EA046086B1 - METHOD FOR OBTAINING DISPERSIONS CONTAINING INHALATION IMMUNOSUPPRESSIVE ACTIVE INGREDIENTS - Google Patents

Description

Technical field

The present invention relates to a method for preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form in an aqueous liquid carrier. In particular, the present invention relates to a method for preparing dispersions containing cyclosporine A in liposomally solubilized form. In addition, the present invention relates to a method for preparing lyophilized pharmaceutical compositions containing an inhaled immunosuppressive active ingredient, as well as lyophilized pharmaceutical compositions that can be obtained by this method.

State of the art

Cyclosporine (the terms Cyclosporin A, CsA and Cyclosporine are used interchangeably herein) is a cyclic oligopeptide with immunosuppressive and calcineurin inhibitory activity. Cyclosporine is characterized by a selective and reversible mechanism of immunosuppression by blocking the activation of T lymphocytes as a result of the production of certain cytokines involved in the regulation of these T cells. This mechanism includes, in particular, inhibition of the synthesis of interleukin-2, which also suppresses the proliferation of cytotoxic T-lymphocytes, which are responsible, for example, for the rejection of foreign tissue. The mechanism of action of cyclosporine inside the cell is the binding of so-called cyclophilins or immunophilins, which belong to a family of proteins that bind cyclosporine with high affinity. Then the resulting complex of cyclosporine and cyclophilin blocks serine-threonine phosphatase-calcineurin. Its level of activity, in turn, allows it to regulate the activation of transcription factors such as NF-KappaB or NFATp/c, which play an important role in the activation of various cytokine genes, including interleukin-2. This results in the blocking of immunocompetent lymphocytes in the G0 or G1 phase of the cell cycle, since these proteins necessary for cell division, such as interleukin-2, are no longer produced. Helper T cells, which enhance the activity of cytotoxic T cells responsible for rejection, are the preferred site of action of cyclosporine. In addition, cyclosporine inhibits the synthesis and release of additional lymphokines responsible for the proliferation of mature cytotoxic T lymphocytes and other lymphocyte functions. The ability of cyclosporine to block interleukin-2 is a key factor in its clinical effectiveness: transplant recipients who are well tolerated have low levels of interleukin-2 production. In contrast, patients with clear signs of rejection reactions demonstrate a lack of inhibition of interleukin-2 production.

The first and currently only cyclosporine on the market (in the 1980s) was cyclosporine A (CsA). CsA in chemical structure is cyclo[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-ocmenoyl]-L-2-aminobutyryl-N-methylglycyl- Nmethyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-O-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-Nmethyl-L-valyl]. The availability of CsA marked the beginning of a new era in transplantation, since with its help it is possible to significantly increase the proportion of transplanted organs that remain functional for a long period of time. The first cyclosporine product (Sandimmun® from Sandoz) has previously demonstrated an approximately 2-fold increase in kidney transplant success rates. A new oral cyclosporine formulation (Neoral® from Sandoz, later Novartis) with higher and more consistent bioavailability, introduced in the 1990s, allowed for more precise dosing and further increased surgical success rates. Even with several new developments of active agents, CsA still remains a widely used agent in organ transplantation.

Currently, lung transplantation can only be successfully performed in patients receiving CsA. Since the introduction of this active agent into clinical practice, the number of lung transplants in all countries of the world has increased sharply. This applies to both single and double lung transplants. Lung transplantation is generally indicated for patients with end-stage lung disease when medical treatment has failed and life expectancy is short due to the disease. Single lung transplantation is indicated, for example, in the case of some forms of emphysema and fibrosis, such as idiopathic pulmonary fibrosis. Double lung transplantation is performed in the case of cystic fibrosis (cystic fibrosis), primary pulmonary hypertension, emphysema with general failure, frequent severe infections, as well as idiopathic pulmonary fibrosis with complications due to recurrent infections. With a successful lung transplant, patients' quality of life can be improved to near normal levels. However, unlike heart, kidney and liver transplants, life expectancy after lung transplant is still relatively low, averaging only 5 years. This may be due to, among other things, the lack of an effective route for administering the active agent cyclosporine to all patients due to systemic side effects such as renal dysfunction, elevated serum creatinine and urea levels, renal damage with structural changes such as interstitial fibrosis, elevated bilirubin levels and serum liver enzymes, hyper

- 1 046086 trichosis, tremor, fatigue, headache, hypertrophic gingivitis, gastrointestinal complaints such as anorexia, abdominal pain, nausea, vomiting, diarrhea, gastritis, gastroenteritis, paresthesia, burning in the arms and legs, arterial hypertension, increased levels of fats in the blood, acne, itching, allergic skin reactions, hyperglycemia, anemia, hyperuricemia, gout, increased body weight, edema, stomach ulcers, cramps, menstrual irregularities, hyperkalemia, hypomagnesemia, flushing, erythema, itching, muscle cramps, muscle pain, myopathy, etc.

Thus, it would be desirable that, for example, after lung transplantation or in the case of certain other indications, it would be possible to administer CsA in a targeted and tissue-specific manner to ensure only modest systemic bioavailability of the active agent and to minimize the effect of the active agent on healthy tissue.

A suitable dosage form can also be used for the treatment and prevention of diseases such as asthma, idiopathic pulmonary fibrosis, sarcoidosis, alveolitis and parenchymal lung disease (see Drugs for the treatment of respiratory diseases, edited by Domenico Spina, Clive p. Page et. al., Cambridge University Press, 2003, ISBN: 0521773210). New therapeutic aspects also provide the possibility of local treatment of possible autoimmune diseases such as neurodermatitis, psoriasis, nonspecific eczema, proliferations or mutations in the skin, as well as treatment after skin grafting. An area of interest is ophthalmology, for example for the treatment of post-corneal transplantation, keratoconjunctivitis or other infectious eye diseases that have an insufficient response to anti-inflammatory therapy, such as steroids. This form is also suitable for the treatment of keratitis in animals, such as dogs.

Topical administration of cyclosporine has previously been attempted, for example in the form of oil eye drops at a concentration of 1 and 2% (dosage form according to the German Federal Medicines Act using refined peanut oil as a solubilizer) or in aerosol form. However, this method of administration has generally not been effective, mainly due to the very low solubility of the active agent in water, which makes the effective use of the drug very difficult. Thus, some solubilization excipients that can be used orally are not suitable for pulmonary use due to intolerance. For example, Sandimmun® Optoral capsules (Novartis), which contain cyclosporine A, formulate a microemulsion concentrate with ethanol, propylene glycol and significant amounts of surfactants and therefore form a dosage form that can cause severe toxic effects if inhaled. Also, Sandimmun® (Novartis) infusion solution concentrate, which is intended for infusion, is not suitable for inhalation: the only excipients in the drug are ethanol and poly(oxyethylene)-40-castor oil. The drug can only be used for infusion, since it is already diluted with 0.9% sodium chloride solution or 5% glucose solution in a ratio of 1:20 to 1:100. The results are large volumes of solutions that are used by infusion rather than inhalation.

WO 2007/065588 A1 describes liquid pharmaceutical compositions containing a therapeutically effective dose of cyclosporine; aqueous liquid carrier; a first solubilizing agent selected from the group of phospholipids; and a second solubilizing agent selected from the group of nonionic surfactants. The described composition is suitable for oral, parenteral, nasal, mucosal, topical and especially pulmonary administration in aerosol form.

WO 2016/146645 A1 describes liposomal dosage forms of cyclosporine, which preferably contain single-layer liposomes. The liposomes preferably have an average diameter of no more than about 100 nm as z-average when analyzed by photon correlation spectroscopy and a polydispersity coefficient of no more than about 0.5 when analyzed by photon correlation spectroscopy.

The proposed composition can be presented in solid form for dilution with an aqueous solvent immediately before inhalation. The solid composition can be prepared by any method suitable for removing solvent from a liquid composition. Preferred examples of methods for preparing such a solid composition include lyophilization and spray drying. To protect the active ingredient during the drying process, it may be advantageous to use lyoprotective and/or bulking agents such as sugar or sugar alcohol, in particular sucrose, fructose, glucose, trehalose, mannitol, sorbitol, isomalt or xylitol. However, most importantly, the sugar is added to a pre-formulated composition containing CsA encapsulated in liposomes.

Considering the high potencies of many macrocyclic immunosuppressive active ingredients, such as cyclosporine A, tacrolimus, sirolimus and/or everolimus, and the potentially serious adverse side effects that can be observed for these potent compounds when dosed incorrectly, it is important that the content

- 2 046086 these active ingredients in pharmaceutical compositions containing such macrocyclic immunosuppressive compounds could be precisely controlled during the production process. This is necessary to avoid potentially serious side effects, as described above, due to overdose of these compounds or to avoid lack of effectiveness due to insufficient dosage, which can also lead to serious complications such as transplant rejection.

It is therefore extremely important that the content of the macrocyclic immunosuppressive active agent can be determined and controlled at each stage of the manufacturing process of a pharmaceutical composition containing such active ingredients.

However, it is mainly the very low solubility in aqueous solution of many macrocyclic immunosuppressants, especially cyclosporine A and tacrolimus, that is considered a problematic factor. These compounds are usually prepared by fermentation and further purified by crystallization. The physical properties of these highly purified compounds unfortunately tend to change over time, for example through processes of aggregation, agglutination or thickening, which produce larger particles that may have different, often less favorable dispersion or dissolution characteristics. This, particularly when not freshly prepared material is used, may increase the risk of inaccurate concentrations of dispersed or dissolved active ingredients during the manufacturing process and in the finished dosage form, respectively.

It is therefore an object of the present invention to provide an improved method for the preparation of pharmaceutical compositions containing an inhaled immunosuppressive active ingredient, such as cyclosporine A, tacrolimus, sirolimus, everolimus or others, the characteristics of which are substantially independent of the physical properties present in the macrocyclic immunosuppressive active ingredient when delivery and application.

Additional objects of the present invention will be apparent from the description provided, including example implementations and claims.

Brief description of the invention

In a first aspect, the present invention relates to a method for preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form in an aqueous liquid carrier, said method comprising the steps of:

a) providing a mixture containing an inhaled immunosuppressive macrocyclic active ingredient;

a membrane-forming substance selected from the group of phospholipids;

a solubility improver selected from the group of nonionic surfactants;

optionally one or more excipients; and an aqueous liquid carrier;

b) dispersing the mixture in the manner provided in step a) to obtain an intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; And

c) homogenizing the intermediate aqueous dispersion obtained in step b) to form a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form.

In a second aspect, the present invention relates to a method for preparing a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, wherein the lyophilized pharmaceutical composition contains an inhaled immunosuppressive macrocyclic active ingredient in a liposomally solubilized form, and the method includes preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in a liposomal solubilized form. form in an aqueous liquid carrier, according to the method according to the first aspect of the invention; and additionally includes the stage

d) removing the aqueous liquid carrier, at least partially, by lyophilization to obtain a lyophilized pharmaceutical composition.

In a third aspect of the present invention, there is provided a lyophilized pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form for reconstitution in an aqueous liquid carrier, the composition being or can be prepared by the method of the second aspect of the present invention.

In a fourth aspect, the present invention relates to a lyophilized pharmaceutical composition according to the third aspect of the invention for use as a medicament for pulmonary administration.

Detailed Description of the Invention

The terms consist of, consists of and consisting of in this document represent so-called limited expressions, i.e. indicate the presence of only the specified components. The terms contain, contains and containing in this context are so-called open-ended expressions, meaning that one may or may not be present as well.

- 3 046086 or more additional components.

The term active pharmaceutical ingredient (also referred to herein as API) refers to any type of pharmaceutically active compound or derivative useful for preventing, diagnosing, stabilizing, treating, or generally inhibiting the progression of a pathological condition, disorder, or disease.

The term therapeutically effective amount as used herein refers to a dose, concentration, or potency suitable to provide the desired pharmacological effect. In the context of the present invention, the term therapeutically effective also refers to prophylactic activity. The therapeutic dose should be determined depending on the individual case of use. Taking into account the nature and severity of the disease, the route of administration, and the growth and condition of the patient, the therapeutic dose should be determined in a manner known to those skilled in the art.

In the context of the present invention, a pharmaceutical composition is a preparation of at least one API and at least one adjuvant, which, in the simplest case, may be, for example, an aqueous liquid carrier such as water or saline.

Terms in the singular do not exclude the plural; those. Terms in the singular are to be understood to refer to the plural unless the context clearly states or requires otherwise. That is, all references to features or limitations of the present invention in the singular form refer to the corresponding features or limitations in the plural form and vice versa, unless specifically stated otherwise or unless otherwise clearly implied by the context intended. Therefore, terms in the singular form have the same meaning as the terms at least one or as one or more, unless otherwise noted. For example, the term ingredient refers to a mixture of ingredients, etc.

When used in the context of this invention, the term approximately or approximately characterizes the variability acceptable in the pharmaceutical industry and inherent in drug products, for example, variations in content due to manufacturing changes and/or degradation of the product over time. This term allows for any deviations that, for pharmaceutical products, allow the evaluated product to be considered as bioequivalent to the product in question in terms of its declared effectiveness for mammals.

Essentially, approximately, approximately, to a large extent, etc. with respect to a characteristic or value, correspond to the exact characteristic or exact value, as well as any characteristic or value that is generally within the normal range or variability acceptable in the art in question. For example, substantially free of water means that no water is specifically added to the composition, but this does not exclude the presence of residual moisture.

In the context of the present invention, a colloidal aqueous solution preferably means a solution without an organic solvent, consisting of essentially unilamellar liposomes having an average diameter of not more than 100 nm and/or a polydispersity index (PI) of not more than 0.50, in which the active agent. Preferably, water or, more specifically, saline is the only liquid solvent contained in such a preparation. Moreover, it is preferable that such a preparation is an aqueous solution or an aqueous colloidal solution, i.e. single-phase liquid system. Such a system essentially does not contain dispersed particles larger than the colloidal particles. Generally, particles smaller than about 1 μm are considered colloidal particles that do not form a separate phase and do not result in the formation of a physical phase boundary. Sometimes particles slightly larger than 1 µm are also considered colloidal. However, preferably, colloidal aqueous solutions in the context of the present invention are substantially free of particles that are not clearly colloidal, ie, for example, particles having a diameter of 1 μm or more.

According to a first aspect, the present invention provides a method for preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form in an aqueous liquid carrier, comprising the steps of:

a) providing a mixture containing an inhaled immunosuppressive macrocyclic active ingredient;

a membrane-forming substance selected from the group of phospholipids;

a solubility improver selected from the group of nonionic surfactants;

optionally one or more excipients; and an aqueous liquid carrier;

b) dispersing the mixture in the manner provided in step a) to obtain an intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; And

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c) homogenizing the intermediate aqueous dispersion obtained in step b) to form a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form.

The method of this first aspect of the present invention is suitable for preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form. The term dispersion as used herein is to be understood in a broad sense and means, unless otherwise specified, a system in which dispersed particles in the form of a dispersed phase are dispersed in a continuous phase, in particular in a liquid continuous phase, especially in an aqueous solution in the form of a continuous phase. The inhaled immunosuppressive macrocyclic active ingredient used in the context of the present invention is to be interpreted broadly and contains immunosuppressive macrocyclic active ingredients that can be administered to a human or animal, preferably a human or warm-blooded animal, in particular a human, by inhalation, while during such inhalation, the immunosuppressive macrocyclic active ingredient or API is transported to at least a portion or region of the respiratory system of said animal or human, in particular to the lungs of said animal or human.

In the context of the present invention, the immunosuppressive macrocyclic active ingredient may be, for example, cyclosporine A (hereinafter also referred to as CsA), tacrolimus, sirolimus and/or everolimus, in particular cyclosporine A and/or tacrolimus, more particularly cyclosporine A, wherein cyclosporine A in terms of chemical structure is cyclo[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-aminobutyryl-N- methylglycyl-Nmethyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-O-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-Nmethyl-L-leucyl- N-methyl-N-methyl-L-methyl-L-valyl] (CAS number 59865-13-3) and a cyclic peptide with immunosuppressive activity.

In the context of the present invention, cyclosporine A can be used in any capacity or origin that is suitable for the preparation of a pharmaceutical composition. For example, cyclosporine A can be produced by standard fermentation methods and in particular embodiments can be used in any suitable form, for example, any particle size or powder form relative to the process of the present invention.

The method according to the present invention allows for the production of an inhaled immunosuppressive macrocyclic ingredient, for example cyclosporin A, tacrolimus, sirolimus and/or everolimus, or a mixture containing one or more of these compounds, in particular cyclosporin A, in liposomally solubilized form. The term liposomally solubilized form as used herein means that at least one inhalable immunosuppressive macrocyclic ingredient as described above, in particular CsA, is included or incorporated into liposome-forming structures that will be formed by other ingredients, in particular a membrane-forming substance selected from the group of phospholipids , and a solubility improver selected from the group of nonionic surfactants, as described in more detail below. However, liposome-forming structures as defined herein may or may not have a continuous or closed bilayer membrane. In certain embodiments, the liposome-forming structures may be at least partially present in a single-layer form or, preferably, may be predominantly present in a single-layer form. The term monolayer as used herein means that the respective liposome-forming structures comprise only a single layer formed by a single lipid bilayer membrane rather than multiple lipid bilayer membranes in a layered structure.

According to process step a), the present invention provides a mixture containing as a first ingredient at least one inhaled immunosuppressive macrocyclic active ingredient as described above, in particular CsA. As a second ingredient, the mixture obtained in step a) contains a membrane-forming substance selected from the group of phospholipids, or a mixture of two or more different membrane-forming substances selected from the group of phospholipids.

The term membrane-forming substance as used herein means that the substance can form a lipid bilayer membrane by self-assembly in an aqueous liquid carrier, such as water or saline, and/or can form liposomes in an aqueous liquid carrier under conditions or circumstances described in more detail below.

Preferred phospholipids contained in the liposome-forming structures of the present invention are, in particular, mixtures of natural or enriched phospholipids, for example, lecithins, such as the commercially available Phospholipon® G90, 100 or Lipoid 90, S 100. Accordingly, in certain embodiments, the membrane-forming agent a substance selected from the group of phospholipids is a mixture of natural phospholipids.

Phospholipids are amphiphilic lipids that contain phosphorus. Such substances, also known as phosphatides, play an important role in nature, especially as substances

- 5 046086 bilayer-forming components of biological membranes, and in the pharmaceutical industry, where phospholipids, which are chemically derived from phosphatidic acid, are widely used. The latter are (usually doubly) acetylated glyceryl-3-phosphate, in which the fatty acid residues can have different lengths. Phosphatidic acid derivatives are, for example, phosphocholines or phosphatidylcholines, in which the phosphate group is further esterified with choline, as well as phosphatidylethanolamine, phosphatidylinositols, etc. Lecithins are natural mixtures of various phospholipids, which usually contain a large proportion of phosphatidylcholines. Preferred phospholipids according to the present invention are lecithins, as well as pure or enriched phosphatidylcholines, such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

In additional specific embodiments, the membrane-forming substance selected from the group of phospholipids that must be in the mixture in process step a) according to the present invention is lecithin containing unsaturated fatty acid residues. In further specific embodiments, the membrane-forming agent selected from the group of phospholipids is a lecithin selected from the group consisting of soy lecithin, LLipoid 90, Lipoid S75, Lipoid S100, Phospholipon® G90, Phospholipon®100, or similar lecithin. In further specific embodiments, the membrane-forming substance selected from the group of phospholipids is selected from Lipoid S100, Lipoid S75, in particular Lipoid S100.

The third ingredient of the mixture, which is used in process step a) according to the present invention, is a solubility improver selected from the group of non-ionic surfactants, or a mixture of two or more different solubility improvers selected from the group of non-ionic surfactants . Nonionic surfactants contain, like other surfactants, at least one relatively hydrophilic and at least one relatively lipophilic region of the molecule. They are monomeric, low molecular weight nonionic surfactants, as well as nonionic surfactants having an oligomeric or polymeric structure. Examples of suitable nonionic surfactants useful as solubility improvers included in the mixture obtained in step a) according to the present invention include polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, such as, for example, polyoxyethylene sorbitan oleate, sorbitol fatty acid esters, poloxamers, vitamin E-TPGS (D-atocopheryl polyethylene glycol succinate 1000) and tyloxapol.

In specific embodiments, the solubility enhancer selected from the group of nonionic surfactants may be selected from the group of polysorbates and vitamin E-TPGS, preferably selected from the group of polysorbates. In a particularly preferred embodiment, the solubility enhancer selected from the group of nonionic surfactants is polysorbate 80.

As an optional fourth ingredient, the mixture which is obtained in process step a) according to the present invention may optionally contain one or more auxiliary substances. Suitable excipients, as specified herein, are known to those skilled in the art. For example, the mixture obtained in step a) according to the present invention may optionally contain pH adjusting agents to regulate the pH, such as physiologically acceptable bases, acids or salts, optionally in the form of buffer mixtures. In this context, the term physiologically acceptable does not mean that one of the excipients must be tolerated as an independent agent and in undiluted form, which does not apply, for example, to a sodium hydroxide solution, but means that the substance must be tolerated in the concentration in which it is contained in the lyophilized pharmaceutical composition, especially after reconstitution.

Suitable pH adjusting agents or pH buffers can be selected, inter alia, taking into account the intended method of application. Examples of potentially useful excipients of this group include sodium hydroxide solution, basic salts of sodium, calcium or magnesium, such as, for example, citrates, phosphates, acetates, tartrates, lactates, etc., amino acids, acid salts, such as hydrogen phosphates or dihydrogen phosphates , especially sodium, in addition organic and inorganic acids, such as, for example, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, cromoglycic acid, acetic acid, lactic acid, tartaric acid, succinic acid, fumaric acid, lysine, methionine , sodium or potassium acid hydrogen phosphates, etc.

In further specific embodiments, the mixture that is obtained in process step a) according to the present invention may contain one or more additional excipients that are selected from chelating agents, for example, disodium edetate dihydrate, sodium calcium EDTA, preferably disodium edetate dihydrate.

In addition, the mixture obtained in step a) according to the present invention may or may not contain osmotically active auxiliary substances as an adjuvant to bring

- 6 046086 mixture to the desired osmolality after dilution, which is important in some applications, for example, in particular for inhalation, to ensure good tolerance. These adjuvants are often referred to as isotonic agents, and their addition does not necessarily result in an isotonic composition after reconstitution, but may provide isotonicity close to physiological osmolality to achieve the maximum possible physiological tolerance.

A particularly commonly used isotonic agent is sodium chloride, but it is not suitable in all cases. In an advantageous embodiment of the present invention, the mixture from process step a) does not contain sodium chloride, except, of course, the natural amount of sodium chloride that may also be contained in pharmaceutical grade water. In another embodiment, the mixture from process step a) contains essentially a neutral salt as an isotonicizing agent, which is not sodium chloride, but is, for example, sodium sulfate or sodium phosphate. However, it should be noted that the isotonic agent may also be contained in an aqueous liquid carrier, for example, in the form of an aqueous solution of sodium chloride (saline). However, in such a case, salts other than sodium salts may also be preferred. Thus, some salts of calcium and magnesium are known to have a beneficial or supportive effect when inhaled solutions of the active agent, possibly due to the fact that they inhibit the local irritation caused by this method of administration, and also because they have a bronchodilator effect, which is currently reported in the clinical literature (eg, Hughes et al., Lancet, 2003, 361 (9375):2114-7), and/or because they inhibit the adhesion of microorganisms to proteoglycans of the respiratory mucosa, providing indirect support for mucociliary clearance as the body's natural defense mechanism from pathogens (K.W. Tsang et al., Eur. Resp., 2003, 21, 932-938). Preferred compounds may be, for example, magnesium sulfate, which has very good pulmonary tolerance and can be used without problems by inhalation, as well as calcium chloride (1-10 mmol).

In further specific embodiments, suitable excipients that can be added to the mixture obtained in step a) of the present invention contain saccharides or sugars, such as disaccharides, as described in more detail below.

As an additional ingredient, the mixture which is obtained in step a) of the present invention contains an aqueous liquid carrier or an aqueous liquid medium. The aqueous liquid carrier or medium may be water or an aqueous solution of pharmaceutically acceptable salts or isotonic agents and may preferably be sterile. However, in other preferred embodiments, the sterile aqueous liquid carrier is water, preferably sterilized or sterile water, such as water suitable for injection.

In specific embodiments of the method according to the present invention, the amount of membrane-forming substance selected from the group of phospholipids, preferably lecithin, which is added to the mixture according to step a), exceeds the amount of solubility enhancing substance selected from the group of nonionic surfactants. In exemplary embodiments, the weight ratio of membrane-forming agent selected from the group of phospholipids, preferably lecithin, to solubility improver selected from the group of nonionic surfactants, preferably polysorbate, is selected in the range of from about 15:1 to about 9:1, preferably from about 14:1 to about 12:1, such as about 13:1.

In further specific embodiments, the weight ratio of membrane-forming agent(s) selected from the group of phospholipids and solubility enhancing agent(s) selected from the group of nonionic surfactants, on the one hand, to the inhalable macrocyclic active ingredient, in In particular, the CsA to be contained in the mixture according to step a) on the other hand is from about 5:1 to about 20:1, preferably from about 8:1 to about 12:1, and more preferably about 9:1.

In further specific embodiments, the weight ratio of a membrane-forming agent selected from the group of phospholipids, preferably lecithin, to a solubility enhancing agent selected from the group of nonionic surfactants, preferably polysorbate, and to an inhaled immunosuppressive macrocyclic active ingredient, particularly CsA, is from about 15:1:1.5 to about 5:0.3:0.5, and preferably about 9:0.7:1.

In additional specific embodiments, the weight ratio of a membrane-forming agent selected from the group of phospholipids described above to an inhaled immunosuppressive macrocyclic active ingredient, particularly CsA, is selected in the range of about 8:1 to about 11:1, preferably from about 8.5: 1 to about 10:1, for example about 9:1.

In illustrative embodiments, the mixture that is produced in process step a) of the present invention may contain an inhaled immunosuppressive macrocyclic active ingredient, particularly CsA, at a concentration selected in the range of from about 1 g/L to about 7 g/L, or from about 2 g/L to about 6 g/L, or about 3 to about 5 g/L, such as 4 g/L.

In additional illustrative embodiments, the mixture obtained in step a) according to

- 7 046086 of the present invention may contain a membrane-forming substance selected from the group of phospholipids as described above, in particular Lipoid, for example Lipoid S100, in a concentration selected in the range from about 20 g/l to about 60 g/l, or from about 30 g/L to about 50 g/L, or from about 30 g/L to about 40 g/L.

In further illustrative embodiments, the mixture obtained in process step a) according to the present invention may contain a solubility improver selected from the group of nonionic surfactants as described above, in particular a polysorbate, for example polysorbate 80, at a concentration selected in the range from about 1 g/L to about 5 g/L, in particular selected in the range from about 2 g/L to about 4 g/L or from about 2.5 g/L to about 3.5 g/L .

In specific embodiments, the mixture which is obtained in process step a) according to the present invention may contain at least one saccharide or sugar, in particular at least one disaccharide, as an excipient. The disaccharide that may be contained in the mixture according to process step a), in particular embodiments of the invention, may be selected from the group consisting of sucrose (sucrose; the terms sucrose and sucrose are used interchangeably herein and are used synonymously to refer to β-Dfructofuranosyl- α-O-glucopyranoside; CAS number 57-50-1), lactose (β-O-galactopyranosyl-(1^4)-Glucose; CAS number 63-42-3) and trehalose (α-O-glucopyranosyl-(1 ^1)-a-O-glucopyranoside; CAS number 99-20-7). In specific embodiments, the mixture obtained in process step a) contains sucrose as an excipient.

In additional specific embodiments of the mixtures obtained in process step a) according to the present invention, the ratio of the weight of the selected at least one saccharide or disaccharide, preferably sucrose, to the weight of cyclosporin A in the mixture obtained in process step a) according to the present invention is selected in a range of from about 10:1 to about 30:1, or from about 20:1 to about 30:1, or from about 20:1 to about 27.5:1, or from about 22.5:1 to about 27, 5:1.

Accordingly, in additional illustrative embodiments, the mixture that is obtained in process step a) according to the present invention may contain a disaccharide, in particular sucrose, trehalose and/or lactose, in particular sucrose, in a concentration selected from the range of about 60 g/l to about 140 g/L, or from about 80 g/L to about 120 g/L, or from about 90 g/L to about 110 g/L.

To prepare the mixture in process step a) according to the present invention, selected ingredients such as those described above can be added to a suitable vessel, for example a mixing vessel, and can be mixed using standard methods until a homogeneous mixture is obtained. In certain embodiments, ingredients may be added to the vessel simultaneously or sequentially as needed. For example, a selected aqueous liquid carrier or carrier such as sterilized water may be added to the container as a first ingredient.

In additional illustrative embodiments, selected excipients can then be added to an aqueous liquid carrier, particularly water, and mixed until sufficient or complete dissolution is achieved. Additional ingredients can be added completely and immediately or sequentially to the resulting mixture or solution. For example, a selected membrane-forming substance selected from the group of phospholipids may be added and the resulting mixture mixed until a sufficiently homogeneous dispersion is obtained. Thereafter, a selected solubility enhancer selected from the group of non-ionic surfactants can be added to the mixture and mixed until a sufficiently homogeneous dispersion is formed. Finally, in exemplary embodiments, the selected inhaled immunosuppressive macrocyclic active ingredient, particularly CsA, can be added and mixed until a sufficiently homogeneous dispersion is obtained.

The preparation of the mixture in process step a) according to the present invention can be carried out at approximately room temperature or, depending on the specific components to be added or dissolved, above or below room temperature, typically at a temperature selected in the range of about 0°C. or from about 5°C to about 45°C, or up to about 50°C. In specific embodiments, certain components, particularly excipients or salts, may be added at elevated temperatures, for example, in the range of about 35°C to about 50°C or from about 40°C to about 45°C, and then the temperature may be reduced, for example, to room temperature or lower, for example to a temperature selected in the range of about 0°C, or from about 5°C to about 25°C, or from about 15°C to about 25°C, depending on additional ingredients or components to be added or dissolved. In some cases, for example, to completely dissolve certain ingredients, additional amounts of water or a selected aqueous liquid carrier may be added to the mixture or may be removed from the mixture by methods known to one skilled in the art.

- 8 046086

The resulting liquid aqueous mixture can then be subjected to process step b) as described below.

According to step b) of the process according to the present invention, the mixture obtained in step a) is dispersed to form an intermediate aqueous dispersion containing at least one inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporine A, in an aqueous liquid carrier. To carry out the dispersion according to step b), the mixture obtained according to step a) as described above can be transferred into a vessel suitable for dispersing the mixture, or, in alternative embodiments of the invention, the dispersion can be carried out in the same vessel with the same device that and at stage a). Suitable vessels may be, for example, vessels made of a material suitable for the preparation of pharmaceutical compositions, for example suitable polymeric materials or preferably stainless steel. Depending on the total final volume of the mixture to be dispersed, the vessel may have a volume typically ranging from about 100 L to about 1000 L, often from about 400 L to about 700 L. In certain embodiments, the selected vessel volume generally exceeds the final total volume of the dispersible mixture, in certain embodiments by at least 50% or even at least 10% of the total final volume of the dispersible mixture. In further specific illustrative embodiments, the vessel may have an (inner) diameter ranging from about 700 mm to about 1400 mm, from about 700 mm to about 1000 mm, or from about 850 mm to about 950 mm, or up to about 920 mm.

Typically, dispersion according to step b) of the process according to the present invention is carried out using a dispersant suitable for dispersing liquid mixtures, in particular aqueous liquid mixtures containing additional components as described above, in quantities and concentrations also described in detail above. The selected dispersant may be, for example, portable or, in other words, immersed in and removed from the aqueous mixture to be dispersed. However, in additional embodiments, the dispersant may be firmly attached or incorporated into the vessel in which the dispersion is performed. In additional embodiments, the dispersant may be placed between two vessels as an integral dispersant, as described in more detail below.

In specific embodiments, the dispersion according to step b) is carried out using a rotor-stator dispersant. Rotor-stator dispersants are known to those skilled in the art and are commercially available, for example devices manufactured by IKA-Werke GmbH & Co KG, Germany, such as the ULTRA-TURRAX® UTE batch dispersants. Rotor-stator dispersants typically comprise a rotor configured to rotate at high speed in a corresponding stator, with both the rotor and stator immersed in the mixture to be dispersed. In particular embodiments, the rotor and/or stator comprise a plurality of teeth. Typically, these teeth are firmly attached to the base of the rotor or stator and are preferably oriented parallel to the major axis of rotation of said rotor or the main dispersant drive shaft connecting the dispersant motor to the rotor. The teeth may be arranged along the respective circumference of the rotor and/or stator, respectively, typically in the form of rows of teeth. It should be noted that both the rotor and/or stator may have only one row of teeth or multiple rows of teeth, which are then typically arranged concentrically with respect to the main axis of rotation of the dispersant. The mixture to be dispersed, as the rotor rotates, is typically forced through the gaps between the teeth or rows of teeth of the rotor and/or stator, resulting in a shear force acting on the mixture to be dispersed.

In additional specific embodiments, the dispersion according to step b) according to the present invention can also be carried out using a built-in dispersant. Integrated dispersants are commercially available, for example devices manufactured by IKA-Werke GmbH & Co KG, Germany, such as the ULTRA-TURRAX® UTL integrated dispersants. Such built-in dispersants provide, for example, continuous dispersion of the mixture or dispersion of the mixture from the first vessel A into the second vessel B and vice versa, resulting in the mixture to be dispersed being continuously charged into the (outer) dispersant.

As an indicator of the shear force acting on the dispersible mixture, a shear rate gradient or the so-called shear rate can be used. The shear rate gradient used herein, especially when using a rotor-stator dispersant, having units of [1/s] or [s ^-1 ], can be determined by dividing the rotor peripheral speed, measured in [m/s], as described in more detail below, by the distance or so-called gap width between the rotor and stator in [m], obtained from formula (I)

Fr = v _u / ds (I) where F _r is the shear rate gradient;

vu is the rotor peripheral speed; and ds is the width of the gap between the stator and the rotor, and the peripheral speed of the rotor is calculated by formula (II)

- 9 046086 v _u = d · π · n/60 (II) where d is the rotor diameter; and n is the rotor rotation speed in rpm (revolutions per minute).

In particular embodiments that employ a rotor-stator dispersant as described above, the rotor typically may have a diameter ranging from about 50 mm to about 150 mm, or from about 60 mm to about 140 mm. In particular, in cases where a submersible or batch disperser is used, as described above, the rotor may have a diameter in the range of from about 80 mm to about 120 mm, or in the range from about 90 mm to about 110 mm, for example from about 95 mm to about 105 mm. In additional specific embodiments that utilize an integral dispersant, as described in more detail below, the rotor may have a diameter selected in the range of about 100 mm to about 140 mm, or from about 110 mm to about 130 mm. In further embodiments, a corresponding stator is typically configured to be arranged around a corresponding rotor. In this regard, the (inner) diameter of the space in which the corresponding rotor is placed usually corresponds to the diameter of the rotor, taking into account twice the gap between the rotor and the stator. In specific embodiments, said gap typically does not exceed about 4 mm, about 3 mm, or about 2 mm, and may be selected in the range of from about 0.5 to about 2 mm, or from about 0.5 to about 1.5 mm, or from about 0.7 to about 0.9 mm. In additional specific embodiments of the invention, especially in cases where an integral dispersant is used, the gap (the size of the annular gap) can be selected in the range of from about 0.1 mm to about 0.6 mm, or from about 0.2 mm to about 0 .5 mm, or from about 0.3 to about 0.4 mm.

In specific embodiments, the dispersion of step b) is performed with a shear rate (shear rate) gradient of at least 22,000 1/s (twenty-two thousand 1/s), or at least 24,000 1/s, or at least 25,000 1/s With. In additional specific embodiments, the dispersion of step b) can be performed with a shear rate gradient (shear rate) selected in the range of from about 22,000 1/s to about 120,000 1/s or from about 22,000 1/s to about 100,000 1/s . In additional specific embodiments of the invention, especially in cases where a submersible or batch dispersant is used, dispersion according to process step b) can be performed with a shear rate gradient from about 24,000 1/s to about 80,000 1/s, or from about 60,000 1/s, or from about 25,000 1/s to about 55,000 1/s, or from about 22,000 1/s, or from about 25,000 1/s to about 40,000 1/s, or from about 25,000 1/s to about 38,000 1/s, or from about 27,000 1/s, or from about 28,000 1/s to about 37,000 1/s, or from about 30,000 to about 37,000 1/s.

In additional specific embodiments of the invention, especially in cases where an integral dispersant is used, dispersion according to process step b) can be performed with a shear rate gradient from about 40,000 1/s to about 110,000 1/s, or from about 100,000 1/s, or from about 45,000 1/s to about 90,000 1/s, or from about 50,000 1/s, or from about 55,000 1/s to about 85,000 1/s, or from about 60,000 1/s to about 85,000 1/s, or from about 65,000 1/s to about 75,000 1/s.

In specific embodiments, the rotor and stator may have multiple teeth, as described above. The number of teeth connected to the stator and to the rotor can be selected independently of each other. In particular embodiments, the number of rotor and stator teeth is at least about 10, or at least about 15, or at least about 20. The number of rotor teeth can generally be selected over a wide range, often in excess of 50, 75 or even 100 teeth. In particular embodiments of the invention, especially in cases where a submersible or batch disperser is used, the rotor may have a number of teeth selected in the range of from about 10 to about 75, or from about 10 or 15 to about 40 or 50, or from about 20 to approximately 35. The number of stator teeth as a whole can be independently selected over a wide range, often exceeding 50, 75 or even 100 teeth. In particular embodiments of the invention, especially in cases where a submersible or batch dispersant is used, the stator has a number of teeth selected in the range of from about 10 to about 75, or from about 10 or 15 to about 40 or 50, or from about 20 to about 35. In these cases, the individual teeth may be spaced a certain distance apart, typically about 15 or 10 mm or lower, for example in the range of about 1 mm to about 8 mm or from about 2 mm to about 6 mm, for example approximately 4 mm.

In additional specific embodiments of the invention, especially when using an integral dispersant, the rotor may have a number of teeth selected in the range of from about 10 to about 75, or from about 20 or 30 to about 60 or 50, or from about 35 to about 45. In these cases, the stator may have a number of teeth selected in the range of from about 30 to about 80, or from about 35 or 40 to about 75 or 70, or from about 45 or 40 to about 60. In these cases, the individual teeth may be located on a certain distance apart, typically about 10 or 5 mm or lower, for example in the range of about 0.5 mm

- 10 046086 to about 5 mm or from about 1 mm to about 2.5 mm. In particular, in cases where an integral dispersant is used, the distance between the stator teeth relative to each other and the rotor teeth relative to each other may differ, for example, from about 1.0 mm to about 2.0 mm, for example 1.6 mm for the stator and from about 1.5 mm to about 2.5 mm, for example 2.0 mm for the rotor.

As a further indicator of the shear forces exerted on the mixture to be dispersed according to process step b) according to the present invention, the shear frequency can be used. The shear frequency used herein, especially for a rotor-stator dispersant, having units of [1/s] or [s- ¹ ], is determined by multiplying the rotor speed [1/s] by the number of rotor teeth and the number of stator teeth. according to formula (III)

Fs P ' Spotop ' S Stator (Ш) where FS is the shift frequency;

n is the rotor speed, measured in [1/s]; a

S _Potop and Stator - the number of teeth of the rotor and stator, respectively.

In specific embodiments, the dispersion according to process step b) according to the present invention is carried out at a shear frequency of at least 42,000 1/s, or at least 44,000 1/s, or at least 45,000 1/s. In additional specific embodiments of the invention, especially in cases where a submersible or batch dispersant is used, the dispersion according to step b) is carried out at a shear frequency selected in the range of from about 42,000 1/s to about 140,000 1/s, or from about 42,000 1/s from to about 120,000 1/s, or from about 45,000 1/s to about 100,000 1/s. In additional specific embodiments, in these cases, the shear rate gradient may be selected in the range of from about 50,000 1/s to about 140,000, or to about 100,000 1/s, or from about 50,000 1/s to about 80,000 1/s, or from about 55,000 1/s to approximately 75,000 1/s or up to approximately 70,000 1/s.

In additional specific embodiments, dispersion according to process step b) according to the present invention, especially in cases where a submersible or batch dispersant is used, can be performed by combining a certain shear rate gradient with a certain shear frequency, for example, a shear rate gradient (shear rate) of at least or at least 45000 1/s. In additional specific embodiments of the invention in which a submersible dispersant is used, the dispersion of step b) can be performed at a shear rate gradient selected in the range of from about 28,000 1/s to about 37,000 1/s or from about 30,000 1/s to about 37,000 1/s and at a shear frequency of from about 50,000 1/s to about 80,000 1/s or from about 55,000 1/s to about 75,000 1/s or up to about 70,000 1/s.

In additional specific embodiments of the invention, especially in cases where an integral dispersant is used as described above, the dispersion according to step b) can be performed at a shear frequency selected in the range from about 100,000 1/s to about 200,000 1/s, or from from about 110,000 1/s to about 190,000 1/s, or from about 120,000 1/s to about 180,000 1/s. In additional specific embodiments, in such cases, the shear rate gradient may be selected in the range of from about 130,000 1/s to about 170,000 or from about 140,000 1/s to about 160,000 1/s.

In additional specific embodiments that utilize an integral dispersant as described above, the dispersion of step b) can be performed with a shear rate gradient selected in the range of about 55,000 1/s to about 85,0001/s, or from about 60,000 1/s to about 85,000 1/s, or from about 65,000 1/s to about 75,000 1/s, and an offset frequency selected in the range from about 120,000 1/s to about 180,000 1/s, or from about 130,000 1/s from to about 170,000, or from about 140,000 1/s to about 160,000 1/s.

As mentioned above, the dispersion according to step b) can be carried out using a submersible dispersant or, in other words, a dispersant which is stationary in the dispersing vessel or which can be removably immersed in the dispersible mixture. However, in alternative embodiments, dispersion according to step b) can be performed using an integral dispersant, as described above. However, in additional embodiments, dispersion according to step b) of the process of the present invention can be performed using either a submersible dispersant (or a plurality of submersible dispersants simultaneously) or an integral dispersant. In additional specific embodiments, the submersible dispersant and the inline dispersant may be used in series. For example, in specific embodiments, dispersion according to step b) of the present invention may be carried out first using a submersible dispersant, particularly under the conditions detailed above, followed by dispersion using an integral dispersant under conditions also detailed above. In additional specific embodiments of the invention, the conditions selected for (partial) dispersion carried out using

- 11 046086 submersible dispersant, especially taking into account the selected shear rate, shear frequency and/or rotor speed, differ from the conditions of (partial) dispersion carried out using an integrated dispersant.

As discussed above, the rotor of the rotor-stator dispersant that can be used in process step b) according to the present invention may have a plurality of teeth, which may be located around the perimeter or circumference of the rotor, the plurality of teeth preferably having the same distance to their respective adjacent ones. teeth in a row. In addition, the rotor may or may not have multiple rows of teeth, which are typically arranged concentrically with respect to the main axis of rotation of the rotor. Accordingly, in certain embodiments of the invention, especially in cases where a submersible dispersant is used, the rotor may have multiple rows of teeth, such as 2 to 4 rows of teeth or 2 to 3 rows of teeth.

In addition, the corresponding stator may or may not also have a plurality of rows of teeth, which are typically arranged concentrically with respect to the main axis of rotation of the stator. Accordingly, in particular embodiments, the stator has multiple rows of teeth, such as 2 to 4 rows of teeth or 2 to 3 rows of teeth. In further specific embodiments, the rotor and stator together have a total of 2 to 8, in particular 3 to 6 rows of teeth. In additional specific embodiments of the invention, especially in cases where an integral dispersant is used, the rotor and stator may also have multiple rows of teeth, such as a total of 3 to 12 rows of teeth, or a total of 6 to 10 rows of teeth, such as a total of 8 rows of teeth for the rotor and stator.

Dispersing according to process step b) of the present invention to produce an intermediate aqueous dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier is typically carried out at high speed of rotation of the dispersant rotor, typically at a rotation speed of up to about 10,000 rpm or up to approximately 8000 rpm (revolutions per minute). In particular embodiments of the invention, especially in cases where a submersible disperser is used, dispersion can be carried out at a speed (of the disperser rotor) selected in the range of about 2000 rpm or from about 3000 rpm to about 6000 rpm, or from about 3000 rpm, or about 4000 rpm to about 5500 rpm. Depending on the diameter of the selected rotor, this results in a rotor peripheral speed of at least about 10 m/s. In particular embodiments, dispersion according to step b) is carried out with a rotor peripheral speed selected in the range of from about 15 m/s to about 40 m/s or from about 15 m/s to about 30 m/s.

In additional specific embodiments of the invention, especially in cases where an integral dispersant is used, dispersion can be carried out at a rotation speed (of the disperser rotor) selected in the range of about 2000 rpm, or from about 3000 rpm to about 6000 rpm , or from about 3000 rpm or about 3500 rpm to about 4500 rpm.

To achieve such peripheral speeds, in particular embodiments, the dispersion according to step b) is carried out using a dispersant with a motor having a power of at least 2 kW, in particular with a motor having a power selected in the range from about 2 kW to about 10 kW or from approximately 3 kW to approximately 8 kW.

To obtain an intermediate aqueous dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier, the dispersion according to step b) is carried out for a period of time usually exceeding 10 minutes or 15 minutes or 1 hour or even 2 hours. In many cases, especially when using only the submersible dispersant or the integral dispersant, the dispersion according to step b) is carried out for a (total) period of time of at least about 1 hour or at least about 3 hours, for example, for a period selected from the range from about 1 hour to about 8 hours, or from about 3 hours to about 8 hours, or from about 3 hours to about 5 hours, or from about 1 hour to about 4 hours.

In additional specific embodiments of the invention, as described in detail above, dispersion according to step b) of the process according to the present invention can be carried out using a submersible dispersant, as well as an integral dispersant, wherein the submersible dispersant and the internal dispersant are used sequentially. In specific embodiments, dispersion may be initiated using a submersible dispersant (or a plurality of submersible dispersants) and then the inline dispersant is used. In such cases, the dispersion time can also be reduced. For example, dispersion using a submersible dispersant can be carried out for a period of time up to about 1 hour or up to about 30 minutes, such as from about 10 minutes to about 30 minutes, followed by dispersion using an inline dispersant for a period of time up to 6 hours or up to 4 h, for example for a period of time from about 1 hour to about 4 hours, which can help effectively reduce the overall duration of the dispersion step.

In additional specific embodiments, the dispersion according to step b) can be carried out at a temperature (of the dispersible mixture) selected from the range of about 15°C

- 12 046086 to about 35°C or from about 15°C to about 30°C, or from about 15°C to about 25°C, or from about 20°C to about 25°C. To avoid temperature rise, the mixture can be cooled by standard methods if necessary. In additional specific embodiments, the dispersion according to step b) is carried out at atmospheric pressure.

The resulting intermediate aqueous dispersion containing at least one inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, in an aqueous liquid carrier obtained according to process step b), which already contains the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form, in particular liposomally solubilized forms of CsA (L-CsA) to a certain extent. However, to complete the solubilization of the selected inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, and to further reduce the particle size of the formed liposomes, the resulting mixture is subjected to further homogenization according to the next step of process c) as described below.

According to step c) of the process according to the present invention, the intermediate aqueous dispersion obtained in step b) is homogenized to form a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form, preferably at least from about 90% to about 100% of the inhalable immunosuppressive active ingredient, in particular CsA, from the total amount of said compound added in step a), is present in liposomally solubilized form.

According to process step c), the resulting intermediate aqueous dispersion is then subjected to homogenization conditions to obtain a colloidal dispersion of an inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, in liposomally solubilized form. In preferred embodiments of the invention, the step of homogenizing the intermediate aqueous dispersion obtained in step b) to form a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form involves high pressure homogenization known to those skilled in the art. In exemplary embodiments, the high pressure homogenization of step c) can be performed using a plunger-valve type homogenizer, which may contain one or more, for example one to six plungers or two to four plungers, in particular three plungers. In additional embodiments, such a plunger-valve type homogenizer may comprise a homogenization valve, in particular a ceramic homogenization valve, for example, NanoVALVE (supplier GEA, Italy). In additional specific embodiments, the homogenization pressure is generated in a two-stage differential pressure, as described in more detail below. Exemplary homogenizers suitable for performing high pressure homogenization according to step c) include, but are not limited to, homoMicrofluidics M-110EH or Ariete NS3006L high pressure (GEA, Italy).

In additional embodiments, the high pressure homogenization of step c) may be performed once or several times in repetition. In particular, high pressure homogenization can be performed several times, for example about 5 to about 15 times. In addition, high pressure homogenization can be carried out at any suitable pressure, typically at a pressure up to about 1500 bar, or at a pressure from about 50 bar to about 1500 bar, or at a pressure selected from about 100 bar to about 1000 bar. Preferably, the high pressure homogenization can be carried out several times, for example from about 5 times to about 15 times, at a pressure of from about 100 bar to about 1000 bar, optionally under reduced pressure.

In additional embodiments, the homogenization according to step c) of the process according to the present invention can be carried out in a two-stage differential pressure, in which the first stage uses a relatively lower pressure and the second stage uses a relatively higher pressure. Examples of pressure ranges for the first stage may be selected within the pressure ranges as described generally above, for example in the range from about 50 bar to about 200 bar, in particular from about 75 bar to about 125 bar, for example about 100 bar. Examples of pressure ranges in the second stage may be selected within the pressure ranges described above, for example in the range from about 500 bar to about 1500 bar, in particular from about 750 bar to about 1250 bar, for example about 1000 bar. As described above, when carried out with a two-stage pressure differential, the homogenization step can be performed several times, for example from about 5 times to about 15 times. In additional embodiments, the high pressure homogenization of step c) can be carried out at temperatures from about 35 to 37°C (temperature of the dispersion to be homogenized), in particular at a temperature in the range from about 2°C to about 35°C, or from about 2°C to about 25°C, or from about 5 or 7°C to about 25°C. In specific embodiments, the homogenization of the intermediate aqueous dispersion obtained in step b) of the process according to the present invention, or at least a portion thereof, can be carried out at temperatures below room temperature, for example, at temperatures ranging from about 2°C to example

- 13 046086 but 10°C. In these cases, as well as in cases where homogenization is carried out at higher temperatures, as described above, a heat exchanger can be used to cool the aqueous dispersion during homogenization.

After completion of the homogenization according to process step c) of the present invention, a homogenized dispersion is obtained containing a colloidal dispersion of an inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, in liposomally solubilized form, consisting essentially of unilamellar liposomes having an average diameter of not more than 100 nm, for example from about 30 nm to about 70 nm, and/or a polydispersity index (PI) of not more than 0.50 or even not more than 0.40, for example from about 0.15 to about 0.2, which preferably takes the form of a clear opalescent solution, containing no visible particles.

After completion of the homogenization according to step c), optional step c1), the resulting homogenized dispersion containing the inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, in liposomally solubilized form, can, if necessary, be sterilized, for example, by sterilizing filtration. Suitable filters for such filtration to remove or reduce potential bioburden or microbial contaminants include, but are not limited to, 0.2 micron pore size Fluorodyne®EX (PALL) and other filters.

In further specific embodiments of the invention, it may be preferable to filter the intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in the aqueous carrier liquid obtained in step b) before homogenizing according to step c). Accordingly, the process according to this first aspect of the present invention may include, as an additional step b1), filtering the intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in the aqueous liquid carrier obtained in step b), before homogenizing the prepared filtered intermediate aqueous dispersion according to step c) .

In accordance with these embodiments of the invention, the intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, in the aqueous liquid carrier obtained in step b) is filtered before being further homogenized at high pressure as described above for step c). This optional additional filtration may prevent mechanical stress and potential damage to the high pressure homogenizer due to potential residual particles in the intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient, particularly cyclosporine A. Filtration can be accomplished using readily available filters or filter media which are suitable for contact with pharmaceutical compounds or compositions, such as steel or a suitable polymeric material. In specific embodiments, filtration according to this optional step b1) is performed using a filter with an average pore width in the range of about 75 μm, or from about 100 μm to about 300 μm, or from about 150 μm to about 250 μm, or from about 200 μm to about 250 microns, or from about 220 microns to about 250 microns, or to about 230 microns, for example about 225 microns.

The process according to this first aspect of the invention produces a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, in liposomally solubilized form, as a product of process step c) as described above. The inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, preferably in a therapeutically effective amount, may consist of liposome-forming structures formed by a membrane-forming substance selected from the group of phospholipids and a solubility improver selected from the group of non-ionic surfactants, as described above .

In specific embodiments, the inhaled immunosuppressive macrocyclic active ingredient, particularly cyclosporine A, is at least partially included (or embedded) in a bilayer membrane in liposome-forming structures. The term is included herein when used in relation to CsA, which is a lipophilic compound, means that CsA is located or incorporated into the inner lipophilic portion of the lipid bilayer membrane, but not into the outer hydrophilic surface of the lipid bilayer membrane (where the term surface may refer to both surface or, more specifically, the inner or outer surface of a bilayer membrane forming liposome-forming structures).

In preferred embodiments, the inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporine A, is advantageously included (or embedded) in a bilayer membrane in liposome-forming structures. In illustrative embodiments, at least about 90%, or even at least about 95%, or even at least about 97.5% of the total amount of the inhaled immunosuppressive macrocyclic acid

- 14 046086 active ingredient, in particular CsA, is contained in the bilayer membranes of liposome-forming structures formed in the process according to the present invention. In additional illustrative embodiments, at least about 90%, or from about 95% to about 97.5%, or up to about 99%, or up to 99.5%, or even up to 99.9% of the total amount of the inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, is included in the bilayer membranes of liposome-forming structures formed in the process according to the present invention.

According to a second aspect, the present invention provides a method for preparing a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, the lyophilized pharmaceutical composition comprising an inhaled immunosuppressive macrocyclic active ingredient in a liposomally solubilized form, the method comprising preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in a liposomal solubilized form. form in an aqueous liquid carrier, according to the method according to the first aspect of the invention described above; and additionally includes the stage

d) removing the aqueous liquid carrier, at least partially, by lyophilization to obtain a lyophilized pharmaceutical composition.

In other words, the present invention also provides a method for preparing a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, wherein the lyophilized pharmaceutical composition contains an inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, in liposomally solubilized form, and the method includes the steps

a) providing a mixture containing an inhaled immunosuppressive macrocyclic active ingredient, in particular CsA; a membrane-forming substance selected from the group of phospholipids;

a solubility improver selected from the group of nonionic surfactants;

optionally one or more excipients; and an aqueous liquid carrier;

b) dispersing the mixture according to step a) to obtain an intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier;

b1) optionally filtering the intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in the aqueous liquid carrier obtained in step b);

c) homogenizing the intermediate aqueous dispersion obtained in step b) or b1) to form a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form;

c1) optionally sterilizing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form; And

d) removing the aqueous liquid carrier, at least partially, under lyophilization conditions to obtain a lyophilized pharmaceutical composition.

It should be understood that all features, embodiments of the invention, starting materials, process conditions and combinations thereof described above for the process of the first aspect of the invention also apply to the process of this second aspect of the invention.

In specific embodiments of this second aspect of the invention, the dispersion according to process step b) can be performed using a submersible dispersant and an integral dispersant as described above, preferably using a submersible dispersant and an internal dispersant in sequence.

The method of this second aspect of the invention includes, in addition to the process steps of the first aspect of the invention, an additional step d) in which the aqueous liquid carrier is at least partially removed under lyophilization conditions to form a lyophilized pharmaceutical composition.

Lyophilization according to process step d) can be carried out using standard technologies known to those skilled in the art, for example using a LyoStar MNL-055-A/LSACC3E lyophilizer or a GEA Lyovac® GT 400-D. Lyophilization to obtain lyophilized pharmaceutical compositions according to the present aspect of the invention can be carried out continuously, for example, at constant pressure and temperature, or preferably can be carried out in stages, each stage of the lyophilization protocol or process can be carried out at a certain pressure, temperature and for a certain period of time . In exemplary embodiments, the lyophilization process or cycle may include up to 20 or from about 2 to about 15, preferably from about 5 to about 15 sequential steps.

- 15 046086

Each step can be carried out, for example, at a temperature of from about 40°C to about -60°C, preferably from about 20°C to about -50°C, at a constant temperature or at temperatures that can be increased or decreased with a certain gradient. In addition, each lyophilization step can be carried out under reduced pressure, for example, at a pressure below external atmospheric pressure, for example, from about 0.005 mbar to about 800 mbar, preferably from about 0.009 mbar to about 0.500 mbar, or to about 0.400 mbar, or to approximately 0.300 mbar.

It should be noted that in addition to the lyophilization described above, the portion of the liquid aqueous carrier to be removed according to process step d) can also be removed by other methods known to those skilled in the art, for example by distillation under reduced pressure, especially before lyophilization. In addition, it may be preferable to prepare aliquots of the dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form obtained in method step c) in a smaller volume containing a specified amount of the active ingredient.

It should be noted here that while the lyophilization according to additional process step d) may, in particular embodiments of the invention, be carried out in the presence of a disaccharide selected from the group consisting of sucrose, lactose and trehalose, wherein at least one disaccharide is present in the amount At least 40% by weight relative to the total weight of the lyophilized composition, said disaccharide is added not to the mixture obtained in process step c), but to the original mixture obtained in process step a).

In a third aspect of the present invention there is provided a lyophilized pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, in liposomally solubilized form for reconstitution in an aqueous liquid carrier, which is or can be obtained by the method of the second aspect of the invention. In certain embodiments, such a lyophilized pharmaceutical composition may comprise an inhaled immunosuppressive macrocyclic active ingredient, preferably CsA;

a membrane-forming substance selected from the group of phospholipids;

a solubility improver selected from the group of nonionic surfactants; and optionally one or more excipients.

In specific embodiments of the invention, and depending on the amount of the above ingredients, lyophilized pharmaceutical compositions that can be prepared by the method according to the second aspect of the invention may contain an inhaled immunosuppressive macrocyclic active ingredient, particularly CsA, in an amount of from about 2 wt.% to about 4% by weight, preferably from about 2.2% by weight to about 3.4% by weight, or even more preferably from about 2.4% by weight to about 3.4% by weight, or from about 2.4% by weight .% to about 3.0 wt%, or from about 2.5 wt% to about 2.9 wt%, or from about 2.6 wt% to about 2.8 wt%, or from about 2.65 wt.% to about 2.75 wt.%, in each case based on the weight of the lyophilized composition.

In additional specific embodiments, the content of a membrane-forming substance selected from the group of phospholipids, preferably Lipoid S100, in the lyophilized composition can be from about 10 or 15 wt.% to about 30 wt.%, and preferably from about 20 wt.% to about 30 wt. %, and even more preferably from about 23 wt.% to about 27 wt.% based on the total weight of the lyophilized composition.

In further specific embodiments, the content of the solubility enhancer selected from the group of nonionic surfactants is preferably selected in the range of from about 0.01 wt.% to about 5 wt.%, or from about 0.1 wt.% to about 4 wt%, or from about 0.5 wt% to about 3.5 wt%, or from about 1 wt% to about 3 wt%, preferably from about 1.5 wt% to about 2, 5 wt.%, or from about 1.6 wt.% to about 2.3 wt.%, or from 1.7 wt.% to about 2.1 wt.%, or from about 1.8 wt.% to about 2.0% by weight, in each case relative to the total weight of the lyophilized composition.

The lyophilized pharmaceutical composition according to this third aspect of the invention, which can be prepared by the method according to the second aspect of the invention, may or may not further contain residual water after lyophilization, which may bind to the surfaces of liposome-forming structures or which may be contained in the internal cavity of potentially hollow liposome-forming structures, such as described above. In preferred embodiments, the amount of residual water contained in the lyophilized composition is up to about 5 wt%, or up to about 3 wt%, or preferably up to about 2 wt%, based on the total weight of the lyophilized pharmaceutical composition.

In further specific embodiments, the lyophilized pharmaceutical composition according to the third aspect of the invention may comprise

a) liposome-forming structures that contain

- 16 046086

i) a therapeutically effective amount of cyclosporine A (CsA);

ii) a membrane-forming substance selected from the group of phospholipids; and iii) a solubility improver selected from the group of non-ionic surfactants; And

b) at least one disaccharide selected from the group consisting of sucrose, lactose and trehalose, wherein the at least one disaccharide is present in an amount of at least 40% by weight relative to the total weight of the lyophilized composition.

It should be noted that in connection with this aspect of the invention, all features, embodiments, ingredients, process parameters and combinations thereof described in connection with the first and second aspects of the invention also apply to this third aspect of the invention, as well as all additional aspects inventions.

In some embodiments, the at least one disaccharide is present in an amount of from at least about 40 wt.% to about 95 wt.%, or up to about 90 wt.%, or up to about 85 wt.%, or up to about 80 wt. %, all values are given relative to the total weight of the lyophilized composition. In additional specific embodiments, the lyophilisates according to this third aspect preferably contain sucrose and/or lactose, in particular sucrose, in an amount selected from the range of about 50 wt.% to about 80 wt.% or up to about 75 wt.%, relative to or based on the total weight of the lyophilized composition. In further preferred embodiments, the lyophilized pharmaceutical compositions according to this aspect contain at least one disaccharide, preferably sucrose, trehalose and/or lactose, in particular sucrose, in an amount selected in the range of about 60 wt.% to about 75 wt.%, even more preferably selected in the range of from about 65 wt.% to about 70 wt.% based on the total weight of the lyophilized composition.

A lyophilized pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, in a liposomally solubilized form for reconstitution in an aqueous liquid carrier, preferably contains an inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, in a therapeutically effective amount and is suitable as a medicinal product, especially for pulmonary use by inhalation.

Accordingly, the fourth aspect of the present invention provides a lyophilized pharmaceutical composition which is or can be prepared by the method of the second aspect of the invention for use as a medicament for pulmonary use, in particular for pulmonary use by inhalation.

In illustrative embodiments, such lyophilized pharmaceutical composition in accordance with the third and fourth aspects of the invention contains or, together with additional optional excipients, primarily consists of or preferably contains (all values relative to the total weight of the lyophilized pharmaceutical composition), wt.%: Inhalation immunosuppressive macrocyclic active ingredient 1-7

Disaccharide40-80

Phospholipid10-40

Nonionic surfactant 0.01-10

Buffer agent1-6

Chelating agent 0.05-0.5 and the sum of the components is 100 wt.% by weight of the finished lyophilized pharmaceutical composition.

In additional illustrative embodiments, such lyophilized pharmaceutical composition in accordance with the third and fourth aspects of the invention contains, or together with additional optional excipients, essentially consists of or preferably contains (all values relative to the total weight of the lyophilized pharmaceutical composition), wt.%: Cyclosporin A 2- 4

Disaccharide40-80

Phospholipid10-40

Nonionic surfactant 0.01-10

Buffer agent1-6

Chelating agent 0.05-0.5

- 17 046086 and the sum of the components is 100 wt.% by weight of the finished lyophilized pharmaceutical composition.

It should be noted that the values and ranges given above are based on a lyophilized and completely anhydrous composition. However, for practical reasons, the lyophilized composition, in addition to the components listed above, may or may not contain a residual amount of water ranging from about 0 wt.% to about 5 wt.% based on the weight of the lyophilized pharmaceutical composition.

In a further illustrative embodiment, such lyophilized pharmaceutical compositions contain or, together with additional optional excipients, essentially consist of or preferably contain (all values relative to the total weight of the lyophilized pharmaceutical composition), wt.%:

Cyclosporine A 2-4 Disaccharide 50-75 Phospholipid 15-40 Nonionic surfactant 0.1-4 Buffer agent 2-6 Chelating agent 0.05-0.5

wherein the sum of the components constitutes 100 wt.% of the finished lyophilized pharmaceutical composition and said lyophilized composition, in addition to the components listed above, may or may not contain a residual amount of water in the range of from about 0 wt.% to about 2 wt.% based on the weight of the lyophilized pharmaceutical composition.

In a preferred illustrative embodiment, the lyophilized pharmaceutical composition contains or, together with additional optional excipients, essentially consists of or consists of or preferably contains (all values are based on the total weight of the lyophilized pharmaceutical composition), wt.%:

Cyclosporine A 2.5-3 Disaccharide 60-75 Phospholipid 20-30 Nonionic surfactant 1-3 Buffer agent 3-5 Chelating agent 0.05-0.2

wherein the sum of the components constitutes 100 wt.% of the finished lyophilized pharmaceutical composition and said lyophilized composition, in addition to the components listed above, may or may not contain a residual amount of water in the range of from about 0 wt.% to about 2 wt.% based on the weight of the lyophilized pharmaceutical composition.

The lyophilized pharmaceutical composition of the third and fourth aspects of the invention can be reconstituted (redispersed) in an aqueous liquid carrier, preferably in a sterile aqueous liquid carrier, to form a colloidal solution or dispersion. In preferred embodiments, pulmonary administration of the lyophilized pharmaceutical composition occurs after said reconstitution in an aqueous liquid carrier to form a colloidal solution or dispersion.

As described above, compositions containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form, which can be prepared by the methods of the present invention in the form of a dispersion or lyophilisate, can be used as medicinal products, in particular after lyophilization and reconstitution in an aqueous liquid carrier, as above, for example for the prevention and treatment of autoimmune diseases, skin diseases, after transplantation or diseases of the sensory organs (eyes, nose, ears), ailments and pulmonary diseases, for example asthma, chronic obstructive bronchitis, parenchymal, fibrotic and interstitial diseases or inflammation, lung cancer, and preferably for the prevention and treatment of acute or chronic transplant rejection reactions, as well as diseases caused by them, such as bronchiolitis obliterans, in particular after lung, bone marrow or stem cell transplantation, in particular after lung transplantation. In addition, they can be used to increase the effectiveness of other drugs, in particular cytotoxic drugs, and an additive or synergistic effect with cyclosporine may be achieved due to the effect of efflux pump inhibition. In some cases, nasal, oral, ophthalmic, mucosal, parenteral or topical administration of the composition according to the present invention may be advantageous. Administration can be accomplished by applying

- 18 046086 nia, drip, spray on the surface or inside the body, which in preliminary tests on animals showed particularly good tolerance.

However, preferably, the pharmaceutical compositions which can be prepared according to the present invention, especially in lyophilized and/or reconstituted form, are suitable for the treatment of pulmonary diseases, in particular asthma, refractory asthma, chronic obstructive bronchitis, parenchymal, fibrotic and interstitial lung diseases and inflammation , and preferably for the prevention and treatment of acute and chronic organ transplant rejection reactions after lung transplantation, as well as diseases caused by them, such as bronchiolitis obliterans.

As mentioned above, pharmaceutical compositions that can be prepared in accordance with the methods of the present invention described in detail above in the first and second aspects of the present invention are suitable as drugs for pulmonary administration. Pulmonary administration can be carried out after reconstitution or, in particular, after redispersing the lyophilized pharmaceutical composition, which can be obtained as described above in the second aspect of the present invention, in an aqueous liquid carrier, preferably in a sterile aqueous liquid carrier, to form a colloidal solution or dispersion , preferably to form a colloidal dispersion.

In preferred embodiments, pulmonary administration of the lyophilized pharmaceutical composition for use described above is accomplished by inhalation. In further preferred embodiments, pulmonary administration occurs after conversion of a pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient, particularly CsA, in liposomally solubilized form into an aerosol, for example, by nebulization or aerosolization. After reconstitution or, in particular, after dispersion in an aqueous liquid carrier, the pharmaceutical compositions obtained by the methods according to the present invention can be advantageously aerosolized and administered using a nebulizer capable of converting a solution, colloidal preparation or suspension, such as the proposed composition containing a liposomally solubilized inhalable immunosuppressive macrocyclic active ingredient, in particular CsA, in a large number of drops capable of reaching the periphery of the lungs. In particular, a jet nebulizer, an ultrasonic nebulizer, a piezoelectric nebulizer, an electrohydrodynamic nebulizer, a membrane nebulizer, an electronic nebulizer, or an electronic vibrating membrane nebulizer can be used. Examples of suitable nebulizers include SideStream® (Philips), AeroEclipse® (Trudell), LC Plus® (PARI), LC Star® (PARI), LC Sprint® (PARI), I-Neb® (Philips/Respironics), IH50 (Beurer), MicroMesh® (Health & Life, Schill), Micro Air® U22 (Omron), Multisonic® (Schill), Respimat® (Boehringer), eFlow® (PARI), AeroNebGo® (Aerogen), AeroNeb Pro® ( Aerogen) and AeroDose® (Aerogen).

However, preferably, a piezoelectric nebulizer, an electrohydrodynamic nebulizer, a membrane nebulizer, an electronic nebulizer, or an electronic vibrating membrane nebulizer can be used. In such cases, suitable nebulizers include the I-Neb® (Philips/Respironics), IH50 (Beurer), MicroMesh® (Health & Life, Schill), Micro Air® U22 (Omron), Multisonic® (Schill), Respimat® ( Boehringer), eFlow® (PARI), AeroNebGo® (Aerogen), AeroNeb Pro® (Aerogen) and AeroDose® (Aerogen).

In preferred embodiments, pulmonary administration of a pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient, particularly CsA, in reconstituted form for use according to this aspect of the invention is accomplished using an ultrasonic or electronic vibrating membrane nebulizer, preferably using a vibrating membrane nebulizer such as eg device type eFlow®, AeroNeb Pro or -Go or I-Neb.

In further preferred embodiments, for targeted delivery of a CsA formulation, especially in liposomally solubilized form, as described above, to the lower respiratory tract, wherein said composition for use in this aspect of the present invention is aerosolized using an electronic vibrating membrane nebulizer. In a particularly preferred embodiment, the lyophilized pharmaceutical composition in reconstituted form for use in accordance with the present invention is aerosolized using an eFlow® nebulizer (PARI Pharma GmbH).

The eFlow® nebulizer nebulizes liquid dosage forms, such as pharmaceutical compositions, which can be produced using the methods of the present invention in reconstituted form using a perforated vibrating membrane to produce an aerosol with a low ballistic moment and a high content of droplets having a respirable size range, typically less than 5 µm. The eFlow® device is designed to nebulize drug faster and more efficiently due to higher nebulization rates, lower wastage and higher levels of drug available in delivered dose (DD) and inhaled dose (RD) compared to conventional nebulizers such as jet nebulizers. nebulizers.

- 19 046086

As described above, a pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, which can be prepared in accordance with the methods of the present invention, can be dispersed (reconstituted) in an aqueous liquid carrier such as water or saline, preferably saline solution (aqueous sodium chloride solution with a concentration of 0.25% w/v), to obtain an opalescent dispersion or solution. In additional embodiments, such liquid liposome dispersions contain substantially no visible particles. The liposomes contained in the dispersion preferably have an average diameter, or more specifically a z-average diameter, of no more than about 100 nm when analyzed by photon correlation spectroscopy on a Malvern ZetaSizer. In a preferred embodiment, the liquid liposomal dispersion contains liposomes with a z-average diameter, as measured by photon correlation spectroscopy (Malvern ZetaSizer), in the range of from about 40 nm to about 100 nm, and even more preferably in the range from about 40 nm to about 70 nm .

In further specific embodiments, said liposomal dispersions have a polydispersity index (PI) as measured by photon correlation spectroscopy of up to about 0.50, preferably up to about 0.4, and even more preferably in the range of from about 0.1 to about 0. 3.

In additional embodiments, liquid liposome dispersions as described above may have an osmolality in the range of about 300 mOsmol/kg to about 550 mOsmol/kg, preferably in the range of about 430 mOsmol/kg to about 550 mOsmol/kg, or from about 370 mOsmol/kg. /kg to approximately 470 mOsmol/kg. The pH of liquid liposome dispersions according to this aspect of the invention is preferably from about 6.0 to 7.0, preferably from about 6.2 to about 6.8. In additional embodiments, after a 1:10 dilution, the liquid liposome dispersion of this aspect of the invention has a turbidity of up to 200 NFU (nephelometric unit of turbidity), preferably in the range of about 55 NFU to about 90 NFU.

It has been unexpectedly discovered that liquid liposome dispersions of a pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, which can be obtained by reconstituting lyophilized pharmaceutical compositions obtained by the method according to the second aspect of the invention, in particular, which were obtained by redispersing the lyophilized pharmaceutical compositions according to the third aspect of the present invention containing a disaccharide selected from the group consisting of sucrose, lactose and trehalose, in an amount of at least 40 wt.% in an aqueous liquid carrier, contain liposomes that are equal in size to or only slightly larger than liposomes in an appropriate dispersion prior to lyophilization as described below. Thus, the methods of the present invention provide, after lyophilization and reconstitution in an aqueous liquid carrier, liquid liposome aqueous dispersions containing liposomes with a median diameter, measured as the z-average diameter by photon correlation spectroscopy (Malvern ZetaSizer), that is equal to or has a value up to 20% greater, preferably only up to 10% greater than the z-average diameter of the liposomes used to prepare the lyophilized pharmaceutical composition of the present invention, before lyophilization, preferably equal to or up to 20% greater than the size of the liposomes obtained by the method according to the second aspect of the present invention, before lyophilization.

Moreover, it has surprisingly been found that the method according to the first aspect of the invention makes it possible to obtain a dispersion containing an inhaled macrocyclic active ingredient, in particular cyclosporin A, in liposomally solubilized form with a precise content of active ingredient corresponding to the amount of said active ingredient introduced during preparation, especially in cases when the intermediate filtration stage is enabled. Thus, in specific embodiments of the invention, the content of the inhaled macrocyclic active ingredient, in particular, cyclosporin A, in liposomally solubilized form contained in the dispersion obtained in accordance with the method of the first aspect of the present invention, contains at least about 95% or at least at least about 97%, or at least about 98%, such as from about 98%, or from about 99% to about 100%, or from about 98% to about 99.95%, or from about 98.5% to about 99.9% of the amount of inhaled macrocyclic active ingredient, in particular cyclosporine A, in the initial mixture obtained in step a) of the method according to the first aspect of the present invention.

Below is a list of numbered embodiments included in the present invention.

1. A method for producing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form in an aqueous liquid carrier, comprising the following steps:

a) providing a mixture containing

- 20 046086 inhaled immunosuppressive macrocyclic active ingredient;

a membrane-forming substance selected from the group of phospholipids;

a solubility improver selected from the group of nonionic surfactants;

optionally one or more excipients; and an aqueous liquid carrier;

b) dispersing the mixture in the manner prescribed in step a) to obtain an intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; And

c) homogenizing the intermediate aqueous dispersion obtained in step b) to form a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form.

2. Method according to claim 1, characterized in that the dispersion according to step b) is carried out using a rotor-stator dispersant.

3. Method according to claim 1 or 2, characterized in that the dispersion according to step b) is carried out at a shear rate gradient (shear rate) of at least 22000 1/s.

4. Method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out using a submersible dispersant.

5. The method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out at a shear rate gradient (shear rate) selected in the range from about 25,000 1/s to about 40,000 1/s.

6. Method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out at a shear frequency of at least 42,000 1/s.

7. The method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out at a shear frequency selected in the range from about 50,000 1/s to about 80,000 1/s.

8. The method according to any of the preceding claims, wherein the dispersion according to step b) is carried out at a shear rate gradient selected in the range from about 28,000 1/s to about 37,000 1/s, and at a shear frequency selected in the range from about 50,000 1 /s to approximately 80,000 1/s.

9. Method according to any one of claims 2 to 8, characterized in that the rotor diameter is from about 60 mm to about 140 mm.

10. A method according to any one of claims 2 to 9, wherein the rotor has a plurality of teeth selected from a range of from about 10 teeth to about 40 teeth.

11. A method according to any one of claims 2 to 10, characterized in that the stator has a plurality of teeth selected in the range from about 10 teeth to about 40 teeth.

12. Method according to any one of claims 2 to 11, characterized in that the rotor and/or stator have multiple rows of teeth.

13. Method according to any one of claims 2 to 12, characterized in that the rotor and/or stator have from 2 to 8 rows of teeth or from 3 to 6 rows of teeth.

14. Method according to any one of claims 2 to 13, characterized in that the dispersion according to step b) is carried out in a container whose diameter is in the range from about 700 mm to about 1000 mm.

15. Method according to any one of claims 2 to 14, characterized in that the dispersion according to step b) is carried out at a rotation speed in the range from about 2000 rpm to about 6000 rpm.

16. Method according to any one of claims 2 to 15, characterized in that the dispersion according to step b) is carried out at a rotation speed in the range from about 3000 rpm to about 5500 rpm.

17. Method according to any one of claims 1 to 3, characterized in that the dispersion according to step b) is carried out using a built-in dispersant.

18. The method according to claim 17, characterized in that the dispersion according to step b) is carried out at a shear rate gradient (shear rate) selected in the range from about 45,000 1/s to about 90,000 1/s.

19. The method according to claim 16 or 17, characterized in that the dispersion according to step b) is carried out at a shear frequency selected in the range from about 100,000 1/s to about 200,000 1/s.

20. The method according to any one of claims 17 to 19, characterized in that the dispersion according to step b) is carried out at a shear frequency selected in the range from about 120,000 1/s to about 180,000 1/s.

21. The method according to any one of claims 17 to 20, characterized in that the dispersion according to step b) is carried out at a shear rate gradient selected in the range from about 55,000 1/s to about 85,000 1/s, and at a shear frequency selected in range from about 130,000 1/s to about 170,000 1/s.

22. A method according to any one of claims 17 to 21, characterized in that the rotor diameter is from about 100 mm to about 140 mm.

23. A method according to any one of claims 17 to 22, characterized in that the rotor has a plurality of teeth selected in the range from about 30 teeth to about 50 teeth.

- 21 046086

24. A method according to any one of claims 17 to 23, characterized in that the stator has a plurality of teeth selected in the range from about 30 teeth to about 80 teeth.

25. Method according to any one of claims 17 to 24, characterized in that the rotor and/or stator have a plurality of rows of teeth.

26. Method according to any one of claims 17 to 25, characterized in that the rotor and/or stator have from 3 to 12 rows of teeth together or from 6 to 10 rows of teeth combined.

27. Method according to any one of claims 17 to 26, characterized in that the dispersion according to step b) is carried out at a rotation speed in the range from about 2000 rpm to about 6000 rpm.

28. Method according to any one of claims 17 to 27, characterized in that the dispersion according to step b) is carried out at a rotation speed in the range from about 3500 rpm to about 4500 rpm.

29. Method according to any one of claims 2 to 28, characterized in that the dispersion according to step b) is carried out with a rotor peripheral speed of at least 10 m/s.

30. The method according to any one of claims 2 to 29, wherein the dispersion according to step b) is carried out with a rotor peripheral speed selected in the range from about 15 m/s to about 40 m/s.

31. Method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out using a dispersant with a motor having a power of at least 2 kW.

32. The method according to any of the preceding claims, wherein the dispersion according to step b) is carried out using a dispersant with a motor whose power is selected in the range from about 2 kW to about 10 kW.

33. The method according to any of the preceding claims, wherein the dispersion according to step b) is carried out using a submersible and built-in dispersant.

34. The method according to claim 33, characterized in that a submersible and built-in dispersant are used sequentially.

35. The method according to claim 33 or 34, characterized in that dispersion is started using a submersible dispersant, and then a built-in dispersant is used.

36. Method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out for at least about 3 hours.

37. A method according to any one of the preceding claims, characterized in that the dispersion according to step b) is carried out for a period ranging from about 3 hours to about 8 hours.

38. A method according to any one of the preceding claims, characterized in that the dispersion according to step b) is carried out for a period ranging from about 3 hours to about 5 hours.

39. The method according to any one of claims 33 to 38, characterized in that the dispersion according to step b) is carried out using a submersible dispersant for a period of time up to about 1 hour, followed by dispersion using a built-in dispersant for a period of time from about 1 hour to approximately 4 hours

40. The method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out at a temperature (of the mixture) selected from the range from about 15°C to about 30°C.

41. The method according to any of the preceding claims, wherein the dispersion according to step b) is carried out at ambient pressure.

42. The method according to any of the preceding claims, which includes as an additional step b1) filtering the intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in the aqueous liquid carrier obtained in step b), before homogenizing the prepared filtered intermediate aqueous dispersion according to step c ).

43. The method according to claim 41, characterized in that the filtration according to step b1) is carried out using a filter with an average pore width in the range from about 200 μm to about 250 μm.

44. The method of any one of the preceding claims, wherein the inhaled immunosuppressive macrocyclic active ingredient is cyclosporine A (CsA) or tacrolimus.

45. The method of any one of the preceding claims, wherein the inhaled immunosuppressive macrocyclic active ingredient is cyclosporine A (CsA).

46. The method according to any of the preceding claims, wherein the mixture obtained in step a) contains an inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporine A (CsA), in a concentration selected in the range from about 1 g/l to about 7 g /L, or from about 2 g/L to about 6 g/L, or from about 3 to about 5 g/L, for example 4 g/L.

47. The method according to any of the preceding paragraphs, characterized in that the membrane-forming substance selected from the group of phospholipids is a mixture of natural phospholipids.

48. The method according to any of the preceding paragraphs, characterized in that the membrane-forming substance selected from the group of phospholipids is lecithin containing unsaturated fatty acid residues.

- 22 046086

49. The method according to any of the preceding claims, characterized in that said membrane-forming substance selected from the group of phospholipids is a lecithin selected from the group consisting of soy lecithin, Lipoid S75, Lipoid S100, Phospholipon® G90, 100 or similar lecithin.

50. The method according to any of the preceding claims, characterized in that the mixture presented in step a) contains a membrane-forming substance selected from the group of phospholipids, in a concentration selected in the range from about 20 g/l to about 60 g/l, or from about 30 g/L to about 50 g/L, or from about 30 g/L to about 40 g/L.

51. The method according to any one of the preceding claims, wherein the solubility enhancing agent selected from the group of nonionic surfactants is selected from the group of polysorbates.

52. The method according to any of the preceding claims, wherein the solubility enhancing agent selected from the group of nonionic surfactants is polysorbate 80.

53. The method according to any of the preceding claims, characterized in that the mixture obtained in step a) contains a solubility enhancing substance selected from the group of nonionic surfactants in a concentration selected from the group from about 1 g/l to about 5 g/L, or from about 2 g/L to about 4 g/L, or from about 2.5 g/L to about 3.5 g/L.

54. The method according to any of the preceding claims, characterized in that the weight ratio of phospholipid to polysorbate is selected in the range from 15:1 to 9:1, preferably from about 14:1 to about 12:1, for example about 13:1,

55. The method according to any of the preceding claims, characterized in that the mass ratio between the (sum of) phospholipid and nonionic surfactant, on the one hand, and the inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, on the other hand, is selected in the range from about 5:1 to about 20:1, preferably about 8:1 to about 12:1, and more preferably about 9:1.

56. The method of any one of the preceding claims, wherein the weight ratio of phospholipid (lecithin), nonionic surfactants and inhaled immunosuppressive macrocyclic active ingredients, in particular CsA, is from about 15:1:1.5 to 5:0.3 :0.5, and preferably about 9:0.7:1.

57. The method according to any of the preceding claims, characterized in that said inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, is at least partially embedded (or included) in a bilayer membrane in liposome-forming structures.

58. The method according to any of the preceding claims, characterized in that the inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, is predominantly (for example, in an amount of at least about 90% or even at least from about 95% to about 97.5% ) is included in a bilayer membrane in liposome-forming structures.

59. The method of any one of the preceding claims, wherein at least from about 90%, or from about 95% to about 97.5%, or up to about 99%, or 99.5%, or even 99.9% of the total an amount of an inhaled immunosuppressive macrocyclic active ingredient, in particular CsA, is incorporated into the bilayer membranes of liposome-forming structures formed in a specific manner according to the present invention.

60. The method according to any of the preceding claims, wherein said mixture obtained in step a) contains at least one excipient selected from the group consisting of sugars, buffers and chelating agents.

61. Method according to any of the preceding claims, characterized in that said mixture obtained in step a) contains at least one disaccharide as an auxiliary substance.

62. The method according to any of the preceding claims, wherein the mixture presented in step a) contains at least one disaccharide selected from the group consisting of sucrose, lactose and trehalose as an excipient.

63. The method according to any of the preceding claims, wherein the mixture obtained in step a) contains at least one disaccharide selected from the group consisting of sucrose, lactose and trehalose as an excipient in a concentration selected in the range of 60 g /L to about 140 g/L, or from about 80 g/L to about 120 g/L, or from about 90 g/L to about 110 g/L.

64. The method according to any of the preceding claims, wherein said mixture obtained in step a) contains sucrose.

65. The method according to any of the preceding claims, characterized in that the aqueous liquid carrier contains an aqueous solution of sodium chloride (saline solution) or sterilized water.

66. The method according to any one of the preceding claims, wherein the step of homogenizing the intermediate aqueous dispersion obtained in step b) to obtain a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form comprises high pressure homogenization.

- 23 046086

67. The method of claim 66, wherein the high pressure homogenization is carried out at a pressure selected from the range of about 100 bar to about 1000 bar.

68. Method according to claim 66 or 67, characterized in that the high pressure homogenization according to step c) is carried out using a plunger-valve type homogenizer.

69. A method of preparing a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, wherein the lyophilized pharmaceutical composition contains an inhaled immunosuppressive macrocyclic active ingredient in a liposomally solubilized form, where the method includes preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in a liposomal solubilized form in an aqueous liquid carrier according to the method according to any of the preceding paragraphs; and additionally includes the stage

d) removing the aqueous liquid carrier, at least partially, by lyophilization to obtain a lyophilized pharmaceutical composition.

70. The method according to claim 69, characterized in that said lyophilized pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form contains an inhaled immunosuppressive macrocyclic active ingredient;

a membrane-forming substance selected from the group of phospholipids;

a solubility improver selected from the group of nonionic surfactants; and optionally one or more excipients.

71. A lyophilized pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form for reconstitution in an aqueous liquid carrier, wherein said composition is or can be obtained by the method of claim 69 or 70.

72. Lyophilized pharmaceutical composition according to claim 71, containing

a) liposome-forming structures that contain a therapeutically effective amount of cyclosporine A (CsA);

a membrane-forming substance selected from the group of phospholipids; and a solubility improving agent selected from the group of nonionic surfactants; And

b) at least one disaccharide selected from the group consisting of sucrose, lactose and trehalose, wherein the at least one disaccharide is present in an amount of at least 40% by weight of the total weight of the lyophilized composition.

73. Lyophilized pharmaceutical composition according to claim 71 or 72 for use as a medicinal product for inhalation.

74. Lyophilized pharmaceutical composition for use according to claim 73, characterized in that inhalation use is carried out after diluting (dispersing) the lyophilized pharmaceutical composition according to claim 71 or 72 in an aqueous liquid carrier to obtain a colloidal solution or dispersion.

75. Lyophilized pharmaceutical composition for use according to claim 73 or 74, characterized in that inhalation use is carried out after converting said composition into an aerosol, for example, by spraying.

76. Lyophilized pharmaceutical composition for use according to any one of claims 29-31, characterized in that inhalation use is carried out by inhalation.

77. Lyophilized pharmaceutical composition for use according to any one of claims 29 to 32, characterized in that pulmonary administration is carried out using an ultrasonic or electronic vibrating membrane nebulizer, preferably using a vibrating membrane nebulizer, such as, for example, an eFlow® type device , AeroNeb Pro or -Go or I-Neb.

78. Lyophilized pharmaceutical composition for use according to any one of claims 73-77 for the prevention and treatment of asthma, refractory asthma, chronic obstructive bronchitis, parenchymal, fibrotic and interstitial pulmonary diseases and inflammation, and preferably for the prevention and treatment of acute and chronic transplant rejection reactions organs after lung transplantation, as well as diseases caused by them, such as bronchiolitis obliterans.

The following examples are shown to illustrate the present invention, but are not intended to limit it in any way.

Examples

Example 1. Preparation of a dispersion containing cyclosporine A in liposomally solubilized form.

1.1. Step a: obtaining a mixture of starting ingredients.

1.1.1. Approximately 70% (~104 L) of water for injection was loaded into the reaction vessel. Water degasi

- 24 046086 was done by introducing nitrogen gas and raising the temperature to 40 to 45°C. 18.0 kg of sucrose, 450.0 g of sodium dihydrogen phosphate dihydrate, 612.0 g of sodium hydrogen phosphate decahydrate and 36.0 g of sodium edetate were added and approximately 5% (8.0 L) water for injection was used for washing. The mixture was stirred until a visually clear solution was obtained.

1.1.2. Cool the solution to 5-25°C and add 6480.0 g of soy lecithin Lipoid S100 and mix until a homogeneous mixture is obtained. 504.0 g of polysorbate 80 HP (Tween 80) was then added with moderate stirring to avoid foam formation and the container containing the polysorbate was washed using approximately 100 ml of water for injection. After this, 720.0 g of cyclosporine and approximately 5% (8 L) water for injection were added.

1.2. Step b: dispersing the initial mixture of ingredients.

1.2.1. The mixture of ingredients obtained in accordance with step 1.1 above was transferred to a 400 L vessel and dispersed for 8 hours at a rotation speed of 4800 rpm using an Ultra Turrax® UTE 115-P (IKA, Germany) high shear immersion dispersant , equipped with a cylindrical stator with an internal diameter of 102.5 mm and 30 teeth and a rotor (TP4, IKA, Germany) having an external diameter of 101.0 mm and 27 teeth (gap between rotor teeth and between stator teeth: 4.0 mm ), with a motor power of 5.5 kW, providing a shear rate gradient of 33,828 1/s and a shear frequency of 64,800 1/s, until a uniform dispersion is formed. After this, the resulting dispersion was stirred for 3 hours, and then 2 portions of 10 liters (each) of water for injection were added to reduce the formation of foam.

1.3. Step c: homogenization of the intermediate aqueous dispersion.

1.3.1. The resulting dispersion was then transferred to a GEA high pressure homogenizer through a 225 µm stainless steel protective filter (Rigimesh® filter, PALL) and then homogenized under high pressure of 100 bar (first stage) and 1000 bar (second stage), respectively. at temperatures up to 25°C. Homogenization at high pressure was repeated 9 times (cycles). After the eighth homogenization, approximately 8 L of water for injection was added.

1.4. Stage d: bioburden reduction.

1.4.1. The resulting homogenized dispersion was then filtered through a 0.2 μm bioburden reduction filter (Fluorodyne ® EX; PALL) and transferred to a filling/storage tank.

1.5. Product characteristics.

1.5.1. The resulting homogenized dispersion had a cyclosporin A content of 100% of the total amount of cyclosporin A added in step 1.1.2.

Example 2. Preparation of a dispersion containing cyclosporine A in liposomally solubilized form.

2.1. Step a: obtaining a mixture of starting ingredients.

2.1.1. The preparation of the original mixture of ingredients described in paragraph 1.1 above was exactly repeated.

2.2. Step b: dispersing the initial mixture of ingredients.

2.2.1. The mixture of ingredients obtained according to step 2.1 above was transferred to a 400 L tank and dispersed for 80 min at a rotation speed of 3000 rpm using an Ultra Turrax® UTE 115-P (IKA, Germany) high shear submersible disperser equipped with a cylindrical stator with an internal diameter of 102.5 mm and 30 teeth; a rotor (TP4, IKA, Germany) with an external diameter of 101.0 mm and 27 teeth (the gap between the rotor teeth, as well as between the stator teeth: 4.0 mm), with a motor power of 5.5 kW, providing a shear rate gradient of 21153 1/s and a shear frequency of 40500 1/s, until a uniform dispersion is formed. After this, the resulting dispersion was stirred for 3 hours, and then 2 portions of 10 liters (each) of water for injection were added to reduce the formation of foam.

2.3. Step c: homogenization of the intermediate aqueous dispersion.

2.3.1. The resulting dispersion was then transferred to a GEA high pressure homogenizer through a 40 µm protective filter (HDC II 40 µm filter (all polypropylene), PALL) and then homogenized under high pressure of 100 bar (first stage) and 1000 bar (second stage ), respectively, at a temperature of 25°C. Homogenization at high pressure was repeated 9 times (cycles). After the eighth homogenization, approximately 8 L of water for injection was added.

2.4. Stage d: bioburden reduction.

2.4.1. The resulting homogenized dispersion was then filtered through a 0.2 μm bioburden reduction filter (Fluorodyne ® EX; PALL) and transferred to a filling/storage tank.

2.5. Product characteristics.

2.5.1. The resulting homogenized dispersion had a cyclosporine content of 95.47% of the total amount of cyclosporine A added in step 2.1.

- 25 046086

Example 3. Preparation of a dispersion containing cyclosporine A in liposomally solubilized form using a built-in dispersant.

3.1. Step a: obtaining a mixture of starting ingredients.

3.1.1. The preparation of the original mixture of ingredients described in paragraph 1.1 above was exactly repeated.

3.2. Step b: dispersing the initial mixture of ingredients.

3.2.1. The mixture of ingredients obtained according to step 3.1 above was then transferred to a 400 L vessel and dispersed for 4 hours at 4000 rpm using an ultra Turrax® UTL 1000/10 (IKA, Germany) with a built-in high shear dispersant , equipped with a dispersing tool (8SF, IKA, Germany), having a stator with an inner diameter of 120.1 mm and 54 teeth and a tooth gap of 1.6 mm and a rotor with an outer diameter of 119.4 mm and 42 teeth with a tooth gap of 2, 0 mm and a motor power of 7.5 kW, which provides a shear rate gradient of 71413 1/s and a shear frequency of 151200 1/s until a uniform dispersion is formed. After this, the resulting dispersion was stirred for 3 hours, and then 2 portions of 10 liters (each) of water for injection were added to reduce the formation of foam.

3.3. Step c: homogenization of the intermediate aqueous dispersion.

3.3.1. Homogenization of the intermediate aqueous dispersion obtained in step 3.2.1 was exactly repeated as described in step 1.3.1 above.

3.4. Stage d: bioburden reduction.

3.4.1. The resulting homogenized dispersion was then filtered through a 0.2 μm bioburden reduction filter (Fluorodyne ® EX; PALL) and transferred to a filling/storage tank.

3.5. Product characteristics.

3.5.1. The resulting homogenized dispersion had a cyclosporin A content of 100% of the total amount of cyclosporin A added in step 3.1.

Example 4 Preparation of a dispersion containing cyclosporine A in liposomally solubilized form using an immersion dispersant and an integrated dispersant.

4.1. Step a: obtaining a mixture of starting ingredients.

4.1.1. The preparation of the initial mixture of ingredients described in paragraph 1.1 above is repeated in its entirety.

4.2. Step b: dispersing the initial mixture of ingredients.

4.2.1. The mixture of ingredients obtained in accordance with step 4.1 above is then transferred to a 400 L vessel and dispersed for 30 min at a rotation speed of 4800 rpm using an Ultra Turrax® UTE 115-P (IKA, Germany) high shear submersible disperser force, equipped with a cylindrical stator with an internal diameter of 102.5 mm and 30 teeth and a rotor (TP4, IKA, Germany) with an external diameter of 101.0 mm and 27 teeth (gap between rotor teeth and between stator teeth: 4.0 mm) with a motor power of 5.5 kW, which provides a shear rate gradient of 33828 1/s and a shear frequency of 64800 1/s. After this, the resulting mixture is dispersed for 4 hours at a rotation speed of 4000 rpm using a built-in Ultra Turrax® UTL 1000/10 (IKA, Germany) high shear disperser equipped with a dispersing tool (8SF, IKA, Germany) having a stator with an internal diameter of 120.1 mm and 54 teeth and a tooth gap of 1.6 mm and a rotor with an outer diameter of 119.4 mm and 42 teeth with a tooth gap of 2.0 mm with a motor power of 7.5 kW, which provides a speed gradient shift 71413 1/s and shift frequency 151200 1/s, until a uniform dispersion is formed. After this, the resulting dispersion was stirred for 3 hours, and then 2 portions of 10 liters (each) of water for injection were added to reduce the formation of foam.

4.3. Step c: homogenization of the intermediate aqueous dispersion.

4.3.1. The resulting dispersion is then transferred to a GEA high pressure homogenizer through a 225 µm stainless steel protective filter (Rigimesh® filter, PALL) and then homogenized under high pressure of 100 bar (first stage) and 1000 bar (second stage), respectively. at temperatures up to 25°C. The high pressure homogenization procedure was carried out 8 times (cycles). After the sixth homogenization, approximately 8 liters of water for injection are added.

4.4. Stage d: bioburden reduction.

4.4.1. The resulting homogenized dispersion is then filtered through a 0.2 µm bioburden reduction filter (Fluorodyne® EX; PALL) and transferred to a filling/storage tank.

4.5. Product characteristics.

4.5.1. The resulting homogenized dispersion has a cyclosporin A content of 100% of the total amount of cyclosporin A added in step 4.1.

Example 5: Aseptic filling, lyophilization and packaging.

5.1. Glass vials with a filling volume of 10 ml were sterilized in a hot air sterilization tunnel, cooled and filled with 1.35 ml aliquots (5 mg dose) of the dispersion obtained

- 26 046086 according to example 1, as described above, after aseptic sterilization using two sterile filters with a pore size of 0.2 μm, located between the filling/storage tank and the filling heads. The vials were then partially sealed with sterilized lyophilization stoppers and loaded into a lyophilizer (GEA Lyovac FCM) and lyophilized in a 72-hour lyophilization cycle.

5.2. After lyophilization was completed, the vials were automatically sealed tightly in the lyophilization chamber. The vials were unloaded and sealed with flip-off caps. Each vial contained approximately 190 mg of an off-white, uniform, porous lyophilized precipitate containing 5 mg of cyclosporin A in liposomal solubilized form, with a maximum residual moisture level of 2% (w/w) and a shelf life of 3 years.

5.3. The composition of the lyophilized drug obtained as described above is given below in table. 1.

Table 1

Ingredient Amount per standard dose Quantity, % (w/w) Cyclosporine A 5 mg 2.69 Polysorbate 80 3.5 mg 1.88 Lipoid S100 45 mg 24.18 Sucrose 125 mg 67.16 Sodium dihydrogen phosphate dihydrate 3.125 mg 1.68 Disodium hydrogen phosphate dodecahydrate 4.25 mg 2.28 Edetate disodium dihydrate 0.25 mg 0.13 Total 186.125 mg 100.00

Example 6: Reconstitution of a lyophilized composition containing cyclosporin A to produce a colloidal solution of cyclosporin A in liposomally solubilized form for nebulization and inhalation.

6.1. To an aliquot of 186.1 mg of the lyophilized sediment prepared in accordance with Example 5 above containing 5 mg of cyclosporin A was added 1.20 ml of a sterile aqueous solution of sodium chloride at a concentration of 0.25% (w/v) to obtain 1.35 ml of an opalescent aqueous solution of liposomal cyclosporine A for inhalation with a CsA concentration of 4 mg/ml. The delivered volume using the device is 1.25 ml (5 mg L-CsA).

6.2. To obtain the corresponding colloidal solution containing 10 mg of cyclosporin A in liposomal solubilized form, dissolve a 372.3 mg aliquot of the lyophilization sediment obtained in accordance with Example 1 described above in 2.40 ml of a sterile aqueous solution of sodium chloride with a concentration of 0.25 % (wt./vol.) to obtain 2.65 ml of an opalescent aqueous solution of liposomal cyclosporine A for inhalation with a CsA concentration of 4 mg/ml. The delivered volume using the device is 2.50 ml (10 mg L-CsA).

6.3. The composition of the diluted drug obtained as described above is given in the table below. 2.

table 2

Ingredient Amount per standard dose Quantity, % (wt./vol.) Cyclosporine A 5 mg 0.4 Polysorbate 80 3.5 mg 0.28 Lipoid S100 45 mg 3.6 Sucrose 125 mg 10 Sodium Dihydrogen Phosphate Dihydrate 3.125 mg 0.25 Disodium hydrogen phosphate dodecahydrate 4.25 mg 0.34 Edetate disodium dihydrate 0.25 mg 0.02 Sodium chloride 2.8 mg 0.22 Water for injections Up to 1.25 ml Up to 100

Example 7. Preparation of lyophilized compositions containing CsA in liposomally solubilized form, and their diluted liposomal solutions in the presence of lactose or trehalose.

7.1. Following the protocols described above in Example 1 and Example 5, lyophilized compositions containing CsA in liposomally solubilized form were prepared in the presence of trehalose as a disaccharide and lactose monohydrate. Both disaccharides were used in the amount required

- 27 046086 to obtain the corresponding sugar content of 7.5 and 10% (w/v) in the prepared diluted liposome solution. In addition, in addition to the composition described in table. 2, the corresponding liposome solutions were prepared with sucrose contents of 5.0 and 7.5% (w/v). In all cases, opalescent colloidal solutions were prepared with the polydispersity index (PI) and liposome diameter (measured as z-average diameter, Zcp.) shown in Table 1 below. 3.

Table 3

Disaccharide Content [% (w/v)] Zcp. [nm] P.I. Opalescent solution Trehalose 7.5 123.2 0.344 Yes Trehalose 10 103.5 0.181 Yes Lactose monohydrate 7.5 89.1 0.115 Yes Lactose monohydrate 10 88.1 0.101 Yes Sucrose 5 93.0 0.346 Yes Sucrose 7.5 86.1 0.445 Yes Sucrose 10 47.7 0.253 Yes

Example 8. Comparison of the characteristics of aqueous liposome dispersions containing CsA in liposomally solubilized form, before lyophilization and after dilution of the lyophilisate.

8.1. An aqueous dispersion of CsA in liposomal solubilized form containing 10% (wt./vol.) sucrose was prepared as described in example 1. In the same way, an aqueous dispersion of CsA in liposomal solubilized form containing 10% (wt./vol.) sucrose was prepared. ) lactose. In addition, an aqueous dispersion containing 10% (w/v) sucrose was lyophilized as described in Example 5 and diluted using water for injection. The main characteristics of the resulting dispersions are shown below in table. 4.

Table 4

Dispersion containing CsA pH Zcp. [nm] P.I. Osmolality [osmol/kg] 10% (w/v) sucrose solution; before lyophilization/reconstitution 6.62 51.0 0.262 0.436 10% (w/v) sucrose solution; after lyophilization/dilution 6.64 55.3 0.230 0.383 10% (w/v) lactose solution; before lyophilization/reconstitution 6.58 50.5 0.251 0.433

Example 9. Stability of lyophilized compositions containing CsA in liposomally solubilized form; comparison of stability.

9.1. Long-term stability of lyophilized compositions containing cyclosporine A.

9.1.1. A lyophilized pharmaceutical composition containing cyclosporine A (5 mg) was prepared in accordance with Example 1 and Example 5 described above. Aliquots of the lyophilized composition in the form of an off-white, homogeneous, porous precipitate after lyophilization were placed in 6R glass vials, capped and stored at 25°C and 60% relative humidity (RH) for 36 months. Before and after the storage period, aliquots of the material were diluted with saline (0.25% (w/v)) to obtain 1.25 ml of diluted solution and the median (Z-average) liposome diameter, polydispersity coefficient, and cyclosporine A content were determined after 3, 6, 9, 12, 18, 24 and 36 months.

9.1.2. Before and after the above storage period, all parameters were found to be within the relevant acceptance criteria. In particular, the polydispersity index (PI) was no more than 0.50 before and after each storage period. In addition, the median (Z-average) diameter of the liposomes was within a specified range of 40 to 100 nm before and after each storage period. In addition, the content of CsA in the diluted solution was within the acceptance criterion of 95.0 to 105.0%.

9.1.3. The long-term stability study described above was repeated at 30°C and 65% relative humidity (RH). All analyzed parameters described above were within the acceptance criteria (mentioned above) before and after storage for 3, 6, 9 and 12 months.

9.1.4. The long-term stability study described above was repeated using a lyophilized pharmaceutical composition containing cyclosporine A (5 mg) prepared in accordance with Example 1 described above, however, in this case, the lyophilized composition had the sucrose content necessary to obtain a liquid composition with content

- 28 046086 I eat sucrose 7.5 wt.% relative to the total weight of the liquid composition after dilution.

9.1.5. In this case also, it was found that before and after the storage period described above, all parameters were within the relevant acceptance criteria. In particular, the polydispersity index (PI) was no more than 0.50 before and after each storage period. In addition, the median (Z-average) diameter of the liposomes was within a specified range of 40 to 100 nm before and after each storage period. In addition, the content of CsA in the diluted solution was within the acceptance criterion of 95.0 to 105.0%.

9.1.6. The experiments described in paragraphs 9.1.1 to 9.1.3 above were repeated using a lyophilized pharmaceutical composition containing 10 mg of cyclosporin A prepared in accordance with Examples 1 and 5 described above. In this case also, it was found that before and after the storage period described above, all parameters were within the relevant acceptance criteria. In particular, the polydispersity index (PI) was no more than 0.50 before and after each storage period. In addition, the median (Z-average) diameter of the liposomes was within a specified range of 40 to 100 nm before and after each storage period. In addition, the content of CsA in the diluted solution was within the acceptance criterion of 95.0 to 105.0%.

Example 10 Spray experiments and aerosol characteristics.

10.1. 2.5 ml (corresponding to 10 mg of CsA) of the colloidal solution obtained in examples 1, 5 and 6 (sequentially) was converted into an aerosol using a specially adapted electronic nebulizer with a vibrating membrane type PARI eFlow 30 XL, having a mixing chamber and inhalation valves /exhalation operating at a flow rate of 15 l/min, in accordance with European Pharmacopoeia 7.3; 2.9.44.

10.2. The droplet size distribution of the resulting aerosol was determined using laser diffraction on a Malvern MasterSizer X. The measured mean particle diameter was 3.3 μm (standard deviation (SD) 0.1) with a geometric standard deviation of 1.5. The respirable fraction (RF) of particles <5 µm was 65.3% (SD 2.8), the respirable fraction of particles <3.3 µm was 37.7% (SD 2.2).

10.3. For experimental inhalation (adults; flow rate 15 ml/min.), a total amount of 9897 μg of cyclosporine A was used in the form of the diluted liquid preparation described in Example 1 above, which was filled into an electronic vibrating membrane nebulizer (PARI eFlow 30 XL), with which carried out the introduction. The delivered dose (DD) of cyclosporine A was 7339 μg (SD: 471). The inhalable dose (RD) for particle size <5 μm was 6534 μg (66.0%; SD 4.3%); The RD for particle size <3.3 µm was 4461 µg (45.1%; SD 3.2%), and the inhalable dose (RD) for particle size <2 µm was 1080 µg (10.9%; SD 0 ,9%).

Example 11. Preparation of a dispersion containing tacrolimus in liposomally solubilized form.

11.1. Step a: obtaining a mixture of starting ingredients.

11.1.1. Fill approximately 70% of the preparation vessel (~104 L) with water for injection. Degass by introducing nitrogen and increase the temperature to a value of 40 to 45°C. 18.0 kg of sucrose, 450.0 g of sodium dihydrogen phosphate dihydrate, 612.0 g of sodium hydrogen phosphate decahydrate and 36.0 g of sodium edetate are added and approximately 5% (8.0 L) water for injection is used for washing. Stir the mixture until a clear-looking solution is obtained.

11.1.2. Cool the solution to 5-25°C and add 6480.0 g of soy lecithin Lipoid S100 and mix until a homogeneous mixture is obtained. Then add 504.0 g of polysorbate 80 HP (Tween 80) with gentle stirring to prevent foaming and rinse the container in which the polysorbate is stored with approximately 100 ml of water for injection. 720.0 g of tacrolimus and approximately 5% (8 L) water for injection are then added.

11.2. Step b: dispersing the initial mixture of ingredients.

11.2.1. The mixture of ingredients obtained in step 11.1, described above, is transferred into a 400 l vessel and dispersed for 8 hours at a speed of 4,800 rpm using an Ultra Turrax® UTE 115-P immersion dispersant (IKA, Germany) with high shear force, equipped with a cylindrical stator with an internal diameter of 102.5 mm and 30 teeth and a rotor (TP4, IKA, Germany) having an external diameter of 101.0 mm and 27 teeth (gap between rotor teeth and stator teeth: 4.0 mm) with a motor power of 5.5 kW, which provides a shear rate gradient of 33,828 1/s and a shear frequency of 64,800 1/s until a uniform dispersion is formed. The resulting dispersion is stirred for 3 hours, after which 2 portions of 10 liters (each) of water for injection are added to reduce the formation of foam.

11.3. Step c: homogenization of the intermediate aqueous dispersion.

11.3.1. The resulting dispersion is transferred to a GEA high pressure homogenizer through a 225 µm stainless steel protective filter (Rigimesh® filter, PALL) and then homogenized under high pressure of 100 bar (first stage) and 1000 bar (second stage), respectively, at temperatures up to 25°C. The high pressure homogenization procedure is carried out 8 times (cycles). After six

- 29 046086 Approximately 8 liters of water for injection are added to this homogenization.

11.4. Stage d: bioburden reduction.

11.4.1. The resulting homogenized dispersion is then filtered through a 0.2 µm bioburden reduction filter (Fluorodyne ® EX; PALL) and transferred to a filling/storage tank.

11.5. Product characteristics.

11.5.1. The resulting homogenized dispersion is expected to have a tacrolimus content of 100% of the total amount of tacrolimus added in step 11.1.2.

Claims (30)

CLAIM 1. A method for producing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form in an aqueous liquid carrier, comprising the following steps: a) providing a mixture containing an inhaled immunosuppressive macrocyclic active ingredient; a membrane-forming substance selected from the group of phospholipids; a solubility improver selected from the group of nonionic surfactants; and an aqueous liquid carrier; b) dispersing the mixture according to step a) at a shear rate gradient of at least 22,000 1/s to obtain an intermediate aqueous dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; And c) homogenizing the intermediate aqueous dispersion obtained in step b) to form a dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form. 2. Method according to claim 1, characterized in that the dispersion according to step b) is carried out using a rotor-stator dispersant. 3. Method according to claim 1 or 2, characterized in that the mixture obtained in step a) contains one or more auxiliary substances. 4. Method according to any of the preceding claims, characterized in that the dispersion in step b) is carried out using a submersible dispersant. 5. The method according to any of the preceding claims, wherein the dispersion in step b) is carried out at a shear rate gradient selected in the range from 25,000 to 40,000 1/s. 6. Method according to any of the preceding claims, characterized in that the dispersion in step b) is carried out at a shear frequency selected in the range from 50,000 to 80,000 1/s. 7. The method according to any of the preceding claims, wherein the dispersion in step b) is carried out at a shear rate gradient selected in the range from 28,000 to 37,000 1/s, and at a shear frequency selected in the range from 50,000 to 80,000 1/s. 8. Method according to any one of claims 2-7, characterized in that the rotor diameter is from 60 to 140 mm. 9. Method according to any one of claims 2 to 8, characterized in that the dispersion in step b) is carried out at a rotation speed in the range from 2000 to 6000 rpm. 10. Method according to any of the preceding claims, characterized in that the dispersion in step b) is carried out using a built-in dispersant. 11. The method according to claim 10, characterized in that the dispersion according to step b) is carried out at a shear rate gradient selected in the range from 45,000 to 90,000 1/s. 12. Method according to claim 10 or 11, characterized in that the dispersion according to step b) is carried out at a shear frequency selected in the range from 100,000 to 200,000 1/s. 13. Method according to any one of claims 10 to 12, characterized in that the dispersion according to step b) is carried out at a shear frequency selected in the range from 120,000 to 180,000 1/s. 14. Method according to any one of claims 10 to 13, characterized in that the dispersion according to step b) is carried out at a shear rate gradient selected in the range from 55000 to 85000 1/s, and at a shear frequency selected in the range from 130000 to 170000 1/s. 15. Method according to any one of claims 10-14, characterized in that the rotor has a diameter in the range from 100 to 140 mm. 16. Method according to any one of claims 10 to 15, characterized in that the dispersion according to step b) is carried out at a rotation speed in the range from 3500 to 4500 rpm. 17. The method according to any of the preceding claims, wherein the dispersion according to step b) is carried out using a submersible dispersant and a built-in dispersant. 18. The method according to claim 17, characterized in that a submersible dispersant and a built-in dispersant are used sequentially. 19. The method according to claim 17 or 18, characterized in that dispersion is started using a submersible dispersant, followed by the use of a built-in dispersant. - 30 046086 20. The method according to any one of claims 2 to 19, in which the dispersion according to step b) is carried out with a rotor peripheral speed selected in the range from 15 to 40 m/s. 21. Method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out for a period ranging from 1 to 8 hours. 22. Method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out over a period of 3 to 8 hours. 23. A method according to any of the preceding claims, characterized in that the dispersion according to step b) is carried out using an immersion dispersant for a period of time up to 1 hour, followed by dispersion using an integral dispersant for a period of time from 1 to 4 hours. 24. The method according to any of the preceding claims, characterized in that the method includes, as an additional step b1) filtering the intermediate aqueous dispersion containing the inhaled immunosuppressive macrocyclic active ingredient in the aqueous liquid carrier obtained in step b), before homogenizing the prepared filtered intermediate aqueous variance according to step c). 25. Method according to claim 24, characterized in that the filtration according to step b1) is carried out using a filter with an average pore radius in the range from 200 to 250 μm. 26. The method according to any of the preceding claims, which includes, as an additional step c1) sterilization of a homogenized dispersion containing an inhaled immunosuppressive macrocyclic active ingredient, in particular cyclosporine A, in the liposomally solubilized form obtained in step c). 27. The method according to claim 26, characterized in that sterilization is carried out by sterilizing filtration. 28. The method of any one of the preceding claims, wherein the inhaled immunosuppressive macrocyclic active ingredient is cyclosporine A (CsA). 29. A method of preparing a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, wherein the lyophilized pharmaceutical composition contains an inhaled immunosuppressive macrocyclic active ingredient in a liposomally solubilized form, the method comprising preparing a dispersion containing an inhaled immunosuppressive macrocyclic active ingredient in a liposomal solubilized form in an aqueous liquid carrier , according to the method according to any of the preceding paragraphs; and additionally includes the stage d) removing the aqueous liquid carrier, at least partially, by lyophilization to obtain a lyophilized pharmaceutical composition. 30. A lyophilized pharmaceutical composition containing an inhaled immunosuppressive macrocyclic active ingredient in liposomally solubilized form for dilution in an aqueous liquid carrier, wherein said composition is prepared by the method of claim 29, wherein the resulting homogenized dispersion is characterized by an active ingredient content of at least 97% of the total amount of active ingredient added to the original mixture.