pharmaceutical composition comprising tacrolimus as an active agent, wherein the stabilizing agent is included in the composition.

16. A pharmaceutical composition comprising a dispersion of tacrolimus in a carrier that contains tartaric acid.

17. A pharmaceutical composition according to embodiment 16, which comprises from about 0.5 to about 5%, tacrolimus, based on 100% of the total weight of the composition.

18. A pharmaceutical composition according to embodiment 16, which comprises from about 1 to about 4% tacrolimus, based on 100% of the total weight of the composition.

A pharmaceutical composition comprising a solid suspension of tacrolimus in a carrier mixture and a stabilizing agent capable of providing a composition below pH 7 in the composition, wherein the pharmaceutical composition contains 8-epitacrolimus and 8-epitacrolimus is present in a concentration below 0.2 wt%.

20. The pharmaceutical composition according to embodiment 19, wherein the solid dispersion is substantially free of organic solvent.

EP 2 575 769 B1

21. A pharmaceutical composition comprising a dispersion of tacrolimus which contains less than 0.5% 8 -titacrolimus after 12 weeks storage at 25 ° C and a relative humidity of 60%.

22. A pharmaceutical composition comprising a dispersion of tacrolimus which contains less than 0.5% 8 -titacrolimus after 10 months of storage at 25 ° C and a relative humidity of 60%.

23. A pharmaceutical composition comprising a dispersion of tacrolimus which contains less than 0.5% 8 -titacrolimus after 3 weeks of storage at 40 ° C and a relative humidity of 75%.

24. A pharmaceutical composition comprising a dispersion of tacrolimus that contains less than 0.5% 8-ticracimus after 19 weeks of storage at 40 ° C and 75% relative humidity.

25. A pharmaceutical composition comprising a solid dispersion of tacrolimus in a carrier mixture and a stabilizing agent, wherein the composition comprises: (a) no more than 0.5% more 8 -titacrolimus after storage at 40 ° C and 75% relative humidity for 5 weeks compared to the composition before storage, or (b) no more than 0.2% more 8-epitacrolimus after storage at 25 ° C and 60% relative humidity for 5 weeks compared to pharmaceutical compositions prior to storage.

26. A stabilized pharmaceutical composition comprising a solid dispersion of tacrolimus, wherein the composition comprises (a) no more than 0.5% of 8-epitacrolimus, (b) no more than 0.5% of tacrolimus diene, (c) no more 0.5% tacrolimus epimer-C4 diethyl and / or (d) no more than 0.5% tacrolimus regioisomer after storage at 25 ° C and 60% relative humidity for 5 weeks.

27. A stabilized pharmaceutical composition comprising a solid dispersion of tacrolimus, wherein the composition comprises (a) no more than 0.5% of 8-epitacrolimus, (b) no more than 0.5% of tacrolimus diene, (c) no more 0.5% tacrolimus epimer-C4 diene and / or (d) no more than 0.5% tacrolimus regioisomer, after storage at 40 ° C and 75% relative humidity for 5 weeks.

28. A pharmaceutical composition comprising tacrolimus and from about 0.05 to about 0.6% by weight of tartaric acid.

29. A pharmaceutical composition comprising tacrolimus and tartaric acid in a weight ratio of about 19: 0.5 to about 20: 6.

Materials and methods

Materials [0066]

Tacrolimus (provided by Eurotrade)

Lactose monohydrate 200 mesh (from DMV)

Polyethylene glycol 6000, Pluracol® E6000 (from BASF)

Poloxamer 188, Pluronic® F-68 (from BASF) magnesium stearate

Croscarmellose sodium, Ac-Di-Sol® (with FMC)

Micro talk

HPMC, i.e. hypromellose sold by ShinEtsu under the trade name Metolose 90SH (type 2910, 2208), Metolose 60SH (type 2910), in various degrees of polymerization (viscosity 3-100,000 cP).

EP 2 575 769 B1

Resin Bead, AG 501X8 Class 20-50 Mesh Biotechnology [0067] Tablets, capsules and granules can be enteric coated with various types of polymers, such as hydroxypropyl methylcellulose acetate succinate (Aqoat), cellulose acetate phthalate, HPMCP hydroxypropyl methylcellulose phthalate or methacrylic acid copolymers, such as like Eudragit L30D,

Eudragit 100 / S, Eudragit 100 / L.

[0068] The hard gelatin capsules of Prograf © manufactured by Fujisawa Ireland Ltd are composed of:

<td>Components</td><td>mg</td>

<td>Tacrolimus, anhydrous</td><td>1.0</td>

<td>Gelatine</td><td>6.9</td>

<td>Hyprometyloceluloza</td><td>1.0</td>

<td>Lactose monohydrate</td><td>24.7</td>

<td>Magnesium stearate</td><td>0.3</td>

<td>Shellac</td><td>qs</td>

<td>Soya lecithin</td><td>qs</td>

<td>Red iron oxide (E172)</td><td>qs</td>

<td>Titanium dioxide (E171)</td><td>qs</td>

<td>Dimethicone (E900)</td><td>qs</td>

methods

In vitro dissolution tests and pH measurement [0069] The following test methods for the compositions and dosage forms of the invention were used.

Test 1 - dissolution:

[0070] In vitro dissolution test according to USP method A, delayed release articles (USP paddle method, rotation speed: 50 rpm, 37 ° C, after 2 hours in an acid medium, the medium changed to phosphate buffer pH 6.8).

Test 2 - dissolution:

[0071] In vitro dissolution test in an aqueous solution for dissolution at pH adjusted to 4.5 (900 ml of water with 0.005% HPC (hydroxypropylcellulose); 37^aboutC; pH set at 4.5; vane method according to USP; rotational speed: 50 rpm).

EP 2 575 769 B1

PH measurement:

[0072] 300 mg of tacrolimus compositions according to the invention are dispersed in 5 ml of water. A conventional pH meter is used for pH measurement.

[0073] The following examples serve to illustrate the invention and are not intended to limit the scope of the present invention.

EXAMPLE 1

Tacrolimus compositions

A. Composition A of tacrolimus (tacrolimus tablets) (disclosed in Example 2 in WO2005 / 020993 and WO2005 / 020994) [0074]

<td>Components</td><td>%</td><td>mg</td>

<td>Tacrolimus monohydrate</td><td>1.98</td><td>2.00</td>

<td>Lactose monohydrate</td><td>40.50</td><td>40.91</td>

<td>Polyethylene glycol 6000 (PEG 6000)</td><td>33.26</td><td>33,60</td>

<td>Poloxamer 188</td><td>14.40</td><td>14.40</td>

<td>Magnesium stearate</td><td>0.50</td><td>0.51</td>

<td>Talc</td><td>4.50</td><td>4.55</td>

<td>Croscarmellose sodium</td><td>5.00</td><td>5.05</td>

<td>Together</td><td>100.00</td><td>101.01</td>

[0075] Tacrolimus was dissolved in PEG 6000 at a temperature above 80 ° C. Poloxamer 188 was added and the solution was heated to above 80 ° C. Using the feed unit Phast FS1.7, the solution was sprayed onto 200 g of lactose monohydrate in a Phast FB100 fluid bed. The obtained granulate was passed through a Comill, sieve No. 1397, 4500 rpm. and mixed with croscarmellose sodium for 3 minutes in a Turbula mixer.

[0076] Magnesium stearate and talc were sieved through a # 300 sieve and mixed in a Turbula mixer for 3 min. The granulate was mixed with magnesium stearate: talc (1: 9) for 0.5 minutes in a Turbula mixer.

[0077] The resulting mixture was compressed into 6 mm tablets with 2 mg active ingredient (100 mg tablet) in the form of a compound cup.

Average disintegration time: 7 minutes. Hardness: 65 N

B. Composition B of tacrolimus (prolonged release tacrolimus tablets):

[0078]

<td>Components</td><td>mg</td>

<td>Tacrolimus monohydrate</td><td>2.04</td>

<td>Lactose monohydrate</td><td>41.70</td>

EP 2 575 769 B1

<td>Polyethylene glycol 6000 (PEG 6000)</td><td>34.30</td>

<td>HPMC (type 2208; 15,000 cP)</td><td>63.00</td>

<td>Poloxamer 188</td><td>14.70</td>

<td>Magnesium stearate</td><td>1.58</td>

[0079] Tacrolimus was dissolved in PEG 6000 and poloxamer 188 at a temperature above 75 ° C. Using a Phast FS1.7 delivery unit, the solution was sprayed onto 200 g of lactose monohydrate in a Phast FB100 fluid bed. The obtained granulate was sieved through a 710 mesh screen and mixed with HPMC for 3 minutes in a Turbula mixer, then mixed with magnesium stearate for 0.5 minutes in a Turbula mixer. [0080] The resulting mixture was compressed into tablets (157 mg tablets) in the form of a compound cup, each tablet having 2 mg of active ingredient. Hardness: 65 N.

EXAMPLE 2

Stability of Composition A of tacrolimus of Example 1 [0081] Composition A of tacrolimus of Example 1 (2 mg tacrolimus tablets) was stored at 30 ° C / 65% RH (relative humidity). At specific time points (start, 1 month, 2 months, 3 months, 6 months), the composition in the form of 2 mg tablets was subjected to the stability test, i.e. analyzed quantitatively by a conventional HPLC test for tacrolimus and the main degradation product - epimer-C8 ( 8epitakrolimus). The results shown in the table below show a clear degradation of tacrolimus over time and a significant presence of the degradation product - C8 epimer measured as a percentage of the total peak area (HPLC).

<td></td><td colspan="5">Time point (months) for the durability test</td>

<td></td><td>0 months</td><td>1 month</td><td>2 months</td><td>3 months</td><td>6 months</td>

<td>Tacrolimus (mg)</td><td>2.13</td><td>1.97</td><td>2.02</td><td>1.82</td><td>1.48</td>

<td>Epimer-C8 (% of total peak area)</td><td>4.8</td><td>8.5</td><td>11.1</td><td>11.6</td><td>13.0</td>

EXAMPLE 3

Tacrolimus compositions comprising a stabilizing agent (metal chelator) [0082] The composition B of tacrolimus of Example 1 was modified by the addition of a stabilizing agent. [0083] The carrier of the composition B is a mixture of PEG 6000 and poloxamer 188.

[0084] Useful stabilizers were identified and evaluated by examining their ability to dissociate and dissolve in the molten carrier the tacrolimus B composition of Example 1, i.e. 0.5% w / w. of each substance (agent), by weight of the total carrier system, was mixed with PEG / Poloxamer at 80 ° C, see the table below:

EP 2 575 769 B1

<td>Stabilizing agent (substance)</td><td>Physical state in the medium</td>

<td>D-glucuronic acid</td><td>Undissolved</td>

<td>Ethylene di-aminotetraacetic acid (EDTA)</td><td>Undissolved</td>

<td>EDTA, us</td><td>Undissolved</td>

<td>EDTA, Na4, 2H2O</td><td>Undissolved</td>

<td>EDTA, NH4, H2O</td><td>Undissolved</td>

<td>EDTA, Ca, Na2</td><td>Undissolved</td>

<td>Tartaric acid</td><td>dissolved</td>

<td>Tartaric acid, K</td><td>Undissolved</td>

<td>Tartaric acid, K2,</td><td>Undissolved</td>

<td>Tartaric acid, (NH4) 2</td><td>Undissolved</td>

<td>Citric acid, H2O</td><td>dissolved</td>

<td>Citric acid, Na</td><td>Undissolved</td>

<td>Citric acid, Na3</td><td>Undissolved</td>

<td>Citric acid, (NH4) 2</td><td>Undissolved</td>

<td>Citric acid, (NH4) 3</td><td>Undissolved</td>

<td>Oxalic acid, 2H2O</td><td>dissolved</td>

<td>Oxalic acid, Ca, ΧΗ2Ο</td><td>Undissolved</td>

<td>Triethyleneaminopentaacetic acid, Ca, Na3, H2O</td><td>Undissolved</td>

<td>8-hydroxyquinoline 0.005%, ½SO4, ^{1 /}? HO</td><td>Undissolved</td>

[0085] Among the stabilizing agents only citric acid, tartaric acid and oxalic acid were dissolved in the carrier mixture. It is noteworthy that regardless of the solubility of the carrier mixture used, these stabilizing agents (chelators) all recrystallized completely or partially when incorporated into the composition B (added to the carrier during the process of preparing the composition B according to example 1).

[0086] In the first experiment, the use of citric acid as a stabilizing agent was investigated by preparing two samples of the composition (lemon # 1 and lemon 2 #) prepared according to example 1 (composition B) and dissolving 0.05% w / w. and 0.5% w / w citric acid, in a molten carrier. Samples of the composition were stored at 40 ° C, 75% RH (relative humidity) and samples were determined for the degradation product - epimer-C8 (8-epitacrolimus), using standard quantitative HPLC after 0, 5 and 16 weeks, cf. results in the table below.

<td>Sample composition</td><td colspan="3">8-epitacrolimus (percentage of the total peak area)</td>

<td></td><td>0 weeks</td><td>5 weeks</td><td>16 weeks</td>

<td>Lemon # 1 (0.05% wt / wt)</td><td>5.30</td><td>8.77</td><td>11.64</td>

<td>Lemon # 2 (0.5% w / w)</td><td>2.20</td><td>3.14</td><td>3.85</td>

[0087] From the results it is evident that the formation of the 8-epitacrolimus degradation product is significantly higher in a composition containing a small amount (0.05% wt / wt) of citric acid.

[0088] In a second experiment, the use of citric acid, tartaric acid and oxalic acid as a stabilizing agent in the B composition of example 1, respectively was investigated. Three samples of the composition (lemon, # 3, # 4 oxalic, tartaric, # 5) were prepared according to the example 1 (composition B) and dissolving 0.5% w / w. a stabilizing agent in the molten carrier. Samples of the composition were stored at 25 ° C / 60% RH as well as at 40 ° C / 75% RH and the samples determined for the epimer-C8 (8-epitacrolimus) degradation product using quantitative HPLC after 0, 5 and 12 weeks, cf. results in the table below. [0089] For comparison, i.e. to investigate whether deionization of the carrier mixture prior to the preparation of the composition B increased the stability of the composition, a sample of the composition (deionized # 6) without a stabilizing agent, but using a deionized support was prepared according to example 1 (composition B). In the production of carriers, traces of e.g. metal ions will inevitably occur in the carrier due to the use of accessories and excipients containing metal ions.

The support was deionized as follows: 5 g of the resin mixture of the beads were weighed for every 100 g of carrier. PEG 6000 and poloxamer 188 were melted at 70 ° C while stirring. The resin was then added to the carrier and stirring was continued for 1 hour. To remove the resin, the carrier was filtered through a 710 mesh and deionized was ready for use. A sample of deionized # 6 compositions was subjected to the same durability tests as samples of compositions # 3, # 4 and # 5.

<td colspan="2">Sample composition no</td><td>0 weeks</td><td>5 weeks</td><td>12 weeks</td>

<td rowspan="2">Lemon # 3 (0.5% w / w)</td><td>25 ° C / 60% RH</td><td>0.04</td><td>0.51</td><td>0.98</td>

<td>40 ° C / 75% RH</td><td>0.04</td><td>1.67</td><td>2.56</td>

<td rowspan="2">Sorrel # 4 (0.5% w / w)</td><td>25 ° C / 60% RH</td><td>0.10</td><td>0.22</td><td>on,</td>

<td>40 ° C / 75% RH</td><td>0.10</td><td>0.45</td><td>1.01</td>

<td rowspan="2">Wine # 5 (0.5% weight / weight)</td><td>25 ° C / 60% RH</td><td>0.02</td><td>0.10</td><td>0.23</td>

<td>40 ° C / 75% RH</td><td>0.02</td><td>0.42</td><td>0.80</td>

<td rowspan="2">Deionized # 6 (comp.)</td><td>25 ° C / 60% RH</td><td>0.85</td><td>6.76</td><td>10.36</td>

<td>40 ° C / 75% RH</td><td>0.85</td><td>11.43</td><td>16.35</td>

[0091] These results show that in all formulations, degradation is more pronounced at elevated temperature / relative humidity. Furthermore, the degradation in the sample of Comparative Composition # 6 without a stabilizing agent is much higher than in samples of compositions containing the stabilizing agent.

[0092] In conclusion, it has been shown that the use of 0.5% w / w. citric acid in the B formulation of tacrolimus in the example improves the stability of tacrolimus significantly compared to the use of less citric acid (0.05% wt / wt). Surprisingly, a better stabilizing effect is obtained, however, thanks to stabilizing agents (both are metal chelators) - tartaric acid and oxalic acid, each at a concentration of 0.5% w / w.

[0093] Further experiments carried out in the same manner unexpectedly showed that the use of tartaric acid in a concentration ranging from 0.10 to 0.60% w / w. effectively reduced

The formation of epitakrolimus in such a way that the degradation was more pronounced when lower and higher concentrations were used, respectively. However, the quantitative formation of another degradation product with a retention time of 21.5 min was increased in a linear fashion with increasing tartaric acid concentration and appears to be a threshold corresponding to a tartaric acid percentage of 0.10% w / w. up to 0.20% wt / wt In addition, a further degradation product was observed with a retention time of 4.4 minutes, especially at low (0.01% wt / wt) and high concentrations of tartaric acid (0.60% wt / wt).

EXAMPLE 4 Addition of a Stabilizing Agent in a Manufacturing Process [0095] Two samples of compositions # 1 and # 2 were prepared, with the difference that the process step of incorporating a stabilizing agent occurs before or after the addition of tacrolimus to the carrier:

Sample of composition # 1 (addition of tartaric acid followed by tacrolimus): 14 g of PEG 6000 and 6 g of Poloxamer 188 were molten, mixed, transferred to Petri dishes, cooled and ground (carrier mixture). 4 g of a carrier mixture with 6 mg of tartaric acid were mixed, the resulting mixture was heated at 75 ° C with stirring for 15 min. In the following, 160 mg of tacrolimus was added and stirring was continued for 2 hours at 75 ° C. The amount of C8-epimer (8-epitacrolimus) in the sample of composition # 1 was examined using standard quantitative HPLC on the day of preparation (day zero) to 0.04% of the total peak area.

[0096] A sample of composition # 2 (addition of tacrolimus and tartaric acid) was prepared in the same manner as sample of composition # 1 except adding tartaric acid, which was added after the addition of 160 mg of tacrolimus (the addition steps were inverted). The amount of C8-epimer (8-epitacrolimus) in the sample of composition # 1 was tested using standard quantitative HPLC on the day of preparation (day zero) to 1.33% of the total peak area.

[0097] In summary, it has been shown that it is important to stabilize the tacrolimus composition with tartaric acid (being a chelator as well as acid), also in the steps of the manufacturing method.

EXAMPLE 5

Stabilizing agents for use in the tacrolimus composition [0098] The purpose of this experiment was to investigate the use of organic and inorganic acids as stabilizing agents in tacrolimus compositions dispersed in a PEG 6000 / poloxamer vehicle. [0099] Four samples of the composition (# 1, # 2, # 3 and # 4) were prepared by melting together 14 g of PEG 6000 and 6 g of poloxamer 188, mixed, transferring the mixture to a Petri dish, cooling and crumbling. 4 g of the carrier mixture was mixed with a suitable amount of a stabilizing agent according to the table below, the mixture was heated and stirred at 75 ° C for 15 minutes. Then 160 mg of tacrolimus was added with stirring, which was continued for 2 hours at 75 ° C. The pH of the sample of the composition was measured.

EP 2 575 769 B1

<td>Sample composition Nr.</td><td rowspan="2">Stabilizing agent</td><td rowspan="2">pH</td><td colspan="2">Epimer-C8 (% of total peak)</td>

<td></td><td>0 days</td><td>7 days</td>

<td># 1</td><td>Lack</td><td>7.0</td><td>2.57</td><td>3.20</td>

<td># 2</td><td>Tartaric acid, 6 mg</td><td>3.5</td><td>0.04</td><td>0.06</td>

<td># 3</td><td>Acetic acid, 150 μl</td><td>3.2</td><td>1.02</td><td>1.70</td>

<td># 4</td><td>1% H2SO4 / 2-propanol, 300 μl</td><td>3.0</td><td>3.18</td><td>3.53</td>

[0100] The result clearly shows the superiority of tartaric acid at pH 3.5 as the stabilizing agent compared to the inorganic acids tested. In contrast to inorganic acids, tartaric acid has a chelating effect. It is contemplated that the chelating stabilizing agent is advantageous in order to prevent or reduce the formation of the degradation product tacrolimus-epimer-C8. The data clearly show that there is no stabilizing effect in maintaining the low pH as such; a stabilizing agent is needed, preferably with a chelating action.

EXAMPLE 6

Stabilizing agents for use in tacrolimus compositions - using the same molar concentration [0101] The use of organic acids with a chelating action as stabilizing agents in the composition of the invention was tested using acids at the same molar concentration:

<td></td><td>pK:</td><td>Harder. Cząst.g / mol</td><td></td>

<td>Tartaric acid</td><td>2.98</td><td>150.09</td><td></td>

<td>Citric acid</td><td>3.13</td><td>210.14</td><td>monohydrate</td>

<td>Oxalic acid</td><td>1.27 / 4.28</td><td>126.07</td><td>dehydrate</td>

[0102] Four samples of the composition were prepared (# 1 Comparison, # 2 Tartar, # 3 Citron and # 4 Vinegar) in the same manner as described in Example 5. Samples of the composition were stored at 40 ° C / 75% RH and samples were labeled degradation - epimer-C8 (8-epitacrolimus) using standard quantitative HPLC after 0, 10 and 30 days, see results in the table below.

<td colspan="3"></td><td colspan="3">Epimer-C8 (% of total peak)</td>

<td>Sample composition no</td><td>Stabilizing agent</td><td>pH</td><td>0 days</td><td>10 days</td><td>30 days</td>

<td># 1 Comparison</td><td>No</td><td>7.0</td><td>2.06</td><td>11.94</td><td>23,74</td>

<td># 2 Winowy</td><td>Tartaric acid, 6 mg</td><td>3.5</td><td>0.05</td><td>0.62</td><td>1.55</td>

<td># 3 Lemon</td><td>Citric acid, 8.4 mg</td><td>3.2</td><td>0.05</td><td>0.58</td><td>1.23</td>

<td># 4 Sorrel</td><td>Oxalic acid, 5.04 mg</td><td>3.4</td><td>0.04</td><td>0.45</td><td>0.80</td>

[0103] This experiment shows that the stabilizing effect of citric acid (pH 3.2) and oxalic acid (pH 3.4) was better than tartaric acid (pH 3.5). However, oxalic acid is usually less suitable in pharmaceutical compositions for regulatory reasons.

EXAMPLE 7

Tartic acid as stabilizing agent in tacrolimus compositions - effect of pH [0104] The effect of pH on the stabilizing and chelating effect of tartaric acid in the tacrolimus composition according to the invention is examined by preparing seven samples of compositions containing the same amount of tartaric acid (chelating effect), but adapted to different pH values ​​with formic acid or trometamol.

[0105] Seven samples of the composition (# 1 pH 3.6, # 2 pH 2.2, # 3 pH 4.9, # 4 pH 5.8, # 5 pH7,1, # 6 pH 7.6 and # 7 pH 8.0) was prepared in the same manner as described in Example 5. To each sample of the composition except # 2, 150 μl of a solution containing 300 mg of tartaric acid in 10 ml of 2-propanol and a volume of 120.49 mg of trometamol in 10 ml were added. methanol as mentioned in the table below. To sample # 2, 6.012 mg of tartaric acid and 400 ml of formic acid were added. Samples of the composition were stored at 40 ° C / 75% RH and the samples determined for degradation product - epimer-C8 (8-epitacrolimus), using standard quantitative HPLC after 0 and 14 days, see results presented in the table below.

<td>A sample</td><td>Center</td><td>PH adjustment, added</td><td colspan="2">Epimer-C8 (% of total</td>

<td>composition no</td><td>Stability</td><td>Trometamol solution (μθ</td><td>peak)</td><td></td>

<td></td><td></td><td></td><td>0 days</td><td>14 days</td>

<td># 1 pH 3.6</td><td>Tartaric acid</td><td>No</td><td>0.05</td><td>0.38</td>

<td># 3 pH 4.9</td><td>Tartaric acid</td><td>500</td><td>0.25</td><td>2.77</td>

<td># 4 pH 5.8</td><td>Tartaric acid</td><td>580</td><td>0.44</td><td>3.75</td>

<td># 5 pH 7.1</td><td>Tartaric acid</td><td>670</td><td>0.70</td><td>5.16</td>

<td># 6 pH 7.6</td><td>Tartaric acid</td><td>750</td><td>0.89</td><td>5.99</td>

<td># 7 pH 8.0</td><td>Tartaric acid</td><td>830</td><td>1.18</td><td>6.77</td>

[0106] Samples of compositions # 1 and # 2 were samples determined on tacrolimus using standard quantitative HPLC after 0, 11 and 30 days, cf. results in the table below.

<td>Composition of the composition no</td><td rowspan="2">Center Stability</td><td rowspan="2">PH adjustment, added formic acid solution (μθ</td><td colspan="3">Epimer-C8 (% total peak)</td>

<td></td><td>0 days</td><td>11 days</td><td>30 days</td>

<td># 1 pH 3.6</td><td>Tartaric acid</td><td>Nothing</td><td>97.83</td><td>93.70</td><td>92.03</td>

<td># 2 pH 2.2</td><td>Tartaric acid</td><td>400</td><td>83,32</td><td>22,59</td><td>10.25</td>

[0107] The results show that pH issues: at pH 2.2 in the tacrolimus composition, immediate degradation of tacrolimus occurs, and after 30 days almost all of tacrolimus degrades. At pH 4.9 to 8.0, it has been shown that even a small increase in pH has a significant effect on the stability of tacrolimus.

EXAMPLE 8

Tartaric acid as a stabilizing agent in tacrolimus compositions - optimal concentration [0108] Five samples of the composition were prepared using the tacrolimus B composition of Example 1 (composition of the 2 mg tablet of tacrolimus) with the addition of tartaric acid as the stabilizing agent in the amount described in Example 3. mentioned in the table below (% by weight calculated based on the total weight of the carrier). Samples of the composition were stored in HDPE silica gel bottles as a drying agent at 25 ° C / 60% RH for 10 months and the samples were tested for degradation product - epimer-C8 (8-epitacrolimus) using standard quantitative HPLC after the period mentioned in the following table.

<td rowspan="2">Conc. acid tartaric</td><td colspan="11">Epimer-C8 (% of total peak) measured after storage by (time in months)</td>

<td>0</td><td>1</td><td>1.5</td><td>2</td><td>3</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td>

<td>0.05% w.</td><td></td><td>0.05</td><td></td><td></td><td>0.23</td><td>0.35</td><td></td><td>0.53</td><td></td><td></td><td></td>

<td>0.10% w.</td><td></td><td></td><td></td><td></td><td>0.07</td><td>0.07</td><td></td><td>0.14</td><td></td><td></td><td></td>

<td>0.15% w.</td><td>0.05</td><td>0.08</td><td>0.04</td><td></td><td>0.06</td><td></td><td></td><td></td><td>0.19</td><td>0.15</td><td>0.19</td>

<td>0.20% w.</td><td></td><td></td><td></td><td>0.07</td><td></td><td>0.11</td><td></td><td></td><td></td><td></td><td></td>

<td>0.50% w.</td><td>0.04</td><td></td><td></td><td>0.14</td><td>0.22</td><td></td><td>0.30</td><td></td><td></td><td>0.23</td><td>1.43</td>

[0109] The results presented indicate the stabilizing effect of tartaric acid in the range of concentrations from

0.05% wt / wt up to 0.50% wt / wt. which provides long-term stability. These data suggest that, in particular, a concentration ranging from 0.10 to 0.20% w / w. tartaric acid has the highest stabilizing effect on the composition of tacrolimus.

EXAMPLE 9 [0110] Tartaric acid as a stabilizing agent for tacrolimus compositions using various carriers

[0111] In this experiment, the use of a stabilizing agent (0.5% w / w tartaric acid, calculated on the weight of the total carrier composition) in a composition with 5% w / w was investigated. tacrolimus in the vehicle as listed in the table below.

[0112] 11 different carrier systems were used and a composition sample with 5% w / w was prepared for each carrier system. tacrolimus dispersed or dissolved in the carrier system (reference composition) and a sample of a 5% w / w composition. tacrolimus dispersed or dissolved in a carrier system containing 0.5% w / w. tartaric acid as a stabilizing agent (composition according to the invention). Each sample of the composition was stored at 40 ° C / 75% RH and the samples were assayed for the epimer-C8 degradation product (8-epitacrolimus), using standard quantitative HPLC after 7 days as a percentage of the total peak value. For each carrier system, the result in the table below is given as the amount of C8-epimer in the reference composition relative to the amount of C8-epimer in the composition of the invention.

<td>Media (trade name)</td><td>Name of the substrate (y)</td><td>Relative amount C8-epimer</td>

<td>Acconon MC-8</td><td>Macrogol glycerides of caprylocapronates, glycerides mixtures of caprylic and capric acids, oxyethylated glycerides with 8 moles of ethylene oxide (ang.Caprylocaproyl Macrogolglycerides PEG-8 Caprylic / Capric Glycerides)</td><td>2.94</td>

<td>Acconon S-35</td><td>Esters of glycerol and ethoxylated soybean oil (Ephoxylated soybean oil glycerol esters PEG-35 Soy Glycerides_</td><td>9.50</td>

<td>Brij 700 P</td><td>Polyoxyethylene stearyl ether 100, Polyoxyethylene 100 stearyl ether, PEG-100 stearyl ether)</td><td>510.04</td>

<td>Captex 200</td><td>Diester of propylene glycol of caprylic acid and caprine (Propylene Glycol Dicaprylate / Dicaprate)</td><td>4.53</td>

<td>Cremophor A 25</td><td>Macrogol cetaclearyl ether (Macrogol cetastearyl ether)</td><td>320.83</td>

<td>Cremophor ELP</td><td>Polyoxylated castor oil, ricinoleate macrogol glycerol (Polyoxyl 35 castor oil, Macrogol glycerol ricinoleate)</td><td>12,21</td>

<td>Grindsted PGMS SPV</td><td>Propylene glycol ester (Propylene Glycol Ester)</td><td>5.70</td>

<td>Labrafil M1944 CS</td><td>Macrogol glycerides oleates, Oleanyans polyoxyglycerides (EnglishOleoyl Macrogolglucerides, Oleoyl Polyoxylglycerides)</td><td>7.80</td>

EP 2 575 769 B1

<td>Media (trade name)</td><td>Name of the substrate (y)</td><td>Relative amount C8-epimer</td>

<td>LipoPEG 6000 DS</td><td>Polyethylene glycol 150 distearate (PEG150 Distearate)</td><td>35.72</td>

<td>MethoxyPEG 5000</td><td>Methoxy polyethylene glycol 5000 (ang. Methoxypolyethylene Glycol 5000)</td><td>54.90</td>

<td>PEG-dimethylether</td><td>Polyethylene glycol dimethyl ether (PEGdimethylether)</td><td>2.09</td>

[0113] The results presented demonstrate the ability of tartaric acid to increase the stability of tacrolimus compositions dispersed or dissolved in a variety of carrier media and polymers.

[0114] All publications, patents and patent applications cited in the present text are hereby incorporated by reference.