BR112013007373B1 - CYCLOSPORINE EMULSION AND USE OF A CYCLOSPORIN EMULSION - Google Patents

Abstract

cyclosporin emulsion, and its uses. the present invention relates to a cyclosporin emulsion containing: i) a cyclosporin ii) a natural oil (long chain triglyceride) iii) a phosphatidylcholine, iv) glycerol, v) a pharmaceutically tolerable alkali salt of a free fatty acid, vi) a medium chain triglyceride oil vii) optionally hydrochloric acid or sodium hydroxide for pH adjustment viii) water.

Description

field of invention

[001] The present invention relates to a new composition of cyclosporin in the form of an emulsion with a content of medium chain triglycerides. The emulsion is an oil-in-water emulsion, is bioequivalent to the marketed product Sandimmune® but has an improved tolerability profile compared to Sandimmune®.

Background of the invention

[002] Cyclosporine is a widely used immunosuppressant in organ and tissue transplantation. Cyclosporine has low water solubility, and a marketed Sandimmune® infusion product was formulated as a concentrate containing 50 mg/ml of cyclosporine and polyoxylated castor oil (Cremophor EL®) in ethanol and must be diluted with an isotonic chloride solution sodium or a 5% glucose solution prior to administration. However, intravenous administration of a cyclosporine composition containing Cremophor EL® has been associated with hypersensitivity reactions, with reactions ranging from mild skin reactions to anaphylaxis and heart failure. Therefore, there is a need for Cremophor® free cyclosporin compositions as well as ready-to-use compositions.

[003] Cremophor EL is the registered trademark of BASF Corp. for its version of polyethoxylated castor oil. It is prepared by reacting 35 moles of ethylene oxide with each mole of castor oil. The resulting product is a mixture (CAS number 61791-12-6): the main component is the material in which castor oil triglyceride hydroxyl groups have ethoxylated with ethylene oxide to form polyethylene glycol ethers. Minor components are polyethylene glycol esters of ricinoleic acid, polyethylene glycols and polyethylene glycol ethers of glycerol. Cremophor EL is a synthetic, non-ionic surfactant. Its utility comes from its ability to stabilize emulsions of non-polar materials in aqueous systems.

[004] A number of Cremophor products are available from BASF(Cremophor RH 40 (polyoxyl-40 hydrogenated castor oil), Cremophor EL (polyocyl-35 castor oil), Cremophor ELP (extra pure grade of EL), which are polyoxyl castor oil grades, and Cremophor A 6 (macrogol-6-cetostearyl ether) and Cremophor A 25 (macrogol-25-cetostearyl ether), which are cetostearyl ether macrogol grades. has been associated with hypersensitivity reactions, however, it is likely that all Cremophor® products in the polyoxyl castor oil series will have the same side effect when administered to a subject.

Brief description of the drawings

[005] Figure 1 - Figure 1 shows the combined individual graphs of whole blood concentrations after infusion of 5 mg/kg of cyclosporine sampled for 48 hours in 52 healthy subjects.

[006] Figure 2 - Figure 2 shows the confidence interval (90%) for the mean ratios (90% of CicloMulsion / Sanimmune) and intra-individual coefficient of variation (CV) of whole blood pharmacokinetic variables of ciclosporin.

Description of the invention

Ready-to-use cyclosporin compositions for parenteral administration have been described among others in European Patent Application No. EP-B-0 570 829. However, further development of similar compositions has been necessary in order to obtain compositions that are bioequivalent to Sandimmune® product and in order to ensure an appropriate service life.

[008] Further developments are directed towards ensuring formulations which both have sufficient stability and shelf life to satisfy the requirements for commercialization and distribution in commercial pharmaceutical activity while being bioequivalent and using ingredients which are beneficial for such therapeutic targets like mitochondrial metabolism and being non-toxic.

[009] In clinical practice, this also means that one can better achieve the goal of eg Sandimmune® and thus ensure that in clinical practice it is possible to avoid the adverse effect of Cremophor® and ethanol and avoid having to premedicate with corticosteroids and/or antihistamines in cases where the aforementioned pretreatment is contraindicated or the multi-pharmaceutical approach is contraindicated.

The present invention provides a Cremophor-free cyclosporin composition for parenteral administration, namely intravenous administration.

[0011] A composition of the present invention contains: A cyclosporin A natural oil (long chain triglyceride) (eg soybean oil) A phosphatidylcholine (eg egg lecithin) Glycerol A pharmaceutically tolerable alkali salt of a free fatty acid ( eg sodium oleate) medium chain triglyceride oil (eg coconut oil and/or palm oil) Optionally hydrochloric acid or sodium hydroxide for pH adjustment Water.

[0012] A composition of the present invention has an osmolality in the isoosmolar range, that is, the osmolarity is in a range from about 280 mosm/l to about 305 mosm/l, preferably about 295 to 300 mosm/l. l, which corresponds to the plasma value, that is, the isoosmolar value. The use of a composition containing cyclosporin with an isoosmolar osmolality is important in order to ensure good biocompatibility. Many of the known cyclosporin emulsions are hyperosmolar in order to obtain good penetration across the blood-brain barrier when the above compositions are used, for example, in the treatment of stroke. However, the cyclosporin emulsions of the invention can also be used in other important therapeutic areas such as, for example, in connection with cardiac reperfusion injury and immunosuppression, where the use of a hyperosmolar composition can lead to undesirable penetration into the brain of metabolites specific substances, blood components, etc., which in turn can lead to toxic or adverse effects, all of which are undesirable.

[0013] The terms osmolarity, osmolality and tonicity are often used interchangeably. For specific definitions see Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company 1990, pages 1481 to 1498, which is hereby incorporated by reference.

[0014] A composition of the present invention can also be used in the treatment of traumatic brain injury, where the composition crosses the blood-brain barrier due to disruptions therein caused by the brain injury itself and where therefore there is no need for a hyperosmolar composition to break the blood-brain barrier. An isoosmolar composition is highly preferred over a hyperosmolar composition in similar indications.

Cyclosporine

[0015] Cyclosporin can be any therapeutically active cyclosporin or cyclosporin analogues and derivatives. Cyclosporine may have immunosuppressive properties or non-immunosuppressive properties (NICAMs), or it may have any other known or unknown effect (for example, for cardioprotection or neuroprotection or for treatment in cardiovascular diseases such as myocardial infarction, reperfusion, or in neurodegenerative diseases , brain injury, ischemia, trauma, etc. Cyclosporins are known to have protective effects on mitochondria. Cyclosporins are preferred which are suitable for use in immunosuppression Cyclosporine can be a natural or synthetic cyclosporin As will be evident from this The following several chiral carbon atoms are present, therefore, any of these forms - whether alone or in any combination - that have therapeutic utility are encompassed by the term "cyclosporin".

[0016] In the Examples, the following cyclosporin was employed:INN: Cyclosporin Chemical names:a) Cyclo[[(2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(methylamino)-oct-6- enoyl]-L-2-aminobutanoyl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N -methyl-L-leucyl-N-methyl-L-valyl]b) Cyclo[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl] -L-2-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-Lvalyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-Nmethyl-L-leucyl -N-methyl-L-valyl]c) [R-[R*,R*-(E)]]-Cylic(L-alanyl-D-alanyl-N-methyl-L-leucyl-Nmethyl-L-leucyl -N-methyl-L-valyl-3-hydroxy-N,4-dimethyl-L-2-amino-6-octenoyl-L-α-aminobutyryl-N-methylglycyl-N-methyl-Lleucyl-L-valyl-N -methyl-L-leucyl)Other names: Cyclosporine (DCI)Cyclosporin (DCI - E)Compendium names: Cyclosporin (Ciclosporinum) (European Pharmacopoeia) Cyclosporine (United States Pharmacopoeia)CAS number: 59865-13-3Structural formula (see Figure 1) Molecular formula: C62H111N11O12 Mass Relative Molecular (Mr): 1202.61 Chirality: Cyclosporin is a molecule of natural origin that has several chiral carbon atoms.General PropertiesPhysical Form: white or off-white powderSolubility: Soluble in acetone, ethanol, methanol, ether, chloroform, and methylene chloride; slightly soluble in saturated hydrocarbons; practically insoluble in water Specific optical rotation: -185° to -193° (methanol). Phosphatidylcholine

Egg lecithin and/or soy lecithin, particularly egg lecithin, are preferred as phosphatidylcholine suppliers, namely 3-sn-phosphatidylcholine or hydrogenated 3-sn-phosphatidylcholine. Lecithins with a content of more than 60% of 3-sn-phosphatidylcholine and/or partially hydrogenated 3-sn-phosphatidylcholine and/or hydrogenated 3-sn-phosphatidylcholine are particularly suitable. Pharmaceutically tolerable fatty acid salts

[0018] An alkali salt of a free fatty acid with 6 to 26 carbon atoms can be added to adjust the pH value or to facilitate the emulsification and homogenization process. The sodium and potassium salts of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid and linolenic acid are especially preferred. In a preferred modality, sodium oleate is used. Isotonically / isoosmolar adjustment substance

[0019] As seen from the examples here, in this patent application, glycerol has been employed to adjust the osmolarity of the composition to isoosmolar. However, other substances such as sorbitol, xylitol, sodium chloride, glucose may also prove adequate. Natural oils (long chain triglycerides)

[0020] Soybean oil or saffron oil or combinations thereof can be used as natural oils. In a preferred modality, soybean oil is used. MCT oils (medium chain triglycerides)

[0021] As appears in the following parts, it appears that the presence of an MCT oil is important in order to obtain the desired stability of the emulsion, namely with respect to the physical stability of the emulsion. Cyclosporin has very good solubility in MCT oil and MCT oil therefore helps to prevent unwanted precipitation of cyclosporin during storage.

[0022] In addition, MCT oils appear to have a beneficial effect on the brain, for example, there are indications that they may be beneficial in the treatment of Alzheimer's disease and other conditions where stabilization, protection and support of brain function. mitochondria is important / necessary. Mitochondria are present and produce energy in almost every cell in the body. Mitochondrial collapse can be associated with a variety of acute injuries, such as myocardial infarctions and traumatic brain injury, and chronic diseases such as amyotrophic lateral sclerosis, multiple sclerosis, and other neurological disorders. In myocardial infarctions, Reperfusion of the blocked artery can cause reperfusion injury and extra injury and heart muscle deficiency, as well as increased mortality. Mitochondrial protection in heart muscle tissue can moderate the long-term impact of heart attacks.

[0023] A suitable MCT oil is coconut oil, palm oil or a combination thereof. Water

[0024] The water used must have a quality suitable for parenteral products, such as water for injection (pharmacopoeia standard).

manufacturing method

[0025] A suitable method for manufacturing a composition of the invention appears in Figure 2 and in the Examples here in this patent application.

In all the compositions mentioned herein, in this patent application, the osmolarity is adjusted to the isoosmolar value of about 285 to about 305 mosm/l, preferably adjusted from about 295 to about 300 mosm/l.

[0027] More specifically, a composition according to the invention contains: A cyclosporin in a concentration range of from 1 to 15 g/l, A phosphatidylcholine (eg egg lecithin) in a concentration range of to from 5 to 25 g/l, A pharmaceutically tolerable alkali salt of a free fatty acid (eg sodium oleate) in a concentration range of from 0.1 to 1 g/l, Optionally, adjust the pH eg 1M sodium hydroxide in an amount sufficient to achieve a final composition pH value of from about 6 to about 8.8, Glycerol in a concentration range of from 10 to 50 g/l,A natural oil (long chain triglyceride) (eg soy oil) in a concentration range from 50 to 150 g/l,Medium chain triglyceride oil in a concentration range of to from 50 to 150 g/l, eWater up to 1 liter.

[0028] In particular embodiments, a composition according to the invention contains: A cyclosporin in a concentration range of from 1 to 15 g/l, Egg lecithin in a concentration range of from 5 to 25 g /l, A pharmaceutically tolerable alkali salt of a free fatty acid (eg sodium oleate) in a concentration range from 0.1 to 1 g/l. Optionally, pH adjustment eg sodium hydroxide 1 M sodium in an amount sufficient to reach a final pH of the composition of from about 8 to about 8.8, Glycerol in a concentration range of from 10 to 50 g/l, One oil natural (long chain triglyceride) (eg soybean oil) in a concentration range from 50 to 150 g/l, Medium chain triglyceride oil in a concentration range from 50 to 150 g /l, eWater up to 1 liter.

[0029] A cyclosporin in a concentration range of from 1 to 15 g/l, A phosphatidylcholine (eg, egg lecithin) in a concentration range of from 5 to 25 g/l, sodium oleate in a concentration range of from 0.1 to 1 g/l, optionally adjust the pH eg 1 M sodium hydroxide in an amount sufficient to reach a final pH value of the composition of from about from 8 to about 8.8 Glycerol in a concentration range from 10 to 50 g/l A natural oil (long chain triglyceride) (eg soybean oil) in a concentration range of to from 50 to 150 g/l, Medium chain triglyceride oil in a concentration range from 50 to 150 g/l, eWater up to 1 liter.

[0030] A cyclosporin in a concentration range of from 1 to 15 g/l, egg lecithin in a concentration range of from 5 to 25 g/l, sodium oleate in a concentration range of a from 0.1 to 1 g/l, optionally adjust the pH eg 1 M sodium hydroxide in an amount sufficient to achieve a final composition pH value of from about 8 to about 8, 8,Glycerol in a concentration range from 10 to 50 g/l,A natural oil (long chain triglyceride) (eg soybean oil) in a concentration range from 50 to 150 g/l l,Medium chain triglyceride oil in a concentration range from 50 to 150 g/l, eWater up to 1 liter.

[0031] A cyclosporin in a concentration range of from 1 to 15 g/l, A phosphatidylcholine (eg, egg lecithin) in a concentration range of from 5 to 25 g/l, A salt of pharmaceutically tolerable alkali of a free fatty acid (eg sodium oleate) in a concentration range from 0.1 to 1 g/l. Optionally, pH adjustment eg sodium hydroxide to 1 M in one sufficient amount to achieve a final composition pH value of from about 8 to about 8.8, Glycerol in a concentration range of from 10 to 50 g/l, soybean oil in a concentration range from 50 to 150 g/l, medium chain triglyceride oil in a concentration range from 50 to 150 g/l, and Water up to 1 liter.

[0032] A cyclosporin in a concentration range of from 1 to 15 g/l, egg lecithin in a concentration range of from 5 to 25 g/l, A pharmaceutically tolerable alkali salt of a fatty acid free (eg sodium oleate) in a concentration range from 0.1 to 1 g/l. Optionally adjust the pH eg sodium hydroxide to 1 M in an amount sufficient to achieve a value of Final pH of the composition from about 8 to about 8.8, Glycerol in a concentration range from 10 to 50 g/l, soybean oil in a concentration range from 50 to 150 g/l, medium chain triglyceride oil in a concentration range from 50 to 150 g/l, eWater up to 1 liter.

[0033] A cyclosporin in a concentration range of from 1 to 15 g/l, A phosphatidylcholine (eg, egg lecithin) in a concentration range of from 5 to 25 g/l, sodium oleate in a concentration range of from 0.1 to 1 g/l, optionally adjust the pH eg 1 M sodium hydroxide in an amount sufficient to reach a final pH value of the composition of from about from 8 to about 8.8, Glycerol in a concentration range from 10 to 50 g/l, soybean oil in a concentration range from 50 to 150 g/l, chain triglyceride oil medium over a concentration range from 50 to 150 g/l, eWater up to 1 liter.

[0034] A cyclosporin in a concentration range of from 1 to 15 g/l, ovoin lecithin in a concentration range of from 5 to 25 g/l, sodium oleate in a concentration range of from from 0.1 to 1 g/l, optionally adjust the pH eg 1 M sodium hydroxide in an amount sufficient to achieve a final composition pH value of from about 8 to about 8.8 ,Glycerol in a concentration range from 10 to 50 g/l, soybean oil in a concentration range from 50 to 150 g/l, medium chain triglyceride oil in a concentration range of a from 50 to 150 g/l, eWater up to 1 liter.

[0035] Furthermore, the invention relates to compositions having the following compositions: A cyclosporin in a concentration of 5 g/l, A phosphatidylcholine (eg egg lecithin) in a concentration of 12 g/l, A salt of pharmaceutically tolerable alkali of a free fatty acid (eg sodium oleate) at a concentration of 0.3 g/l. Optionally, pH adjustment eg sodium hydroxide to 1 M in an amount sufficient to achieve a value of Final pH of the composition from about 8 to about 8.8, Glycerol at a concentration of 25 g/l, A natural oil (long chain triglyceride) (eg soybean oil) at a concentration of 100 g/l, medium chain triglyceride oil in a concentration range of 100 g/l, Water up to 1 liter.

[0036] A cyclosporin at a concentration of 5 g/l, egg lecithin at a concentration of 12 g/l, A pharmaceutically tolerable alkali salt of a free fatty acid (eg sodium oleate) at a concentration of 0, 3 g/l, Optionally adjust the pH eg 1 M sodium hydroxide in an amount sufficient to achieve a final composition pH value of from about 8 to about 8.8, Glycerol in a concentration 25 g/l, A natural oil (long chain triglyceride) (eg soybean oil) at a concentration of 100 g/l, medium chain triglyceride oil at a concentration range of 100 g/l, Water up to 1 liter.

[0037] A cyclosporine at a concentration of 5 g/l, A phosphatidylcholine (eg egg lecithin) at a concentration of 12 g/l, sodium oleate at a concentration of 0.3 g/l, Optionally adjust pH eg 1M sodium hydroxide in an amount sufficient to achieve a final pH value of the composition of from about 8 to about 8.8, Glycerol at a concentration of 25 g/l, A natural oil (long chain triglyceride) (eg soybean oil) in a concentration of 100 g/l, medium chain triglyceride oil in a concentration range of 100 g/l, Water up to 1 liter.

[0038] A cyclosporin at a concentration of 5 g/l, egg lecithin at a concentration of 12 g/l, sodium oleate at a concentration of 0.3 g/l, Optionally, pH adjustment eg sodium hydroxide 1 M sodium in an amount sufficient to reach a final pH value of the composition of from about 8 to about 8.8, Glycerol at a concentration of 25 g/l, A natural oil (long chain triglyceride) ( eg soybean oil) in a concentration of 100 g/l, medium chain triglyceride oil in a concentration range of 100 g/l, Water up to 1 liter.

[0039] A cyclosporin at a concentration of 5 g/l, A phosphatidylcholine (eg egg lecithin) at a concentration of 12 g/l, A pharmaceutically tolerable alkali salt of a free fatty acid (eg oleate sodium) at a concentration of 0.3 g/l. Optionally adjust the pH eg 1 M sodium hydroxide in an amount sufficient to achieve a final composition pH value of from about 8 to about 8.8,Glycerol at a concentration of 25 g/l,soy oil at a concentration of 100 g/l,medium chain triglyceride oil at a concentration range of 100 g/l,Water up to 1 liter.

[0040] A cyclosporin at a concentration of 5 g/l, egg lecithin at a concentration of 12 g/l, A pharmaceutically tolerable alkali salt of a free fatty acid (eg sodium oleate) at a concentration of 0, 3 g/l, Optionally adjust the pH eg 1 M sodium hydroxide in an amount sufficient to achieve a final composition pH value of from about 8 to about 8.8, Glycerol in a concentration 25 g/l, soybean oil at a concentration of 100 g/l, medium chain triglyceride oil at a concentration range of 100 g/l, Water up to 1 liter.

[0041] A cyclosporine at a concentration of 5 g/l, A phosphatidylcholine (eg egg lecithin) at a concentration of 12 g/l, sodium oleate at a concentration of 0.3 g/l, Optionally adjust pH eg 1M sodium hydroxide in an amount sufficient to achieve a final pH value of the composition of from about 8 to about 8.8. soybeans in a concentration of 100 g/l, medium chain triglyceride oil in a concentration range of 100 g/l, Water up to 1 liter.

[0042] A cyclosporin at a concentration of 5 g/l, egg lecithin at a concentration of 12 g/l, sodium oleate at a concentration of 0.3 g/l, Optionally, pH adjustment eg sodium hydroxide 1 M sodium in an amount sufficient to reach a final pH of the composition of from about 8 to about 8.8, Glycerol at a concentration of 25 g/l, soybean oil at a concentration of 100 g/ l, medium chain triglyceride oil in a concentration range of 100 g/l, Water up to 1 liter.

[0043] In all the compositions mentioned above, the medium chain triglycerides can be coconut oil, palm oil, or combinations thereof.Ingredient Quantity (kg) Quality Standard Cyclosporin 1.50 R1-CEP 1999-034Rev 02Egg lecithin 3.60 Glycerol (water free) 7.50 European Pharmacopoeia Sodium oleate 0.09 Sodium hydroxide at 1 M q,s, European Pharmacopoeia Soy oil (long chain triglyceride) 30.00 European Pharmacopoeia MCT oil (chain triglyceride) average) 30.00 European Pharmacopoeia

[0044] The composition of the emulsion that was tested in a clinical study was produced according to the following formula (300 L batch size):

[0045] Thus, a preferred composition of the invention is as follows:

[0046] MCT oil is coconut oil, palm oil or a combination thereof.

[0047] As appears from the experimental section a composition according to the invention, namely, the composition mentioned above is bioequivalent to the Sandimmune® product, that is, the concentrate diluted to a ready-to-use composition. The study of bioequivalence was carried out in accordance with well-established guidelines (EMEA - European Medicines Agency, London, January 2010. Guideline on the investigation of bioequivalence, European Medicines Agency EMEA), or US Department of Health and Human Services, Food and Drug Administration, March 2003, Guidance for Industry, Bioavailability and Bioequivalence Studies for Orally Administered Drug Product - General Considerations General, US Food and Drug Control Agency (FDA)).

[0048] The study further reveals that a composition according to the invention has a much better safety profile than the Sandimmune® product. Thus, without premedication with corticosteroids, in 19 subjects two severe anaphylactic reactions were observed after administration with Sandimmune®, whereas similar reactions were not observed after administration in 33 subjects with a composition according to the invention. To summarize from the clinical study the table below shows the adverse effects observed. Two serious adverse events (SAEs) were reported. These were anaphylactic and anaphylactoid reactions that occurred after the administration of Sandimmune® injection. The proportion of total serious adverse events was significantly higher in Sandimmune® injection compared to the composition according to the invention.

[0049] Another advantage of a composition of the invention (in addition to bioequivalence with Sandimmune® injection and better safety profile than Sandimmune® injection) is the excellent stability of the composition. It is now believed that the presence of medium-chain triglycerides plays an important role in this regard. Thus, based on stability studies carried out in accordance with ICH (International Conference on Harmonization) guidelines and reported in the Examples, the overall conclusion is that a shelf life of 30 to 36 months is reasonable when stored below 25 °C and protected from light.

[0050] Other advantages are:1. Ready-to-use solution. Sandimmune® Injection is marketed as a 50 mg/ml infusion concentrate that has to be withdrawn by syringe and then injected into a larger volume of saline or 5% glucose solution. The concentrated solution is highly viscous, especially at room temperature or below, which makes it more difficult to extract and empty to the right amount than with a ready-to-use composition. Furthermore, after injection of the infusion concentrate in saline, it is necessary to mix carefully in order to avoid the injection of large lumps of Cremophor® containing cyclosporin into the circulation, which also affects the concentration of cyclosporin administered initially and later in a treatment.

[0051] 2. No non-physiological emulsifier. The content of a composition of the invention is physiological fats and phospholipids that can be metabolized by the human body.

[0052] 3. There is no risk of serious solvent-related hypersensitivity reactions, including anaphylactic reactions and death.

[0053] 4. There is no risk of cytotoxicity, nephrotoxicity or solvent-related cardiotoxicity.

[0054] 5. Eliminates the need for specialized IV tubing required for products containing Cremophor® (in order to prevent the leaching of plasticizers).

[0055] 6. Premedication with corticosteroids or antihistamines is not required.

[0056] 7. There is no risk of penetration of undesirable substance over the blood-brain barrier.

[0057] The emulsions of the invention are suitable for use in the treatment of disease where cyclosporin is indicated. Examples are immunosuppressive disorders, reperfusion injury (organ damage), cardiac reperfusion injury (cardioprotection), renal reperfusion injury (nephroprotection), hepatic reperfusion injury (hepatoprotection), prevention of brain injury (neuroprotection).

[0058] Figure 1 shows the structure of cyclosporin Figure 2 shows a method for manufacturing an emulsion according to the invention Figure 3 shows arithmetic means with standard deviation of the blood concentration of Ciclosporin in participants who were administered CicloMulsion or Sandimmune . n=52 Figure 4 shows the mean blood concentration of Ciclosporin with standard deviation in participants given CicloMulsion (A) or Sandimmune (B) either with or without premedication consisting of 50 mg diphenhydramine orally, 10 mg intravenous dexamethasone and 50 mg intravenous ranitidine.

[0059] The invention is further illustrated in the following non-limiting examples.ExamplesExample 1Manufacture of a composition according to the invention Preparation of emulsion Preparation of aqueous suspension:

[0060] A mixing tank is provided with the appropriate amount of hot water for injection. All raw materials with the exception of oil components are crushed and dispersed in water for injection at 55 to 70 °C. The suspension is dispersed by means of intense mixing. Pre-emulsion preparation:

[0061] A solution of preheated oil components (50 to 60 °C) containing dissolved cyclosporin is added. The aqueous suspension and oil phase are mixed at 55 to 70°C. If necessary, adjust the pH. Homogenization:

[0062] The preparation of the final emulsion occurs by several steps of homogenization. Pressures of 400 ± 30 bar (stage 1) and 100 ± 30 bar (stage 2) are applied. The temperature of the emulsion is between 55 °C and 90 °C. Between the homogenization steps the emulsion is stored inside intermediate storage tanks.

Final emulsion:

[0063] Depending on the capacity of the equipment used (homogenizers and vessel) the emulsion has to be diluted under agitation with the appropriate amount of water for injection. The resulting emulsion is cooled to a temperature of 15 to 25 °C. A sample of the final concentration is taken for in-process control and a pH adjustment can be performed.

Emulsion filtration:

[0064] The emulsion is filtered through a 10 μm absolute pore measuring filter.

Completion and closing:

[0065] The emulsion is filled under an atmosphere of nitrogen into suitable infusion bottles. Filled vials are closed with rubber stoppers and aluminum caps with clear flip-off caps. Sterilization:

[0066] The filled containers are sterilized in a rotating autoclave. Example 2 Composition according to the invention

Exemplo 3Estabilidade de uma composição de acordo com a invenção[0067] The composition of the emulsion that was tested in a clinical study was produced according to the following formula (batch size 300 L):

Example 3 Stability of a composition according to the invention

[0068] The composition of Example 2 was subjected to stability studies in accordance with the guidelines of the ICH (International Conference on Harmonization).

[0069] Lot PP0915013 of Cyclosporin 5 mg/ml Emulsion for Injection is stabled at 25°C/60%RH and 40°C/75%RH in accordance with ICH requirements. 12-month data are currently available.

Exemplo 4Um estudo clínico para comparar a biodisponibilidade e a farmacocinética da ciclosporina[0070] All results, at both temperatures, are well within specification. Cyclosporine appears very stable with no downward trend. Only small amounts of degradation products are found. The following tables summarize the results.

Example 4 A clinical study to compare the bioavailability and pharmacokinetics of cyclosporine

Summary Background

[0071] Cyclosporine is an immunosuppressant widely used in organ and tissue transplantation. Cyclosporine has low water solubility, and the concentrate for solution for infusion was therefore formulated in polyoxyethylated castor oil (Cremophor EL®). However, intravenous administration of cyclosporine preparations containing cremophor has been associated with hypersensitivity reactions, with reactions ranging from mild skin reactions to anaphylaxis and heart failure. A ready-to-use, cremophor-free lipid emulsion of cyclosporin (CicloMulsion, 5 mg/ml cyclosporin) for intravenous use has been developed. The objectives of the present study were to compare the pharmacokinetic and tolerability profile of CicloMulsion with the reference product Sandimmune Injection.

Methods

Caucasian and non-Caucasian male and female, healthy volunteers (n = 52) were investigated according to a two-period, single-dose, randomized, open-label, blind-to-lab crossover design and hidden from the subject. Five mg/kg of each of the two formulations was infused intravenously over 4 hours. Cyclosporine blood concentrations were determined by validated LC-MS/MS analysis. Routine pharmacokinetic comparisons were performed in accordance with the US FDA agency and the European EMA agency using analysis of variance. Point estimates of 90% confidence intervals were calculated for the test/reference least squares geometric mean ratios of relevant variables. Tolerability and safety were calculated by monitoring adverse events, total blood count, vital sign measurements, electrocardiogram, and post-study physical examination.

Pharmacokinetics

[0073] Whole blood cyclosporine concentrations are shown in Figure 1.

[0074] Confidence intervals of pharmacokinetic variables of whole blood cyclosporine

Análise Proporcional de Eventos AdversosTabela 2 - Foram reportados dois eventos adversos graves (SAEs). Estes foram as reações anafilática e anafilactóide que ocorreram depois da administração de Sandimmune® injeção. A proporção de adversos graves totais foi significativamente maior no Sandimmune® injeção comparado comCicloMulsion.

Conclusões[0075] The confidence intervals of pharmacokinetic variables of whole blood cyclosporine are shown in Figure 2 and in Table 1 below: Table 1 - Confidence interval (90%) for the mean ratios (90% of CicloMulsion / Sanimmune) and coefficient of intra-individual variation (CV) of whole blood cyclosporine pharmacokinetic variables.

Proportional Analysis of Adverse EventsTable 2 - Two serious adverse events (SAEs) were reported. These were the anaphylactic and anaphylactoid reactions that occurred after the administration of Sandimmune® injection. The proportion of total serious adverse events was significantly higher in Sandimmune® injection compared to CycloMulsion.

Conclusions

[0076] CicloMulsion, a ready-to-use, cromophor-free intravenous cyclosporine formulation is bioequivalent to Sandimmune® injection.

[0077] CicloMulsion presents an improved safety and tolerability profile.

Serious Cremophor-related hypersensitivity reactions, including anaphylactic reactions and death, can therefore be avoided in patients requiring intravenous cyclosporine.

[0079] CicloMulsion has the following advantages over Sandimmune® i.v. injection:1. Ready-to-use solution2. Physiological emulsifier. The contents of CicloMulsion are physiological fats and phospholipids that can be metabolized by the human body.3. There is no risk of serious hypersensitivity reactions related to cremophor, including anaphylactic reactions and death.4. There is no risk of cytotoxicity, nephrotoxicity or cardiotoxicity related to cremophor.5. Eliminates the need for specialized IV tubing required for products containing cremophor (in order to prevent plasticizer leaching).Clinical study detailsIntroduction

[0080] Cyclosporin is widely used to prevent graft rejection after transplantation. The currently marketed intravenous formulation, Sandimmune® Injection (Sandimmune), uses Cremophor® EL (CrEL) as an emulsifying excipient. CrEL is known to cause hypersensitivity reactions in some patients, ranging from skin reactions to anaphylactic shock and death. We evaluated the pharmacokinetics and tolerability of a new, CrEL-free lipid cyclosporin emulsion, CicloMulsion®, compared to Sandimmune. Fifty-two healthy subjects were treated with 5 mg/kg of each of the two cyclosporine formulations as an intravenous infusion over 4 h. Bioequivalence assessments were performed according to current guidelines. The geometric mean ratios for CicloMulsion / Sandimmune (90% confidence interval) were 0.90 (0.88 to 0.92) for Area Under the Curve (0 h to the last quantifiable concentration) and 0.95 (0. .92 to 0.97) for maximum blood concentration of cyclosporine. For all additional variables analyzed, the 90% confidence intervals were also within the accepted bioequivalence range of 0.80 to 1.25. An anaphylactoid reaction and an anaphylactic reaction, both classified as serious adverse events, have been reported after Sandimmune treatment. No serious adverse events were recorded after treatment with CicloMulsion. The proportion of total adverse events was significantly higher for Sandimmune. We conclude that CicloMulsion is bioequivalent to Sandimmune and has fewer adverse reactions.

[0081] Cyclosporine (CsA) is a potent immunosuppressant first recorded in 1983 that acts by reducing the function of T lymphocytes through the inhibition of calcineurin. CsA is widely used to prevent organ rejection and graft-versus-host disease after solid organ or bone marrow transplantation, and to treat autoimmune conditions such as psoriasis, atopic dermatitis, pyoderma gangrenosum, ulcerative colitis, idiopathic nephrotic syndrome and inflammatory uveitis (1-7). The molecule is highly hydrophobic and requires a lipophilic solvent for administration. The intravenous (IV) form of CsA currently on the market, Sandimmune® Injection (Novartis Pharma Stein AG, Switzerland) (Sandimmune), is a preparation with ethanol and polyoxyethylated castor oil, Cremophor® EL (CrEL).

[0082] CrEL is not inert (8) and there have been a number of reports of serious adverse events after IV CsA administration due to reactions to this vehicle medium (9-16). Hypersensitivity reactions to CrEL have also been reported when used for other intravenous drug preparations such as diazepam and Althesin® (17-21). In rodents, neurotoxicity, cardiotoxicity and nephrotoxicity due to IV CsA dissolved in CrEL (22-25) have been demonstrated and, in a canine model, CrEL has been shown to reduce cardiac output and hepatic blood flow in a non-dose-dependent manner (26). The effects of CrEL include complement activation, histamine release and severe hypersensitivity reactions (8, 17, 19, 21).

[0083] CsA in CrEL requires a dilution step prior to administration. It has been reported that inadequate preparation of formulations containing CrEL causes anaphylactoid reactions (27, 28). An additional concern with the use of ethanol and CrEL is the leaching of plasticizers from polyvinyl chloride (PVC) bags and infusion systems used in routine clinical practice. Consequently, preparation and administration must be done using glass or other non-PVC infusion systems (29, 30).

Concern about the safety of CrEL as a carrier medium for IV drugs has arisen on numerous occasions and several drugs that were previously produced in CrEL preparations are now available only with other carrier media such as lipid emulsions. Known examples are propofol (17, 31) and diazepam (32). Others, such as the chemotherapeutic agent paclitaxel, are available with both CrEL and without CrEL as a solvent (33), and the anesthetic drug Althesin® containing CrEL is no longer marketed for use in humans (17, 21, 34).

[0085] In this study, the aim was to evaluate the pharmacokinetics of a new CsA preparation ready for liver use of CrEL and ethanol for IV administration, CicloMulsion® (NeuroVive Pharmaceutical AB, Lund, Sweden), in relation to the product currently containing CrEL in the market (Sandimmune® Injection) and to assess whether the two formulations are bioequivalent. A ready-to-use preparation without CrEL potentially offers increased patient safety with fewer adverse events due to improper handling or immunological reactions to CrEL.

Study Outline

[0086] This was a single-center, open-label, subject-blind, laboratory-blind, single-dose, randomized, two-treatment, two-period, two-sequence cross-over study of the pharmacokinetics of two CsA formulations IV. The primary objective was to evaluate the pharmacokinetics, and the secondary objective was to compare the tolerability profiles of the two preparations.

[0087] The study protocol, including amendments, subject information sheets, and informed consent documents, were reviewed by the University of the Free State's Ethics Committee of the Faculty of Health Sciences. (Reference number ETOVS 65/09), and by the South African Medicines Control Council (Reference number BE 2009009), and written approval was obtained. The study was carried out in accordance with the Declaration of Helsinki and Directive for Good Clinical Practice (Guideline for Good Clinical Practice) issued by the International Conference on Harmonization. It is designed to comply with the Guidance for Industry (Statistical Approaches to Establishing Bioequivalence) Industry Guide issued by the US FDA (US Department of Health and Human Services, Food and Drug Administration) (35), and the Note for Guidance on the Investigation of Bioavailability and Bioequivalence (Note for Guidance on the Investigation of Bioavailability and Bioequivalence) by the European EMA (The European Medicines Agency) (36).

[0088] The reference formulation of CsA used was Sandimmune® Injection (Novartis Pharma Stein AG, Switzerland, 50 mg/mL CsA, United States Pharmacopeia, USP) containing Cremophor® EL (each 1 mL of concentrate for infusion was diluted to 20 mL of 0.9% saline solution before use). The test product used was CicloMulsion® (NeuroVive Pharmaceutical AB, Lund, Sweden) 5 mg/mL Cremophor and Ethanol free ready-to-use cyclosporin lipid emulsion from the European Pharmacopoeia / United States Pharmacopoeia. Each mL of lipid emulsion contains 100 mg refined soybean oil, 100 mg medium chain triglycerides, 12 mg egg lecithin, 25 mg glycerol, water, and sodium oleate and sodium hydroxide for pH adjustment. Participants

[0089] Healthy male volunteers and healthy non-pregnant, non-lactating female volunteers between 18 and 55 years of age with a body mass index within the range of 19 to 33 kg/m2 were eligible. Additional inclusion criteria were 60 to 100 kg body mass, normal lead-12 electrocardiogram (ECG) and vital signs, clinically acceptable findings on medical history and physical examination, laboratory results within reference ranges (unless the deviation was considered irrelevant for the purposes of the study), intention to undergo pre-study, interim and post-study physical examinations and laboratory investigations, ability to understand and intention to sign the informed consent statement, and abstinence from tobacco during the study and three months before the study. Female participants of childbearing age underwent a pregnancy test prior to each CsA dose and, if positive, were excluded from the study. During the study period, reliable non-hormonal methods of contraception had to be used.

[0090] Exclusion criteria include evidence of psychiatric disorder, history of or current drug abuse (including alcohol), use of any medication within two weeks prior to first administration of study medication, participation in another study with an experimental drug with administration within twelve weeks prior to the present study, major illness during the last three months, donation or blood loss exceeding 500 mL during the eight weeks prior to the first administration of study drug, testing positive for Hepatitis B or C or HIV, positive urine drug screening, vaccination of any type within four weeks of the first dose or vaccination schedule within three months of the last dose, close family member receiving live vaccine during the study or within three months post-study, and hypotension or hypertension during the screening period.

[0091] The history of any of the following diseases was also a criterion for exclusion from the study: any type of malignancy, immunodeficiency, tendency to recurrent infections, known untreated parasitic infection, allergy to any compound in the reference product and in the product of test, or to egg or soy, any bronchospastic diseases, epilepsy, porphyria, psoriasis, atopic dermatitis, hypercholesterolemia, gout, rheumatoid arthritis or kidney disease.

[0092] Care was taken to include both female and male participants of both the Caucasian and non-Caucasian races. Written informed consent was obtained from all participants prior to study enrollment.

Sampling Period

[0093] Subjects were randomized into two treatment sequences: the test product followed by the reference product or vice versa. There was a washout period stipulated for 14 to 21 days between the first and second treatment periods. Participants reported to the physician the night before treatment for laboratory tests, including blood samples, pregnancy testing, and urine drug screening. Subjects were instructed not to eat any citrus fruits and/or apple or pineapple 72 h prior to initiation of the infusion and, within 24 h, no alcohol or any products containing caffeine were allowed. On the day of the clinic, the only food served before drug administration was a standardized breakfast. Through an indwelling IV cannula, subjects received either 5 mg/kg of CicloMulsion (test) or 5 mg/kg of Sandimmune (reference), infused at a constant rate for 4 h with a syringe pump. The recommended dose for inducing immunosuppression with Sandimmune in clinical practice is 3 to 6 mg/kg/day.

[0094] The same arm was used for administration during both treatment periods. All infusion equipment was compatible with both the reference product and the test product. Through an IV cannula in the contralateral arm, a total of 22 blood samples for CsA analysis were obtained pre-dose and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3 , 3.5, 4, 4.5, 5, 6, 7, 8, 10, 14, 18, 24, 30, 36 and 48 hours after the start of the infusion. All samples were collected into vials containing ethylene diamine tetraacetic acid (EDTA), labeled and stored at -70°C until analysis. The concentration of CsA in whole blood was evaluated with liquid chromatography-mass spectrometry / mass spectrometry (LC-MS/MS). The method has been validated in accordance with current US FDA agency guidelines (37). The lowest limit of quantification (LLOQ) was 39.39 ng/mL and the mean CV was 3.5% for CsA. Complete series of calibration standards and quality controls are included within each operation.

Twenty-four hours after the start of study medication administration, subjects were allowed to leave the clinic, with the proviso that they returned for subsequent blood sample collection. Vital signs were monitored during the infusion and measured 2 h after completion of the infusion and after the last blood sample was drawn. Meals and drinks during clinic days were standardized. Water was allowed ad libitum before and after study medication infusion. Alcohol, caffeine, citrus fruits, apples and pineapples were not allowed until the last blood sample was taken in each treatment period.

Tolerability Assessment

[0096] Each subject was carefully monitored for adverse events (AEs) during the infusion, and was asked on the day of the study for any symptoms of similar events. Adverse events were graded as mild, moderate or severe according to the following definitions: Mild: Not causing limitation of usual activities; the subject may experience slight discomfort.

[0097] Moderate: Causing some limitation of usual activities; the subject may experience uncomfortable discomfort.

[0098] Severe: Causing inability to perform usual activities; the subject may experience intolerable discomfort or pain.

[0099] The investigator considered each adverse event with respect to causality to the administered medicinal product as "certain," "probable," "possible," "unlikely," "not related" or "not evaluable." Each adverse event was coded according to the Medical Dictionary for Regulatory Activities (MedDRA) and reported according to strict criteria.

[00100] The proportions of global adverse events and adverse events by organ class were compared between CicloMulsion and Sandimmune using a 95% confidence interval (CI) for the difference between paired proportions and p-values of the McNemar test.

Pharmacokinetic and Bioequivalence Assessment

[00101] Pharmacokinetic variables for CsA were calculated using non-compartmental methods using WinNonlin® Professional Version 5.2 (Pharsight Corporation, Mountain View CA, USA). Statistical analysis was performed using the SAS® Software Version 9.1 program (SAS institute, Cary, NC, USA). All values below the LLOQ before the first positive sample were replaced with zero. For pharmacokinetic evaluation, endpoint values below the LLOQ were ignored.

[00102] CicloMulsion was compared with Sandimmune with respect to a series of pharmacokinetic variables using analysis of variance (ANOVA) with sequence, subject within sequence, product and period effects on log-transformed data. The parameters compared were Area Under the CsA time-concentration curves for hour 0 to the time of the last quantifiable concentration (AUC0-t), hour 0 h extrapolated to infinity (AUC0-w), hour 4 h extrapolated to infinite (AUC4-w), time 0 to 4 h (AUC0-4) and hour 4 to last quantifiable concentration (AUC4-t), maximum blood CsA concentration (Cmax), apparent terminal CsA half-life (t^ .z), blood CsA clearance (CL) and mean length of stay (MRT). Cmax, AUC0-w, AUC0-t and AUC4-w were considered as primary variables and the rest as secondary. Point estimates and 90% confidence intervals for the CicloMulsion / Sandimmune geometric mean ratios of all variables were calculated. The two products were considered bioequivalent if the 90% confidence interval for the primary variables was within the limits of 0.8 and 1.25.

[00103] Based on the FDA and EMA recommended bioequivalence range of 0.80 to 1.25 for Cmax, AUC0-t and AUC0-w, an estimated within-subject coefficient of variation (CV) of 35% was required , and a mean "test/reference" ratio between 0.95 and 1.05, 52 subjects were required to obtain 80% potency at an alpha level of 0.05 to demonstrate bioequivalence (38).

Modifications to Study Planning

[00104] The initial study design did not include any premedication, however, due to a surprisingly high incidence of Serious Adverse Events (SAEs) for the reference product, the remainder of the study was performed with premedication. An amendment to the study protocol was written and approved by the ethics committees named above. For consistency reasons, premedication was used both before CicloMulsion and before Sandimmune even though the adverse events that triggered the onset of premedication were observed after Sandimmune administration. Thirteen subjects completed both treatment periods without premedication. Eighteen subjects received the test product in the first treatment period without premedication, and received the reference product with premedication in the second treatment period. The remainder (21 subjects) received premedication during both treatment periods, and therefore with both the test product and the reference product.

[00105] Premedication consisted of a 50 mg capsule of diphenhydramine orally 1 h before the start of test or reference drug infusion, 10 mg dexamethasone by slow IV injection, and 50 mg IV infusion of ranitidine for 5 minutes approximately 30 minutes prior to each dose of study drug. The study was suspended for the protocol amendments to be approved. This caused the washout period for the 18 subjects who received the test product in the first treatment period without premedication and the reference product with premedication in the second treatment period to be extended to more than six weeks. None of the premedications are known to alter the pharmacokinetic properties of CsA or affect the bioanalytical test.

Results

[00106] Sixty-five volunteers were enrolled in the study and randomized to a treatment sequence. Two subjects were withdrawn prior to the first dosing due to illness, and eleven more were withdrawn prior to completion. Of these eleven, three were due to withdrawal of consent, two due to the decision of the researcher/sponsor (uncertainty of the dose received due to problems with the infusion), and six due to adverse events. None of these individuals were included in the bioequivalence analysis. Fifty-two participants completed the study and were included in the pharmacokinetic evaluation. Demographic data of individuals who completed the study are shown in Table 1. Table 1: Demographics of individuals included in the pharmacokinetic study

[00107] Sixty-three participants received at least one dose of study medication and were included in the global tolerability assessment. Due to an unexpectedly high number of serious adverse reactions to Sandimmune, the study protocol was changed and premedication was introduced as described above. Statistical analysis of the incidence of adverse events was performed only including the thirteen participants who received both the test and reference drug without premedication. Evaluation of Pharmacokinetics and Bioequivalence

[00108] For each time point, arithmetic means with standard deviation were calculated for the whole blood CsA concentrations for the test drug CicloMulsion and for the reference drug Sandimmune. A graphical presentation of CsA concentrations over time is provided in Figure 1. In Figures 2A and B, time curves by concentration are presented separately for individuals receiving and not receiving concomitant premedication. The pharmacokinetic parameters are shown in Table 2. Table 2: Ciclosporin pharmacokinetic parameters in subjects treated with CicloMulsion or Sandimmune as a single intravenous infusion over 4 h at a dose of 5 mg/kg. Arithmetic means, standard deviation (SD) and coefficient of variation (CV).

[00109] The portion of AUC0-^ that was extrapolated was approximately 5% for both study medications, indicating that a reliable estimate of AUC0-^ was obtained. There were no values below the LLOQ present among positive samples.

[00110] Statistical analysis of bioequivalence after dosing with CicloMulsion or Sandimmune is presented in Table 3. The point estimates of the CicloMulsion / Sandimmune geometric mean ratios (90% CI) of the primary variables Cmax and AUC0-t for CsA were of 0.95 (0.92 to 0.97) and 0.90 (0.88 to 0.92), respectively, and AUC0-^ and AUC4-^ for CsA were 0.90 (0.88 to 0 .92) and 0.87 (0.84 to 0.90), respectively. Thus, the 90% confidence interval of all primary variables was within the acceptable range for bioequivalence of 0.80 to 1.25. The 90% confidence interval for all secondary variables also meet the criteria for bioequivalence.Table 3: Bioequivalence assessment of whole blood Ciclosporin exposure after single dose dosing of CicloMulsion (test) and Sandimmune Injection (reference) . Geometric means of log-transformed data, standard deviation (SD), point estimates, and 90% Confidence Interval (CI) of the CicloMulsion / Sandimmune ration Eco-efficient intra-subject variation (CV) injection.

tolerability

[00111] Of 63 subjects, 55 reported one or more adverse events. Adverse events graded at least “possibly” related to study medication were predominantly reported by the medDRA-encoded organ in the Nervous System Disorders and Vascular Disorders classes (Table 4). Nervous system disorders included headache, burning sensation, paresthesia, vertigo and sensory loss. Vascular disorders included hot flash events and orthostatic hypotension. When subjects received Sandimmune without premedication, 84% (16/19) experienced adverse events rated at least possibly related to study medication, compared with 64% (21/33) when they received CicloMulsion. With premedication, the numbers were 76% (31/41) for Sandimmune and 67% (16/24) for CicloMulsion. Proportional analysis of adverse events was performed for subjects treated with both CicloMulsion and Sandimmune without premedication. There was a significantly higher proportion of total adverse events (p=0.003) and vascular disorders (p=0.03) when subjects were treated with Sandimmune. There were no other significantly different proportions between the two formulations.Table 4: Summary of all adverse events at least possibly related to study medication. Number of patients (n) reporting adverse events and incidence in %. Presented with and without premedication for CicloMulsion and Sandimmune Injection.

[00112] Two serious adverse events were recorded. The first affected a 21-year-old male, who in the first treatment period was randomized to receive the reference medication, Sandimmune. Directly after starting the infusion he experienced difficulty breathing and a flushing sensation. The infusion was stopped, treatment with 40% oxygen was started using a Hudson mask, and the individual received 25 mg promethazine and 200 mg hydrocortisone IV. Due to continuous dyspnea, nebulized albuterol inhalation was initiated. Thirty-seven after starting the infusion, the event was considered resolved and oxygen therapy was discontinued. On the day of follow-up, he left the clinic but reported feeling tired for another three days. The event was diagnosed as an anaphylactoid reaction.

[00113] The second of two reported serious adverse events concerned a 42-year-old male who also received the reference drug Sandimmune in the first treatment period. Eleven minutes after the infusion of the medication was started, he developed a cough, facial flushing, and dyspnea. Thoracic auscultation revealed wheezing sounds and peripheral blood oxygen saturation (SpO2) measured by pulse oximetry was 95%. His condition rapidly deteriorated, the subject became pale and sweaty, and a systolic/diastolic blood pressure of 75/42 mmHg was recorded. The infusion was stopped and therapy with 40% oxygen was started using a Hudson mask and IV infusion with Ringer's lactate. Blood pressure was not increasing and SpO2 dropped to 91% after a couple of minutes; the subject was treated with 0.5 mg adrenaline and 25 mg promethazine intramuscularly and 5 mg salbutamol as nebulized inhalation. A 200 mg hydrocortisone infusion was also started. Five minutes after the first medical intervention his blood pressure was normalized; within one hour all drug treatment can be discontinued. After 24 hours, treatment with IV fluids was discontinued and the subject was allowed to leave the clinic. He reported feeling fatigued for another five days but was able to perform all normal activities. The event was diagnosed as an anaphylactic reaction. Both subjects were excluded from the remainder of the study.

[00114] Due to the events described above, as well as an allergic reaction considered moderate and possibly related to Sandimmune that also caused the subject's withdrawal, the study was suspended and an amendment to the study protocol was made with the addition of premedication as per described above.

[00115] Three other subjects did not complete the study due to adverse events. These were all moderate and possibly or probably related to study medication. Of these, two participants received the test product with premedication and one the reference product with premedication. None of the subjects receiving the test drug CicloMulsion without premedication were withdrawn due to adverse events.

[00116] A summary of all adverse events at least possibly related to study medication is presented in Table 4. There were no clinically significant or consistent changes in laboratory values or ECG findings due to administration of CsA. Vital signs and clinical findings remained essentially unchanged throughout the study for all participants with the exception of the two serious adverse events described above.

Discussion

[00117] After a single IV dose of 5 mg/kg, CicloMulsion and Sandimmune met the conventional criteria for bioequivalence. CicloMulsion was the best tolerated of the two CsA formulations.

[00118] Three patients were excluded from the study due to adverse Sandimmune reactions, therefore the study design was changed after a decision by the local authorities. Due to this high incidence of serious adverse events in individuals receiving Sandimmune, premedication was introduced. The pharmacokinetic parameters calculated between subjects who received and did not receive premedication were similar, and bioequivalence with a low intra-subject CV (for most variables below 10%) can be established including premedicated and non-premedicated subjects. medicated. Therefore, protocol changes and the introduction of premedication did not affect the pharmacokinetic profile of CsA.

[00119] In clinical practice, most IV drugs containing CrEL (such as the anticancer agent Paclitaxel) are administered with premedication due to the known risk of hypersensitivity reactions to CrEL or, in the case of Sandimmune, it is often administered as part of a combined corticosteroid regimen. It is reasonable to assume that the actual incidence of adverse Sandimmune reactions is obscured by the protective effect of corticosteroids. A number of reports of reactions to CrEL have been explained by improper dilution of the concentrate for Sandimmune Injection. CrEL has a higher specific gravity than water and a high viscosity and, unless properly mixed, will not be evenly partitioned in the infusion bottle. CrEL and CsA concentrations up to nine times higher than the intended dose have been reported during the first 10 minutes of infusion when improperly mixed (15, 27, 28). With a stable, ready-to-use preparation without the need for dilution, this would not be a problem.

[00120] When Althesin® was withdrawn in the late eighties due to severe side effects due to CrEL, some authors claimed that CrEL should not be used as a solvent for future drugs (39). To date, CsA for IV administration has not been available with any other emulsifying excipient; this is surprising considering the extensive literature reporting serious or even fatal CrEL-related reactions (27, 40)

[00121] There are several previous studies comparing the pharmacokinetics of CsA formulations administered orally, but few for IV administration. In its oral form, CsA has often been found to have wide variation in bioavailability among individuals (41-45) due to a number of factors, such as gastric emptying rate, biliary, pancreatic and intestinal secretion rate, polymorphism in liver enzymes. cytochrome P4503A, and different p-glycoprotein haplotypes expressed in the intestinal wall mucosa. Intra-individual differences are generally explained by dietary factors and clinical condition (42-44, 46, 47).

[00122] There are examples of IV drugs in which the pharmacokinetic profiles were significantly altered when a lipid emulsion was introduced as an emulsifier; known examples are Propofol and Diazepam (17, 48-50). Taking this into account, the estimated intra-individual coefficient of variation (CV) was estimated to be 35% when designing the study. It turned out to be below 10% for all primary variables, supporting the view that most of the variation in intra-subject bioavailability is due to factors that are essentially relevant to CsA formulations administered orally. The intersubject CV% in this study was between 10% and 20% for Cmax, AUC0-t, and AUC0-^ , consistent with previous reports for CsA IV (41, 51, 52). The ongoing debate over the interchangeability of branded and generic formulations of orally administered CsA for reasons of variability should therefore not be extrapolated to IV formulations of the drug.

[00123] From the study, we conclude that CicloMulsion is bioequivalent to Sandimmune and that the ready-to-use, ethanol and CrEl-free formulation IV CsA CicloMulsion is better tolerated.

[00124] 1. Griveas I, Visvardis G, Papadopoulou D, Nakopolou L, Karanikas E, Gogos K et al. Effect of cyclosporine therapy with low doses of corticosteroids on idiopathic nephrotic syndrome. Artifico Organs; 34(3): 234-237.

[00125] 2. Warren RB, Griffiths CE. Systemic therapies for psoriasis:methotrexate, retinoids, and cyclosporine. Clin Dermatol 2008; 26(5): 438-447.

[00126] 3. Fritsche L, Dragun D, Neumayer HH, Budde K. Impact of cyclosporine on the development of immunosuppressive therapy. Transplant Proc 2004; 36(2 Suppl): 130S-134S.

[00127] 4. Naganuma M, Fujii T, Watanabe M. The use of traditional and newer calcineurin inhibitors in inflammatory bowel disease. J Gastroenterol.

[00128] 5. Hijnen DJ, ten Berge O, Timmer-de Mik L, Bruijnzeel-Koomen CA, de Bruin-Weller MS. Efficacy and safety of long-term treatment with cyclosporin A for atopic dermatitis. J Eur Acad Dermatol Venereol 2007; 21(1): 85-89.

[00129] 6. Kacmaz RO, Kempen JH, Newcomb C, Daniel E, Gangaputra S, Nussenblatt RB et al. Cyclosporine for ocular inflammatory diseases. Ophthalmology; 117(3): 576-584.

[00130] 7. Storb R, Antin JH, Cutler C. Should methotrexate pluscalcineurin inhibitors be considered standard of care for prophylaxis of acute graft-versus-host disease? Biol Blood Marrow Transplant; 16(1 Suppl): S18-27.

[00131] 8. Dorr RT. Pharmacology and toxicology of Cremophor ELdiluent. Ann Pharmacother 1994; 28(5 Suppl): S11-14.

[00132] 9. Chapuis B, Helg C, Jeannet M, Zulian G, Huber P, Gumovski P. Anaphylactic reaction to intravenous cyclosporine. N Engl J Med 1985; 312(19): 1259.

[00133] 10. Kahan BD, Wideman CA, Flechner S, Vanburen CT. Anaphylactic Reaction to Intravenous Cyclosporin. Lancet 1984; 1(8367): 5252.

[00134] 11. Leunissen KML, Waterval PWG, Vanhooff JP. Anaphylactic Reaction to Intravenous Cyclosporin. Lancet 1985; 1(8429): 636-636.

[00135] 12. Friedman LS, Dienstag JL, Nelson PW, Russell PS, Cosimi AB. Anaphylactic Reaction and Cardiopulmonary Arrest Following Intravenous Cyclosporine. Am J Med 1985; 78(2): 343-345.

[00136] 13. Volcheck GW, Van Dellen RG. Anaphylaxis to intravenous cyclosporine and tolerance to oral cyclosporine: case report and review. Ann Allergy Asthma Immunol 1998; 80(2): 159-163.

[00137] 14. van Hooff JP, Bessems P, Beuman GH, Leunissen KM. Absence of allergic reaction to cyclosporin capsules in patient allergic to standard oral and intravenous solution of cyclosporin. Lancet 1987; 2(8573): 1456.

[00138] 15. Mackie FE, Umetsu D, Salvatierra O, Sarwal MM. Pulmonary capillary leak syndrome with intravenous cyclosporin A in pediatric renal transplantation. Pediatric Transplant 2000; 4(1): 35-38.

[00139] 16. Howrie DL, Ptachcinski RJ, Griffith BP, Hardesty RJ, Rosenthal JT, Burckart GJ et al. Anaphylactoid reactions associated with parenteral cyclosporine use: possible role of Cremophor EL. Drug Intell Clin Pharm 1985; 19(6): 425-427.

[00140] 17. Baker MT, Naguib M. Propofol: the challenges of formulation. Anesthesiology 2005; 103(4): 860-876.

[00141] 18. Dye D, Watkins J. Suspected anaphylactic reaction to Cremophor EL. Br Med J 1980; 280(6228): 1353.

[00142] 19. Huttel MS, Schou Olesen A, Stoffersen E. Complement-mediated reactions to diazepam with Cremophor as solvent (Stesolid MR). Br J Anaesth 1980; 52(1): 77-79.

[00143] 20. Moneret-Vautrin DA, Laxenaire MC, Viry-Babel F.Anaphylaxis caused by anti-cremophor EL IgG STS antibodies in a case of reaction to althesin. Br J Anaesth 1983; 55(5): 469-471.

[00144] 21. Briggs LP, Clarke RS, Watkins J. An adverse reaction to the administration of disoprofol (Diprivan). Anaesthesia 1982; 37(11): 1099-1101.

[00145] 22. Thiel G, Hermle M, Brunner FP. Acutely impaired function during intravenous administration of cyclosporine A: a cremophore side-effect. Clin Nephrol 1986; 25 Suppl 1: S40-42.

[00146] 23. Verani R. Cyclosporine nephrotoxicity in the Fischer rat. Clin Nephrol 1986; 25 Suppl 1: S9-13.

[00147] 24. Windebank AJ, Blexrud MD, by Groen PC. Potentialneurotoxicity of the solvent vehicle for cyclosporine. J Pharmacol Exp Ther 1994; 268(2): 1051-1056.

[00148] 25. Sanchez H, Bigard X, Veksler V, Mettauer B, Lampert E, Lonsdorfer J et al. Immunosuppressive treatment affects cardiac and skeletal muscle mitochondria by the toxic effect of vehicle. J Mol Cell Cardiol 2000; 32(2): 323-331.

[00149] 26. Bowers VD, Locker S, Ames S, Jennings W, Corry RJ. The hemodynamic effects of Cremophor-EL. Transplantation 1991; 51(4): 847-850.

27. Theis JG, Liau-Chu M, Chan HS, Doyle J, Greenberg ML, Koren G. Anaphylactoid reactions in children receiving high-dose intravenous cyclosporine for reversal of tumor resistance: the causative role of improper dissolution of Cremophor EL . J Clin Oncol 1995; 13(10): 2508-2516.

[00151] 28. Liau-Chu M, Theis JG, Koren G. Mechanism of Anaphylactoid Reactions: preparing a high-dose intravenous cyclosporine leads to a bolus infusion of Cremophor EL and cyclosporine. Ann Pharmacother 1997; 31(11): 1287-1291.

[00152] 29. Gotardo MA, Monteiro M. Migration of diethylhexylphthalate from PVC bags into intravenous cyclosporine solutions. J Pharm Biomed Anal 2005; 38(4): 709-713.

[00153] 30. Venkataramanan R, Burckart GJ, Ptachcinski RJ, Blaha R, Logue LW, Bahnson A et al. Leaching of diethylhexyl phthalate from polyvinyl chloride bags into intravenous cyclosporine solution. Am J Hosp Pharm 1986; 43(11): 2800-2802.

31. Trapani G, Altomare C, Lyso G, Sanna E, Biggio G. Propofol in anesthesia. Mechanism of action, structure-activity relationships, and drug delivery. Curr Med Chem 2000; 7(2): 249-271.

[00155] 32. Mattila MA, Rossi ML, Ruoppi MK, Korhonen M, LarniHM, Kortelainen S. Reduction of venous sequelae of i.v. diazepam with a fat emulsion as solvent. Br J Anaesth 1981; 53(12): 1265-1268.

[00156] 33. Micha JP, Goldstein BH, Birk CL, Rettenmaier MA, Brown JV, 3rd. Abraxane in the treatment of ovarian cancer: the absence of hypersensitivity reactions. Gynecol Oncol 2006; 100(2): 437-438.

[00157] 34. Morgan M, Whitwam JG. Althesin. Anaesthesia 1985;40(2):121-123.

[00158] 35. Guidance for Industry: Statistical Approaches to Establishing Bioequivalence. United States Department of Health and Human Services, Food and Drug Administration (FDA) Rockville, MD, USA. 2001

[00159] 36. Note for Guidance on the Investigation of Bioavailability and Bioequivalence. European Medicines Agency (EMA) CPMP/EWP/QWP/1401/98. London, Great Britain. 2001

[00160] 37. Guidance for Industry: Bioanalytical Method Validation. United States Department of Health and Human Services, Food and Drug Administration (FDA) Rockville, MD, USA. 2001

[00161] 38. Diletti E, Hauschke D, Steinijans VW. Sample size determination for bioequivalence assessment by means of confidence intervals. Int J Clin Pharmacol Ther Toxicol 1992; 30 Suppl 1: S51-58.

[00162] 39. Kanto JH. Propofol, the newest induction agent of anesthesia. Int J Clin Pharmacol Ther Toxicol 1988; 26(1): 41-57.

40. Kloover JS, den Bakker MA, Gelderblom H, vanMeerbeeck JP. Fatal outcome of a hypersensitivity reaction to paclitaxel: a critical review of premedication regimens. Br J Cancer 2004; 90(2): 304-305.

[00164] 41. Gupta SK, Manfro RC, Tomlanovich SJ, GambertoglioJG, Garovoy MR, Benet LZ. Effect of food on the pharmacokinetics of cyclosporine in healthy subjects following oral and intravenous administration. J Clin Pharmacol 1990; 30(7): 643-653.

[00165] 42. Christians U, Klawitter J, Clavijo CF. Bioequivalencetesting of immunosuppressants: concepts and misconceptions. Kidney Int Suppl (115): S1-7.

[00166] 43. Christians U, First MR, Benet LZ. Recommendations forbioequivalence testing of cyclosporine generics revisited. Ther Drug Monit 2000; 22(3): 330-345.

44. Kahan BD, Dunn J, Fitts C, Van Buren D, Wombolt D, Pollak R et al. Reduced inter- and intrasubject variability in cyclosporine pharmacokinetics in renal transplant recipients treated with a microemulsion formulation in conjunction with fasting, low-fat meals, or high-fat meals. Transplantation 1995; 59(4): 505-511.

45. Kees F, Bucher M, Schweda F, Gschaidmeier H, FaerberL, Seifert R. Neoimmun versus Neoral: a bioequivalence study in healthy volunteers and influence of a fat-rich meal on the bioavailability of Neoimmun. Naunyn Schmiedebergs Arch Pharmacol 2007; 375(6): 393-399.

46. Christians U, Schmitz V, Haschke M. Functional interactions between P-glycoprotein and CYP3A in drug metabolism. Expert Opin Drug Metab Toxicol 2005; 1(4): 641-654.

[00170] 47. Kovarik JM, Mueller EA, van Bree JB, Fluckiger SS, Lange H, Schmidt B et al. Cyclosporine pharmacokinetics and variability from a microemulsion formulation--a multicenter investigation in kidney transplant patients. Transplantation 1994; 58(6): 658-663.

[00171] 48. Dutta S, Matsumoto Y, Ebling WF. Propofolpharmacokinetics and pharmacodynamics extracted from a cremophor EL formulation. J Pharm Sci 1997; 86(8): 967-969.

49. Fee JP, Dundee JW, Collier PS, McClean E. Bioavailability of intravenous diazepam. Lancet 1984; 2(8406): 813.

[00173] 50. Fee JP, Collier PS, Dundee JW. Bioavailability of three formulations of intravenous diazepam. Acta Anaesthesiol Scand 1986; 30(4): 337-340.

51. Lee M, Min DI, Ku YM, Flanigan M. Effect of grapefruit juice on pharmacokinetics of microemulsion cyclosporine in African American subjects compared with Caucasian subjects: does ethnic difference matter? J Clin Pharmacol 2001; 41(3): 317-323.

52. Min DI, Lee M, Ku YM, Flanigan M. Gender-dependentracial difference in disposition of cyclosporine among healthy African American and white volunteers. Clin Pharmacol Ther 2000; 68(5): 478-486.

Claims (15)

1. Cyclosporine emulsion, characterized by the fact that it has the following composition: Ingredient Concentration g/LC Cyclosporin 5Egg lecithin 12Glycerol (water-free) 25Sodium oleate 0.3Sodium hydroxide 1 M qSoybean oil (100long chain triglyceride ) MCT oil (100 medium chain triglyceride) Water for injection Ad 1 Lem that the emulsion is isoosmolar and has an osmolarity from 280 to 305 mosm/l. 2. Cyclosporin emulsion according to claim 1, characterized in that it has an osmolarity of from about 295 to about 300 mosm/l. 3. Cyclosporine emulsion according to claim 1 or 2, characterized in that it has a shelf life of at least 30 months when stored below 25 °C and protected from light. 4. Cyclosporine emulsion according to claim 3, characterized in that the shelf life is 36 months. 5. Emulsion according to any one of claims 1 to 4, characterized in that the pH of the emulsion is from about 6 to about 8.8. 6. Emulsion according to any one of claims 1 to 5, characterized in that it is for use in immunosuppression or other conditions susceptible to treatment with cyclosporin, in which the administration of the emulsion to a subject in need of it is done without any premedication with corticosteroids. 7. Emulsion according to any one of claims 1 to 6, characterized in that the MCT oil comprises coconut oil or palm oil or a combination thereof. 8. Emulsion according to any one of claims 1 to 7, characterized in that the MCT oil is coconut oil, palm oil or a combination thereof. 9. Use of a cyclosporine emulsion, as defined in any one of claims 1 to 8, characterized in that it is for the preparation of a composition for reducing the rate of anaphylactic reactions in patients undergoing treatment with cyclosporine, and/or for use in immunosuppressive treatment with cyclosporine, and/or for use in cardiac reperfusion injury (cardioprotection), and/or for use in preventing brain injury (neuroprotection), and/or for use in preventing reperfusion injury (organ protection ), and/or for use in renal reperfusion injury (nephroprotection), and/or for use in hepatic reperfusion injury (hepatoprotection). 10. Cyclosporine emulsion according to any one of claims 1 to 9, characterized in that it is for use in immunosuppressive treatment with cyclosporine. 11. Cyclosporine emulsion according to any one of claims 1 to 9, characterized in that it is for use in cardiac reperfusion injury (cardioprotection). 12. Cyclosporine emulsion, according to any one of claims 1 to 9, characterized in that it is for use in preventing brain damage (neuroprotection). 13. Cyclosporine emulsion, according to any one of claims 1 to 9, characterized in that it is for use for preventing reperfusion injury (organ protection). 14. Cyclosporine emulsion according to any one of claims 1 to 9, characterized in that it is for use in renal reperfusion injury (nephroprotection). 15. Cyclosporine emulsion according to any one of claims 1 to 9, characterized in that it is for use in liver reperfusion injury (hepatoprotection).

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