Process for the preparation of inhalable dispersions containing immunosuppressive active ingredients - Patent Application 20070122997

Abstract

The present invention relates to a process for the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier, the process comprising: a) -inhalable immunosuppressive macrocyclic active ingredients; - membrane-forming substances selected from the group of phospholipids; - solubility enhancers selected from the group of nonionic surfactants; optionally one or more excipients; and - providing a mixture comprising an aqueous liquid carrier; b) dispersing the mixture provided in step a) to form an intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; and c) homogenizing the intermediate aqueous dispersion as formed in step b) to form a dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form.

Description

[Technical Field]

[0001] The present invention relates to a process for the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier. In particular, the present invention relates to a process for the preparation of a dispersion comprising cyclosporin A in liposome-solubilized form. Furthermore, the present invention relates to a process for the preparation of a lyophilized pharmaceutical composition comprising an inhalable immunosuppressive active ingredient, and to the lyophilized pharmaceutical composition obtainable by such a process. [Background technology]

[0002] Cyclosporin (CsA or "cyclosporin" are used synonymously herein) is a cyclic oligopeptide with immunosuppressive and calcineurin-inhibitory activity. It is characterized by a selective and reversible mechanism of immunosuppression by blocking the activation of T lymphocytes through the production of certain cytokines involved in their regulation. It is particularly involved in the inhibition of interleukin-2 synthesis, simultaneously suppressing the proliferation of cytotoxic T lymphocytes, which are responsible for, for example, the rejection of foreign tissues. Cyclosporin acts intracellularly by binding to so-called cyclophilins or immunophilins, which belong to a family of proteins that bind cyclosporin with high affinity. The cyclosporin-cyclophilin complex then blocks the serine-threonine phosphatase calcineurin. This active state consequently controls the activation of transcription factors such as NF-KappaB or NFATp / c, which play a crucial role in the activation of various cytokine genes, including interleukin-2. This controlled activation results in the arrest of immunocompetent lymphocytes in the G0 or G1 phase of the cell cycle, since proteins essential for cell division, such as interleukin-2, cannot be further produced. T helper cells, which increase the activity of cytotoxic T cells responsible for rejection, are the preferred site of attack for cyclosporine. Furthermore, cyclosporine inhibits the synthesis and release of additional lymphokines responsible for the proliferation of mature cytotoxic T lymphocytes and other lymphocyte functions. Cyclosporine's ability to block interleukin-2 is crucial to its clinical effectiveness: transplant patients who tolerate transplants well are characterized by low interleukin-2 production. In contrast, patients with overt rejection do not exhibit any inhibition of interleukin-2 production.

[0003] The first, and so far only, cyclosporine to be commercially available (in the 1980s) was cyclosporine A (CsA). CsA is chemically defined as cyclo-[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl]. Its effectiveness marked the beginning of a new era in transplantation medicine, as it was possible, with the help of cyclosporine, to substantially increase the proportion of transplanted organs that remained functional in the long term. The first cyclosporine drug (Sandimmun®, Sandoz) was already able to approximately double the success rate in kidney transplants. Since the 1990s, new oral combinations of cyclosporine (Neoral®, Sandoz, later Novartis) with higher and more reliable bioavailability have allowed for better dosing and further increased success rates. Despite the development of several new active agents, CsA remains the drug frequently used in transplant medicine.

[0004] Currently, lung transplantation can also be performed successfully if the patient is treated with CsA. Since the introduction of this active agent into clinical treatment, the number of lung transplants performed worldwide has increased dramatically. This applies to both single and double lung transplants. Lung transplantation is usually considered for patients with end-stage lung disease, where drug therapy has failed and the disease has a short life expectancy. Single lung transplantation is indicated in cases of certain forms of emphysema and fibrosis, such as idiopathic pulmonary fibrosis. In cases of cystic fibrosis (mucoviscidosis), primary pulmonary hypertension, emphysema with global dysfunction, and idiopathic pulmonary fibrosis with complications from frequent severe infections and reinfections, both lungs are transplanted. Successful lung transplantation can improve the patient's quality of life again to a nearly normal level. However, in contrast to heart, kidney, and liver transplants, survival after lung transplantation remains relatively short, averaging only 5 years. This may be due, inter alia, to the fact that the active drug, cyclosporine, cannot be administered effectively to all patients due to systemic side effects such as renal insufficiency, elevated serum levels of creatinine and urea, renal damage accompanied by structural changes such as interstitial fibrosis, elevated serum levels of bilirubin and liver enzymes, hirsutism, tremor, fatigue, headache, diffuse hypertrophic gingivitis, anorexia, abdominal pain, nausea, vomiting, diarrhea, gastritis, gastroenteritis, paresthesia, tingling pain in the hands and feet, arterial hypertension, elevated blood fat levels, acne, rash, allergic skin reactions, hyperglycemia, anemia, gout, weight gain, edema, gastric ulcers, cramps, menstrual disorders, hyperkalemia, hypomagnesemia, hot flashes, erythema, itching, muscle cramps, muscle pain, and myopathy.

[0005] Therefore, for example, after lung transplantation, or in certain other indications, it is desirable to administer CsA in a targeted and tissue-specific manner, with the aim of minimizing the effects of the active agent on healthy tissues, resulting in only low systemic bioavailability of the active agent.

[0006] Suitable dosage forms can also be used for the treatment and prevention of diseases such as asthma, idiopathic pulmonary fibrosis, sarcoidosis, alveolitis, and parenchymal lung diseases (see Drugs for the Treatment of Respiratory Diseases, Domenico Spina, Clive P. Page et al., eds., Cambridge University Press, 2003, ISBN 0521773210). Novel therapeutic approaches also arise for the topical treatment of possible autoimmune conditions, including neurodermatitis, psoriasis, nonspecific eczema, skin hyperplasia, or skin degeneration, and for the treatment after skin transplantation. An interesting field of application is in the ophthalmology field, for example, for the treatment of keratoconjunctivitis after corneal transplantation, or other infectious eye diseases that respond partially or insufficiently to anti-inflammatory treatment, for example, with steroids. It is also useful for the treatment of keratitis in animals, such as dogs.

[0007] Attempts have been made to administer cyclosporine topically, for example, in the form of 1% and 2% oily eye drops (prepared according to the German Pharmacopoeia (official formulary) using refined peanut oil as a solubilizer) or as an aerosol. However, this approach has typically failed, primarily due to the extremely low water solubility of the active drug, which significantly complicates effective administration. Therefore, in the case of pulmonary administration, certain adjuvants that can be used for solubilization in oral administration cannot be used due to lack of tolerance. For example, Sandimmun® Optoral capsules (Novartis) containing cyclosporine A contain a microemulsion concentrate with ethanol, propylene glycol, and a significant amount of surfactant. Therefore, they constitute a formulation that may cause serious toxic effects if inhaled. Similarly, Sandimmun® Infusion Concentrate (Novartis) is available for infusion, but it is also not inhalable. The only adjuvants contained therein are ethanol and poly(oxyethylene)-40-castor oil. It can only be used for infusion, as it is pre-diluted in a ratio of 1:20 to 1:100 with 0.9% sodium chloride solution or 5% glucose solution, which allows for large volumes to be administered by infusion but not by inhalation.

[0008] WO 2007 / 065588 discloses a liquid pharmaceutical composition containing a therapeutically effective amount of cyclosporine, an aqueous liquid carrier, a first solubilizing agent selected from the group of phospholipids, and a second solubilizing agent selected from the group of non-ionic surfactants. The disclosed composition is suitable for oral, parenteral, nasal, mucosal, topical, and, in particular, pulmonary application in aerosol form.

[0009] WO 2016 / 146645 discloses a liposomal formulation of cyclosporine, preferably comprising unilamellar liposomes, preferably having a mean diameter of at most about 100 nm, measured as the z-average using photon correlation spectroscopy, and a molecular weight dispersity of at most about 0.5, as measured by photon correlation spectroscopy.

[0010] The formulation may be presented as a solid formulation for reconstitution with an aqueous solvent immediately prior to inhalation. Solid formulations may be prepared by any method suitable for removing solvent from a liquid formulation. Preferred examples of methods for preparing such solid formulations include freeze-drying and spray-drying. To protect the active ingredient during the drying process, it may be useful to incorporate a cryoprotectant and / or bulking agent, such as a sugar or sugar alcohol, particularly sucrose, fructose, glucose, trehalose, mannitol, sorbitol, isomalt, or xylitol. Of particular note, however, is that sugars may be added to preformed formulations containing liposome-encapsulated CsA.

[0011] In view of the high potency of many macrocyclic immunosuppressive active ingredients, such as cyclosporin A, tacrolimus, sirolimus, and / or everolimus, and the potentially serious and undesirable side effects that these potent compounds can have if not administered appropriately, it is extremely important that the content of these active ingredients in pharmaceutical compositions containing the macrocyclic immunosuppressive compounds can be accurately controlled during the manufacturing process, in order to avoid the potentially serious side effects described above due to overdosing of these compounds, or to avoid a lack of efficacy due to underdosing, which can even lead to serious complications such as transplant rejection.

[0012] Therefore, it is of utmost importance that the content of macrocyclic immunosuppressive active drugs can be determined and controlled at each step of the manufacturing process of pharmaceutical compositions containing such active ingredients.

[0013] In this context, the very low solubility of many macrocyclic immunosuppressants (especially cyclosporin A and tacrolimus) in aqueous solutions is considered problematic. These compounds are typically prepared by fermentation and further purified by crystallization. Unfortunately, the physical properties of these highly purified compounds tend to change over time due to, for example, strong agglomeration, weak agglomeration, or clumping processes that result in the formation of larger particles that can have different, often less favorable, dispersion or dissolution behaviors. However, this can increase the risk of inaccurate concentrations of dispersed or dissolved active ingredients during the manufacturing process and in the final dosage form, especially when using materials that are not freshly prepared.

[0014] It is therefore an object of the present invention to provide an improved process for the preparation of pharmaceutical compositions comprising an inhalable immunosuppressive active ingredient, such as, for example, cyclosporin A, tacrolimus, sirolimus, everolimus or others, which is in most cases independent of the physical properties by which the macrocyclic immunosuppressive active ingredient is delivered and disposed.

[0015] Further objects of the present invention will become apparent from the present disclosure, including the examples and claims. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2007 / 065588 Pamphlet [Patent Document 2] International Publication No. 2016 / 146645 Brochure [Non-patent literature]

[0017] [Non-Patent Document 1] Drugs for the treatment of respiratory dis-eases, edited by Domenico Spina, Clive p.Page et.al., Cambridge University Press, 2003, ISBN 0521773210 Summary of the Invention

[0018] In a first aspect, the present invention relates to a process for the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier, the process comprising: a) -inhalable immunosuppressive macrocyclic active ingredients; - membrane-forming substances selected from the group of phospholipids; - solubility enhancers selected from the group of nonionic surfactants; optionally one or more excipients; and - providing a mixture comprising an aqueous liquid carrier; b) dispersing the mixture provided in step a) to form an intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; and c) homogenizing the intermediate aqueous dispersion as formed in step b) to form a dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form.

[0019] In a second aspect, the present invention relates to a process for the preparation of a lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form, for reconstitution in an aqueous liquid carrier, which process comprises the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier according to the process of the first aspect of the invention; d) removing the aqueous liquid carrier at least partially under lyophilization conditions to form a lyophilized pharmaceutical composition.

[0020] In a third aspect, the present invention provides a lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form, for reconstitution in an aqueous liquid carrier, which composition is obtained or obtainable according to a process according to the second aspect of the invention.

[0021] 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 application. DETAILED DESCRIPTION OF THE INVENTION

[0022] The terms "consist of," "consists of," and "consisting of" used in this specification are so-called closed language, which means that only the mentioned components are present. The terms "comprise," "comprises," and "comprising" used in this specification are so-called open language, which means that one or more additional components may or may not be present.

[0023] The term "active pharmaceutical ingredient" (also referred to throughout this document as "API") refers to any type of pharmaceutically active compound or derivative that is useful in the prevention, diagnosis, stabilization, treatment, or generally management of a medical condition, disorder, or disease.

[0024] The term "therapeutically effective amount" as used herein refers to a dose, concentration, or strength that is useful for producing a desired pharmacological effect. In the context of the present invention, the term "therapeutically effective" also includes prophylactic activity. The therapeutic dose should be defined according to the individual case to which it is applied. Depending on the nature and severity of the disease, the route of application, and the height and condition of the patient, the therapeutic dose should be determined by methods known to those skilled in the art.

[0025] In the context of the present invention, a "pharmaceutical composition" is a combination of at least one API and at least one adjuvant, which in the simplest case may be an aqueous liquid carrier such as water or saline, for example.

[0026] The use of "a" or "an" does not exclude plural referents. That is, the singular forms "a," "an," and "the" should be understood to include plural referents unless the context clearly indicates or requires otherwise. In other words, all references to individual features or limitations of this disclosure should include the corresponding plural features or limitations, and vice versa, unless the referenced context expressly dictates otherwise or clearly implies a contrary concept. Thus, the terms "a," "an," and "the" have the same meaning as "at least one" or "one or more," unless otherwise defined. For example, reference to "an ingredient" includes mixtures of ingredients, etc.

[0027] As used herein, the term "about" or "ca." compensates for variability accepted in the pharmaceutical industry and inherent in pharmaceuticals, such as differences in content due to manufacturing variations and / or product degradation over time. This term allows for any variation in pharmaceutical practice that allows the product being evaluated to be considered bioequivalent in mammals to the stated strength of the claimed product.

[0028] "Essentially," "about," "approximately," "substantially," and the like, in connection with a characteristic or value, include the exact characteristic or exact value, as well as any characteristic or value that is normally considered to be within a normal range or variability accepted in the relevant art. For example, "substantially water-free" means that no water is intentionally included in the formulation, but this does not exclude the presence of residual moisture.

[0029] In the context of the present invention, "aqueous colloidal solution" preferably refers to an organic solvent-free solution consisting primarily of unilamellar liposomes with an average diameter of at most 100 nm and / or a molecular weight dispersity (PI) of 0.50 or less, in which the active agent is at least primarily dissolved. Preferably, water, or more specifically, saline, is the only liquid solvent contained in the combination. Furthermore, the combination is preferably an aqueous solution or aqueous colloidal solution, i.e., a single-phase liquid system. Such a system is substantially free of dispersed particles having a particle size larger than that of colloidal particles. By convention, particles smaller than about 1 μm are considered to be colloidal particles that do not constitute a separate phase and do not form a physical interface. Sometimes, particles in the size range slightly larger than 1 μm are also considered to be colloidal. However, preferably, the aqueous colloidal solution used herein is essentially free of particles that do not clearly belong to the colloidal spectrum (i.e., particles with a diameter of 1 μm or more).

[0030] According to a first aspect, the present invention provides a process for the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier, the process comprising: a) -inhalable immunosuppressive macrocyclic active ingredients; - membrane-forming substances selected from the group of phospholipids; - solubility enhancers selected from the group of nonionic surfactants; optionally one or more excipients; and - providing a mixture comprising an aqueous liquid carrier; b) dispersing the mixture provided in step a) to form an intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; and c) homogenizing the intermediate aqueous dispersion as formed in step b) to form a dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form.

[0031] The process according to the first aspect of the present invention is suitable for preparing a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form. The term "dispersion" as used herein should be understood in a broad sense, and unless otherwise specified, refers to a system in which particles dispersed as a dispersed phase are dispersed in a dispersed phase, specifically in a continuous liquid phase, and especially in an aqueous liquid as a continuous phase. The term "inhalable immunosuppressive macrocyclic active ingredient" as used in the context of the present invention should be interpreted in a broad sense and includes immunosuppressive macrocyclic active ingredients that can be administered by inhalation to humans or animals, preferably humans or warm-blooded animals, particularly humans. Meanwhile, during such inhalation, the immunosuppressive macrocyclic active ingredient or API is delivered to at least a portion or part of the animal's or human's respiratory system, specifically to the animal's or human's lungs.

[0032] In the context of the present invention, the immunosuppressive macrocyclic active ingredient may be, for example, cyclosporin A (also referred to herein as "CsA"), tacrolimus, sirolimus and / or everolimus, but in particular cyclosporin A and / or tacrolimus, more in particular cyclosporin A, which is chemically defined as cyclo-[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl] (CAS number 59865-13-3), a cyclic peptide with immunosuppressive activity.

[0033] In the context of the present invention, cyclosporin A may be used of any quality or from any source that is suitable for preparing a pharmaceutical composition. For example, cyclosporin A may be prepared by standard fermentation techniques and, in certain embodiments, may be used in any suitable form, such as any particle size or powder form, in connection with the process according to the present invention.

[0034] The process according to the present invention allows the preparation of inhalable immunosuppressive macrocyclic components, such as cyclosporin A, tacrolimus, sirolimus, and / or everolimus, or mixtures containing one or more of these compounds, particularly cyclosporin A, in liposome-solubilized form. The term "liposome-solubilized form" as used herein means that at least one inhalable immunosuppressive macrocyclic component (particularly CsA) as described above is incorporated into or intercalated with a liposome-forming structure formed by other components, as described in more detail below, particularly a membrane-forming substance selected from the group of phospholipids and a solubility-enhancing substance selected from the group of nonionic surfactants. However, the liposome-forming structure as referred to herein may or may not have a continuous or closed bilayer membrane. In certain embodiments, the liposome-forming structure may be at least partially, or preferably predominantly, unilamellar. The term "unilamellar" as used herein means that the corresponding liposome-forming structure contains only a single layer formed by a single lipid bilayer membrane and does not contain multiple lipid bilayer membranes arranged in layers.

[0035] According to step a) of the process of the present invention, a mixture is provided as a first component, comprising at least one inhalable immunosuppressive macrocyclic active ingredient, in particular CsA, as described above. As a second component, the mixture provided according to step a) comprises 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.

[0036] As used herein, the term "membrane-forming material" means a material that is capable of self-assembling in an aqueous liquid carrier, e.g., water or saline, to form a lipid bilayer membrane, and / or that is capable of forming liposomes in an aqueous liquid carrier under conditions or circumstances described in more detail below.

[0037] Preferred phospholipids comprised by the liposome-forming structures of the invention are in particular natural phospholipids or mixtures of concentrated phospholipids, such as lecithins such as the commercially available Phospholipon® G90, 100 or Lipoid 90, S100. Thus, in a particular embodiment, the membrane-forming substance selected from the group of phospholipids is a mixture of natural phospholipids.

[0038] Phospholipids are amphipathic lipids containing phosphorus. Phospholipids, also known as phosphatides, play an important role in nature, particularly as bilayer-forming components of biological membranes. These phospholipids are frequently used for pharmaceutical purposes and are chemically derived from phosphatidic acid. Chemically derived phospholipids are acylated glycerol-3-phosphates, in which the fatty acid residues can be of different lengths (usually double). Derivatives of phosphatidic acid include, for example, phosphocholines or phosphatidylcholines, in which the phosphate group is additionally esterified with choline, as well as phosphatidylethanolamines and phosphatidylinositols. Lecithin is a natural mixture of various phospholipids and usually contains a high proportion of phosphatidylcholines. Preferred phospholipids according to the present invention are lecithin and pure or concentrated phosphatidylcholines, such as dimyristoylphosphatidylcholine, di-palmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0039] In a further particular embodiment, the membrane-forming substance selected from the group of phospholipids provided in the mixture of step a) of the present invention is a lecithin containing unsaturated fatty acid residues. In an even more particular embodiment, the membrane-forming substance selected from the group of phospholipids is a lecithin selected from the group consisting of soybean lecithin, Lipoid 90, Lipoid S75, Lipoid S100, Phospholipon® G90, Phospholipon® 100 or comparable lecithins. In a further particular embodiment, the membrane-forming substance selected from the group of phospholipids is selected from Lipoid S100, Lipoid S75, in particular Lipoid S100.

[0040] The third component of the mixture provided in step a) of the process of the present invention is a solubility enhancer selected from the group of nonionic surfactants, or a mixture of two or more different solubility enhancers selected from the group of nonionic surfactants. Other surfactants include nonionic surfactants with at least one substantially hydrophilic molecular region and at least one substantially lipophilic molecular region. There are monomeric, low-molecular-weight nonionic surfactants and nonionic surfactants with oligomeric and polymeric structures. Examples of suitable nonionic surfactants suitable as solubility enhancers for inclusion in the mixture of step a) of the present invention include polyoxyethylene alkyl esters, such as polyoxyethylene sorbitan oleate, sorbitan fatty acid esters, poloxamers, vitamin E-TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), and tyloxapol.

[0041] In certain embodiments, the solubility enhancer selected from the group of non-ionic surfactants may be selected from the group of polysorbates and vitamin E-TPGS, and preferably it is selected from the group of polysorbates. In a particularly preferred embodiment, the solubility enhancer selected from the group of non-ionic surfactants is polysorbate 80.

[0042] As an optional fourth component, the mixture provided in step a) of the process of the present invention may optionally contain one or more excipients. Suitable excipients referred to herein are known to those skilled in the art. For example, the mixture provided in step a) of the present invention may optionally contain a pH adjuster for adjusting the pH, such as a physiologically acceptable base, acid, or salt, optionally a buffer mixture. In this context, the term "physiologically acceptable" does not mean that one of the excipients must be acceptable by itself and in undiluted form. This is not the case, for example, with sodium hydroxide solution. However, it does mean that one of the excipients must be acceptable at the concentration at which it is contained in the lyophilized pharmaceutical composition, especially after reconstitution.

[0043] Suitable pH adjusters or buffers for adjusting the pH may be selected, inter alia, with regard to the intended route of application. Examples of potentially useful excipients in this group include sodium hydroxide solution; sodium, calcium, or magnesium basic salts of, for example, citrate, phosphate, acetate, tartrate, and lactate; sodium, calcium, or magnesium basic salts of amino acids; acid salts such as hydrogen or dihydrogen phosphate, especially their sodium salts; and organic and inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, cromoglycic acid, acetic acid, lactic acid, tartaric acid, succinic acid, fumaric acid, lysine, and methionine; and sodium or potassium acid hydrogen phosphates.

[0044] In a further particular embodiment, the mixture provided according to the process of step a) of the present invention may comprise one or more further excipients selected from chelating agents, for example such chelating agents are edetate disodium dihydrate, calcium sodium EDTA, preferably edetate disodium dihydrate.

[0045] Furthermore, the mixture provided according to step a) of the present invention may or may not contain an osmotically active adjuvant as an excipient in order to adjust the osmolality to the desired level after reconstitution. This is important in order to achieve good tolerability, especially in certain applications such as inhalation. To achieve the best possible physiological tolerability, the addition of such adjuvants does not necessarily result in an isotonic composition after reconstitution, but they are often called "tonicity agents", even though they do result in an isotonicity approximating physiological osmolality.

[0046] A particularly frequently used tonicity agent is sodium chloride, although this is not always preferred. In an advantageous embodiment of the present invention, the mixture according to step a) does not contain sodium chloride, except, of course, for the amount of sodium chloride widely distributed in nature that may also be present in pharmaceutical-grade water. In another embodiment, the mixture according to step a) does not contain sodium chloride, but contains an essentially neutral salt as the tonicity agent, such as sodium sulfate or sodium phosphate. It should be noted, however, that the tonicity agent may also be contained in an aqueous liquid carrier, for example, in the form of an aqueous sodium chloride solution (physiological saline). However, in this case, salts other than sodium salts may also be preferred. Thus, certain calcium and magnesium salts are known to have a positive or supporting effect on the inhalation of active agent solutions. This is because these salts themselves may offset the local irritation caused by administration, have the currently assumed bronchodilatory effect in clinical contexts (e.g., Hughes et al., Lancet. 2003; 361(9375): 2114-7), and / or inhibit the adhesion of microorganisms to proteoglycans in the mucous membranes of the respiratory tract, indirectly supporting mucociliary clearance as an organism's natural defense against pathogens (KW Tsang et al., Eur. Resp. 2003. 21, 932-938). For example, magnesium sulfate, which has excellent pulmonary tolerance and can be inhaled without concern, may be advantageous, as can calcium chloride (1-10 mmol).

[0047] In a further particular embodiment, suitable excipients that may be added to the mixture provided according to step a) of the present invention include saccharides or sugars, such as disaccharides, which are described in more detail below.

[0048] As a further component, the mixture provided according to step a) of the present invention comprises an aqueous liquid carrier or an aqueous liquid solvent. The aqueous liquid carrier or solvent may be water or an aqueous solution of a pharmaceutically acceptable salt or an isotonic agent, and may preferably be sterile. In another preferred embodiment, the sterile aqueous liquid carrier is water, preferably sterilized water or sterile water, such as water suitable for injection.

[0049] In a particular embodiment of the process of the present invention, the amount of membrane-forming substance selected from the group of phospholipids, preferably lecithin, provided in the mixture according to step a) is greater than the amount of solubility-enhancing substance selected from the group of nonionic surfactants. In an exemplary embodiment, the weight ratio of membrane-forming substance selected from the group of phospholipids, preferably lecithin, to solubility-enhancing substance selected from the group of nonionic surfactants, preferably polysorbate, is selected in the range of about 15:1 to about 9:1, preferably 14:1 to about 12:1, e.g., 13:1.

[0050] In a further particular embodiment, the weight ratio between the (total amount of) one or more membrane-forming substances selected from the group of phospholipids and, on the one hand, the solubility-enhancing substance selected from the group of nonionic surfactants, and, on the other hand, the inhalable macrocyclic active ingredient (in particular CsA) provided in the mixture according to step a) is selected in the range of from about 5:1 to about 20:1, preferably from about 8:1 to about 12:1, more preferably about 9:1.

[0051] In an even more specific embodiment, the weight ratio of the membrane-forming substance selected from the group of phospholipids (preferably lecithin), the solubility-enhancing substance selected from the group of non-ionic surfactants (preferably polysorbate), and the inhalable immunosuppressive macrocyclic active ingredient (especially CsA) is selected in the range of about 15:1:1.5 to about 5:0.3:0.5, preferably about 9:0.7:1.

[0052] In a further particular embodiment, the weight ratio of the membrane-forming substance selected from the group of phospholipids described above to the inhalable immunosuppressive macrocyclic active ingredient (especially CsA) is selected in the range of about 8:1 to about 11:1, preferably about 8.5:1 to about 10:1, for example about 9:1.

[0053] In an exemplary embodiment, the mixture provided according to process step a) of the present invention may comprise an inhalable immunosuppressive macrocyclic active ingredient (particularly CsA) at a concentration selected within the range of about 1 g / L to about 7 g / L, or about 2 g / L to about 6 g / L, or about 3 g / L to about 5 g / L, for example 4 g / L.

[0054] In a further exemplary embodiment, the mixture provided according to process step a) of the present invention has a concentration selected within the range of about 20 g / L to about 60 g / L, or about 30 g / L to about 50 g / L, or about 30 g / L to about 40 g / L, and may comprise a membrane-forming substance selected from the group of phospholipids as described above, in particular a lipid such as Lipoid S100.

[0055] In an even further exemplary embodiment, the mixture provided according to process step a) of the present invention may comprise a solubility enhancer selected from the group of non-ionic surfactants described above, in particular a polysorbate such as polysorbate 80, at a concentration selected within the range of about 1 g / L to about 5 g / L, in particular at a concentration selected within the range of about 2 g / L to about 4 g / L, or about 2.5 g / L to about 3.5 g / L.

[0056] In certain embodiments, the mixture provided according to process step a) of the present invention may comprise at least one saccharide or sugar, in particular at least one disaccharide, as an excipient. The disaccharide that may be comprised by the mixture according to process step a) may, in certain embodiments, be selected from the group consisting of sucrose (sucrose; as used herein, the terms "sucrose" and "sucrose" have the same meaning, β-D-fructofuranosyl α-D-glucopyranoside; CAS No. 57-50-1), lactose (β-D-galactopyranosyl-(1→4)-D-glucose; CAS No. 63-42-3), and trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside; CAS No. 99-20-7). In certain embodiments, the mixture provided according to process step a) comprises sucrose as an excipient.

[0057] In further particular embodiments of the mixture provided according to process step a) of the present invention, the weight ratio of the selected at least one saccharide or disaccharide (preferably sucrose) to the weight of cyclosporin A in the mixture provided according to step a) of the process of the present invention is selected in the 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 to about 27.5:1.

[0058] Thus, in a further exemplary embodiment, the mixture provided according to step a) of the present invention may comprise a disaccharide, in particular sucrose, trehalose and / or lactose, in particular sucrose at a concentration selected within the range of about 60 g / L to about 140 g / L, or about 80 g / L to about 120 g / L, or about 90 g / L to about 110 g / L.

[0059] To provide a mixture according to step a) of the process of the present invention, the selected components described above may be added to a suitable container, such as a steering vessel, and stirred using standard techniques until a homogeneous mixture is formed. In certain embodiments, the components may be added to the container all at once or sequentially as needed. For example, a selected aqueous liquid carrier or solvent, such as sterile water, may be added to the container as the first component.

[0060] In further exemplary embodiments, the selected excipient may then be added to an aqueous liquid carrier, particularly water, and stirred until sufficient or complete dissolution is achieved. Additional ingredients may be added to the resulting mixture or solution all at once or sequentially, all together. For example, a selected membrane-forming substance selected from the group of phospholipids may be added, and the resulting mixture may be stirred until a sufficiently homogeneous dispersion is formed. Thereafter, a selected solubility-enhancing substance selected from the group of nonionic surfactants may be added to the mixture and stirred until a sufficiently homogeneous dispersion is formed. Finally, according to exemplary embodiments, the selected inhalable immunosuppressive macrocyclic active ingredient (particularly CsA) may be added and stirred until a sufficiently homogeneous dispersion is formed.

[0061] The preparation of the mixture according to process step a) of the present invention may be carried out at about room temperature, or at a temperature above or below room temperature, depending on the particular components to be added or dissolved. This is typically a temperature selected within the range of about 0°C or about 5°C to about 45°C or about 50°C. In certain embodiments, some components, particularly excipients or salts, may be added at elevated temperatures, for example, from about 35°C to about 50°C, or from about 40°C to about 45°C. The temperature may then be lowered to a temperature below room temperature, for example, from about 0°C or about 5°C to about 25°C, or from about 15°C to about 25°C, depending on the additional components or ingredients to be added or dissolved. Optionally, additional amounts of water or a selected aqueous liquid carrier may be added to the mixture, e.g., to complete dissolution of certain components, or may be removed from the mixture by techniques known to skilled persons. The resulting liquid aqueous mixture is then subjected to process step b), described below.

[0062] According to step b) of the process of the present invention, the mixture provided in step a) is dispersed to form an intermediate aqueous dispersion comprising at least one inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporin A) in an aqueous liquid carrier. To carry out the dispersion according to step b), the mixture prepared according to step a) as described above may be transferred to a suitable container for dispersing the mixture; in an alternative embodiment, the dispersion may be carried out in the same container as the apparatus used in step a). Suitable containers may be, for example, containers made of a material suitable for preparing pharmaceutical compositions, such as a suitable polymeric material or, preferably, stainless steel. Depending on the overall final volume of the mixture to be dispersed, the container may typically have a volume ranging from about 100 L to about 1,000 L, often from about 400 L to about 700 L. In certain embodiments, the volume of the container is selected to be substantially greater than at least 50% of the overall final volume of the mixture to be dispersed, or even greater than at least 10% of the overall final volume of the mixture to be dispersed. In more specific exemplary embodiments, the container may have an (inner) diameter in the range of about 700 mm to about 1,400 mm, or of about 700 mm to about 1,000 mm, or of about 850 mm to about 950 mm or about 920 mm.

[0063] Generally, the dispersing according to step b) of the process of the present invention is carried out using a disperser suitable for dispersing liquid mixtures, in particular aqueous liquid mixtures containing the above-described additional components in the amounts and concentrations detailed above. The selected disperser may, for example, be portable, that is, immersed in the aqueous mixture to be dispersed and removed therefrom. However, in a further embodiment, the disperser may be rigidly attached to or integrated with the vessel in which the dispersing is carried out. However, in a further embodiment, the disperser may be located between two vessels as an in-line disperser, as described in more detail below.

[0064] In certain embodiments, the dispersion according to step b) is carried out using a rotor-stator disperser. Rotor-stator dispersers are known to those skilled in the art and are commercially available, for example, from IKA-Werke GmbH & Co. KG (Germany), such as the ULTRA-TURRAX® UTE batch disperser. Rotor-stator dispersers typically comprise a rotor adapted to rotate at high speed within a corresponding stator, while both the rotor and the stator are immersed in the mixture to be dispersed. In certain embodiments, the rotor and / or the stator comprise a large number of teeth. Typically, these teeth are rigidly attached to the base plate of the rotor or stator and are preferably oriented parallel to the main axis of rotation of the rotor or the main drive shaft of the disperser, which is connected to the motor of the disperser comprising the rotor. The teeth may typically be arranged around the corresponding circumference of the rotor and / or stator in the form of a row of teeth. It should be noted that the rotor and / or stator may have only one row of teeth or multiple rows of teeth, which may typically be arranged concentrically about the main axis of rotation of the disperser. As the rotor rotates, the mixture to be dispersed is typically forced through gaps between the teeth or rows of teeth of the rotor and / or stator, thereby exerting shear forces on the mixture to be dispersed.

[0065] In a further particular embodiment, the dispersion according to step b) of the present invention may be carried out using an in-line disperser. In-line dispersers are commercially available, for example, from IKA-Werke GmbH & Co KG (Germany), such as the ULTRA-TURRAX® UTE in-line disperser. Such in-line dispersers allow for continuous dispersion or dispersing of the mixture, for example, from a first container A to a second container B, or vice versa, whereby the mixture to be dispersed is continuously fed into the (external) disperser.

[0066] As a measure of the shear force acting on a mixture being dispersed, the term "shear rate" or, in other words, "shear velocity" may be used. As used herein, the term "shear rate" can be expressed in terms of [1 / s] or [s-1

[0033] When used in particular in connection with a rotor-stator type disperser having dimensions of [m / s], as described in more detail below, the peripheral speed of the rotor measured in [m / s] is calculated as a function of the distance, or in other words, the formula (I): (I) JPEG0007726884000001.jpg750 (in the formula, F R means the shear rate, and v u means the peripheral speed of the rotor, and ds means the width of the gap between the stator and the rotor. In this case, the peripheral speed of the rotor is given by Equation II: (II) d is the diameter of the rotor and n is the rotational speed of the rotor (revolutions per minute, rpm) calculated according to the formula:

[0067] In certain embodiments using the rotor-stator type dispersers described above, the rotors may generally have diameters ranging from about 50 mm to about 150 mm, or from about 60 mm to about 140 mm. In particular cases where the immersion or batch type dispersers described above are used, the rotors may have diameters ranging from about 80 mm to about 120 mm, or from about 90 mm to about 110 mm, for example, from about 95 mm to about 105 mm. In further specific embodiments using an in-line disperser, described in more detail below, the rotors may have diameters selected from the ranges of about 100 mm to about 140 mm, or from about 110 mm to about 130 mm. In further embodiments, the corresponding stators are typically adapted to surround the corresponding rotors. Thus, the (internal) diameter of the space received by the corresponding rotor typically corresponds to the diameter of the rotor plus twice the gap distance between the rotor and the stator. In certain embodiments, this gap usually does not exceed about 4 mm, about 3 mm, or about 2 mm, and may be selected within the range of about 0.5 to about 2 mm, about 0.5 to about 1.5 mm, or about 0.7 to 0.9 mm, particularly when an immersion-type disperser or a batch-type disperser is used. In further specific embodiments, particularly when an in-line disperser is used, the gap (shear gap) may be selected within the range of about 0.1 mm to about 0.6 mm, about 0.2 mm to about 0.5 mm, or about 0.3 mm to 0.4 mm.

[0068] In particular embodiments, the dispersing according to step b) is carried out at a shear rate of at least 22,000 1 / s, or at least 24,000 1 / s, or 25,000 1 / s. In further particular embodiments, the dispersing according to step b) may be carried out at a shear rate selected within the range of from about 22,000 1 / s to about 120,000 1 / s, or from 22,000 1 / s to about 100,000 1 / s. In further particular embodiments, particularly when an immersion or batch disperser is used, the dispersion according to process step b) may be carried out at a shear rate of from about 24,000 1 / s to about 80,000 1 / s or 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 1 / s to about 37,000 1 / s.

[0069] In further particular embodiments, particularly when an in-line disperser is used, the dispersing according to process step b) may be carried out at a shear rate of from about 40,000 1 / s to about 110,000 1 / s or 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.

[0070] The rotor and stator may each have a plurality of teeth, as described above, in certain embodiments. The number of teeth connected to the stator and rotor may be selected independently of one another. In certain embodiments, the number of teeth on the rotor and stator, respectively, is at least about 10, or at least about 15, or at least about 20. The number of teeth on the rotor may generally be selected within a wide range, often exceeding 50, 75, or even 100, respectively. In certain embodiments, particularly when an immersion or batch disperser is used, the rotor may have some number of teeth selected within a range of about 10 to about 75, or about 10 or 15 to about 40 or 50, or about 20 to about 35. The number of teeth on the stator may generally be selected independently within a wide range, often exceeding 50, 75, or even 100, respectively. In certain embodiments, particularly when an immersion or batch type disperser is used, the stator has a number of teeth selected within the range of about 10 to about 75, or about 10 or 15 to about 40 or 50, or about 20 to about 35. In these cases, the individual teeth may be spaced apart from one another, typically by no more than about 15 mm or 10 mm, such as selected within the range of about 1 mm to about 8 mm, or about 2 mm to about 6 mm, such as about 4 mm.

[0071] In more specific embodiments, particularly when an in-line disperser is used, the rotor may have a number of teeth selected from the range of about 10 to about 75, or about 20 or 30 to about 60 or 50, or about 35 to about 45. In these cases, the stator may have a number of teeth selected from the range of about 30 to about 80, or about 35 or 40 to about 75 or 70, or about 45 or 40 to about 60. In these cases, the individual teeth may be spaced apart from one another, typically by about 10 mm or 5 mm or less, e.g., selected from the range of about 0.5 mm to about 5 mm, or about 1 mm to about 2.5 mm. Particularly when an in-line disperser is used, the distance between the stator teeth and the rotor teeth may differ, e.g., from about 1.0 mm to about 2.0 mm, e.g., 1.6 mm, for the stator, and from about 1.5 mm to about 2.5 mm, e.g., 2.0 mm, for the rotor.

[0072] As a further measure of the shear force acting on the mixture dispersed according to step b) of the process of the present invention, the term "shear frequency" may be used. As used herein, the term "shear frequency" refers to [1 / s] or [s -1

[0033] When used in particular in connection with a rotor-stator type disperser having dimensions of formula (III): (III) JPEG0007726884000003.jpg687 (in the formula, F s represents the shear frequency, n is the rotor rotation speed measured in [1 / s], and S ロータ and S ステータ may be determined by multiplying the rotor rotation speed [1 / s] by the number of rotor teeth and the number of stator teeth according to the formula (where r denotes the number of rotor and stator teeth, respectively).

[0073] In certain embodiments, the dispersing according to step b) of the process of 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 further particular embodiments, particularly when an immersion-type disperser or a batch-type disperser is used, the dispersing according to step b) is carried out at a shear frequency selected within the range of from about 42,000 1 / s to about 140,000 1 / s, or from about 42,000 1 / s to about 120,000 1 / s, or from about 45,000 1 / s to about 100,000 1 / s. In even more specific embodiments, in these cases, the shear rate may be selected within the range of about 50,000 1 / s to about 140,000 or about 100,000 1 / s, or about 50,000 1 / s to about 80,000 1 / s, or about 55,000 1 / s to about 75,000 1 / s or about 70,000 1 / s.

[0074] In even more particular embodiments, the dispersion according to step b) of the process of the present invention may be carried out at a combination of a particular shear rate and a particular shear frequency, particularly when an immersion or batch disperser is used, such as a shear rate (shear rate) of at least 22,000 1 / s, or at least 24,000 1 / s, or at least 25,000 1 / s, and 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 a further specific embodiment in which an immersion-type disperser is used, the dispersion according to step b) may be carried out at a shear rate selected within the range of about 28,000 1 / s to about 37,000 1 / s, or about 30,000 1 / s to about 37,000 1 / s, and a shear frequency of about 50,000 1 / s to about 80,000 1 / s, or about 55,000 1 / s to about 75,000 1 / s or about 70,000 1 / s.

[0075] In further specific embodiments, particularly when an in-line disperser as described above is used, the dispersion according to step b) may be carried out at a shear frequency selected within the range of about 100,000 1 / s to about 200,000 1 / s, or about 110,000 1 / s to about 190,000 1 / s, or about 120,000 1 / s to about 180,000 1 / s. In even more specific embodiments, in these cases, the shear rate may be selected within the range of about 130,000 1 / s to about 170,000 1 / s, or about 140,000 1 / s to about 160,000 1 / s.

[0076] In even more specific embodiments in which the in-line disperser described above is used, the dispersion according to step b) may be carried out at a shear rate selected from the range of about 55,000 1 / s to about 85,000 1 / s, or about 60,000 1 / s to about 85,000 1 / s, or about 65,000 1 / s to about 75,000 1 / s, and a shear frequency selected from the range of about 120,000 1 / s to about 180,000 1 / s, or about 130,000 1 / s to about 170,000 1 / s, or about 140,000 1 / s to about 160,000 1 / s.

[0077] As already mentioned above, the dispersion according to step b) may be carried out using an immersion-type disperser, i.e., a disperser that is fixedly arranged in the dispersing vessel or that can be removably immersed in the mixture to be dispersed. However, in an alternative embodiment, the dispersion according to step b) may be carried out using an in-line disperser as described above. However, in a further embodiment, the dispersion according to step b) of the present invention may be carried out using both an immersion-type disperser (or several immersion-type dispersers simultaneously) and an in-line disperser. In a further particular embodiment, an immersion-type disperser and an in-line disperser may be used sequentially. For example, in a particular embodiment, the dispersion according to step b) of the present invention may first be carried out using an immersion-type disperser under the conditions as described in detail above, and then the dispersion may be carried out using an in-line disperser, also under the conditions as described in detail above. In a further particular embodiment, the conditions selected for the (partial) dispersion carried out using an immersion-type disperser are different from those for the (partial) dispersion carried out using an in-line disperser, in particular with respect to the selected shear rate, shear frequency and / or rotor rotation speed.

[0078] As explained above, the rotor of a rotor-stator type disperser that can be used in step b) of the process of the present invention may have multiple teeth that can be arranged around the circumference or periphery of the rotor, and the multiple teeth preferably have the same distance from each neighboring tooth in a row. Furthermore, the rotor may or may not have multiple rows of teeth that are typically arranged concentrically with respect to the main axis of rotation of the rotor. Thus, in certain embodiments, particularly when an immersion type disperser is used, the rotor may have multiple rows of teeth, such as two to four rows of teeth, or two or three rows of teeth.

[0079] Additionally, the corresponding stator may or may not also have multiple rows of teeth, typically arranged concentrically about the stator's primary axis of rotation. Thus, in certain embodiments, the stator has two to four rows of teeth, or multiple rows of teeth, such as two or three rows of teeth. In even more specific embodiments, the combined rotor and stator have a total of two to eight rows of teeth, particularly three to six rows of teeth. In even more specific embodiments, particularly when an in-line disperser is used, the rotor and stator may also have multiple rows of teeth, such as three to twelve rows of combined teeth, or six to ten rows of combined teeth, e.g., eight rows of teeth for the combined rotor and stator.

[0080] Dispersion according to step b) of the process of the present invention to provide an intermediate aqueous dispersion containing an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier is typically carried out at a high rotational speed of the disperser rotor, typically up to about 10,000 rpm or up to about 8,000 rpm (revolutions per minute). In certain embodiments, particularly when an immersion-type disperser is used, dispersion may be carried out at a rotational speed (of the disperser rotor) selected within the range of about 2,000 rpm or about 3,000 rpm to about 6,000 rpm, or about 3,000 rpm or about 4,000 rpm to about 5,500 rpm. Depending on the rotor diameter selected, such a rotational speed results in a rotor peripheral speed of at least about 10 m / s. In certain embodiments, dispersion according to step b) is carried out at a rotor peripheral speed selected within the range of about 15 m / s to about 40 m / s, or about 15 m / s to about 30 m / s.

[0081] In further specific embodiments, particularly when an in-line disperser is used, the dispersion may be carried out at a rotation speed (of the rotor of the disperser) selected within the range of about 2,000 rpm or about 3,000 rpm to about 6,000 rpm, or about 3,000 rpm or about 3,500 rpm to about 4,500 rpm.

[0082] To achieve such a peripheral speed, in a particular embodiment, the dispersion according to step b) is carried out using a disperser equipped with a motor having a power of at least 2 kW, in particular a motor having a power selected within the range of about 2 kW to about 10 kW, or about 3 kW to about 8 kW.

[0083] To prepare an intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier, the dispersing according to step b) is typically carried out for a period of 10 or 15 minutes, or 1 hour, or even more than 2 hours. Often, particularly when an immersion-type disperser or an in-line disperser is used alone, the dispersing according to step b) is carried out for a (total) period of at least about 1 hour or at least about 3 hours, for example, for a period selected within the range of about 1 hour to about 8 hours, or about 3 hours to about 8 hours, or about 3 hours to about 5 hours, or about 1 hour to about 4 hours.

[0084] In a further specific embodiment, dispersion according to step b) of the process of the present invention as described in detail above may be carried out using an immersion disperser and an in-line disperser, but the immersion disperser and the in-line disperser are used consecutively. In a specific embodiment, dispersion may begin using an immersion disperser (or multiple immersion dispersers) followed by an in-line disperser. In these cases, the dispersion time may also be shortened. For example, dispersion using an immersion disperser may be carried out for a period of up to about 1 hour or up to about 30 minutes, for example, for a period of about 10 minutes to about 30 minutes, followed by dispersion using an in-line disperser for a period of up to 6 hours or up to 4 hours, for example, for a period of about 1 hour to about 4 hours. This may help to effectively shorten the overall duration of the dispersion process.

[0085] In further particular embodiments, the dispersing according to step b) may be carried out at a temperature (of the mixture to be dispersed) selected within the range of about 15° C. to about 35° C., or about 15° C. to about 30° C., or about 15° C. to about 25° C., or about 20° C. to about 25° C. If necessary, the mixture may be cooled using standard techniques to avoid a temperature increase. In further particular embodiments, the dispersing according to step b) is carried out at ambient (atmospheric) pressure.

[0086] The resulting intermediate aqueous dispersion comprising at least one inhalable immunosuppressive macrocyclic active ingredient (especially CsA) in an aqueous liquid carrier obtained according to process step b) already contains some of the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form, particularly liposome-solubilized CsA (L-CsA). However, to complete the solubilization of the selected inhalable immunosuppressive macrocyclic active ingredient (especially CsA) and to further reduce the particle size of the formed liposomes, the resulting mixture is subjected to further homogenization according to the next process step c) described below.

[0087] According to step c) of the process of the present invention, the intermediate aqueous dispersion formed in step b) is homogenized to form a dispersion containing the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form, wherein preferably at least about 90% to about 100% of the inhalable immunosuppressive active ingredient (particularly CsA) of the total amount of said compound as incorporated according to step a) is present in liposome-solubilized form.

[0088] According to process step c), the resulting intermediate aqueous dispersion is then subjected to homogenization conditions to produce a colloidal dispersion of the inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporin A) in liposomally solubilized form. In a preferred embodiment, homogenizing the intermediate aqueous dispersion formed in step b) to form a dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposomally solubilized form comprises high-pressure homogenization, as known to those skilled in the art. In an exemplary embodiment, the high-pressure homogenization according to step c) may be carried out using a piston-gap homogenizer, which may comprise one or more plungers (e.g., 1 to 6, or 2 to 4, particularly 3). In further embodiments, such a piston-gap homogenizer may comprise a homogenization valve, particularly a ceramic homogenization valve such as NanoVALVE (available from GEA, Italy). In further particular embodiments, the homogenization pressure may be applied in a two-stage pressure cascade, as described in more detail below. Exemplary homogenizers suitable for carrying out high pressure homogenization according to step c) include, but are not limited to, high pressure homogenizers Microfluidics M-110EH or Ariete NS3006L (GEA, Italy).

[0089] In a further embodiment, the high-pressure homogenization according to step c) may be carried out once or repeatedly multiple times. Specifically, the high-pressure homogenization may be carried out repeatedly, for example, about 5 to about 15 times. Furthermore, the high-pressure homogenization may be carried out at any suitable pressure, typically up to about 1,500 bar, or at a pressure in the range of about 50 to about 1,500 bar, or at a pressure selected within the range of about 100 to about 1,000 bar. Preferably, the high-pressure homogenization may be carried out repeatedly, for example, about 5 to 15 times at a pressure in the range of about 100 to about 1,000 bar, optionally under reduced pressure.

[0090] In a further embodiment, the homogenization according to step c) of the process of the present invention may be carried out in a two-stage pressure cascade, applying a relatively low pressure in a first stage and a relatively high pressure in a second stage. Exemplary pressure ranges for the first stage may generally be selected within the pressure ranges described above, e.g., from about 50 to about 200 bar, such as about 100 bar, particularly from about 75 to about 125 bar. Exemplary pressure ranges for the second stage may generally be selected within the pressure ranges described above, e.g., from about 500 to about 1,500 bar, such as about 1,000 bar, particularly from about 750 to about 1,250 bar. As noted above, when carried out in a two-stage pressure cascade, the homogenization step may be carried out repeatedly, e.g., from about 5 to about 15 times. In further embodiments, the high-pressure homogenization according to step c) may be carried out at a temperature of up to about 35° C. to 37° C. (temperature of the dispersion to be homogenized), and in particular at a temperature in the range of about 2° C. to about 35° C., or about 2 to about 25° C., or about 5° C. or about 7 to about 25° C. In certain embodiments, the homogenization of the intermediate aqueous dispersion as subjected to step b) of the process of the present invention, or at least a portion of such homogenization, may be carried out at a temperature below room temperature, for example at a temperature in the range of about 2° C. to about 10° C. In these cases, as well as when homogenization is carried out at the higher temperatures described above, a heat exchanger may be used to cool the aqueous dispersion during homogenization.

[0091] After completion of the homogenization according to process step c) of the present invention, a homogenized dispersion is obtained comprising a colloidal dispersion of an inhalable immunosuppressive macrocyclic active ingredient (in particular cyclosporin A) in liposomally solubilized form, consisting mainly of unilamellar liposomes having an average diameter of at most 100 nm, for example from about 30 nm to about 70 nm, and / or a dispersity index (PI) of 0.50 or less, or even 0.40 or less, for example from about 0.15 to about 0.2, which preferably appears as a clear, opalescent solution free of particles visible to the naked eye.

[0092] After completion of homogenization according to step c), according to optional step c1), the resulting homogenized dispersion containing the inhalable immunosuppressive macrocyclic active ingredient (in particular cyclosporin A) in liposome-solubilized form may be sterilized, if necessary, for example by sterile filtration. Suitable filters for such filtration to remove or reduce potential bioburden or microbial contaminants include, but are not limited to, Fluorodyne® EX (PALL) filters having a pore size of 0.2 μm.

[0093] In a further particular embodiment, it may be useful to filter the intermediate aqueous dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier formed in step b) prior to the homogenization according to step c). Thus, the process according to the first aspect of the invention may comprise, as a further step, b1) filtering the intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier formed in step b) prior to homogenizing the resulting filtered intermediate aqueous dispersion according to step c).

[0094] According to these embodiments, the intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporine A) in an aqueous liquid carrier formed in step b) is filtered before being further processed by high-pressure homogenization as described above in connection with process step c). Such optional additional filtration may be beneficial to avoid mechanical stress in the high-pressure homogenizer and potential damage from potentially remaining particles in the intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporine A). Filtration may be carried out using readily available filters or filter materials suitable for contact with pharmaceutical compounds or compositions, such as steel or suitable polymeric materials. In certain embodiments, the filtration according to this optional step b1) is carried out using a filter having an average pore width in the range of about 75 μm, or about 100 μm to about 300 μm, or about 150 μm to about 250 μm, or about 200 μm to about 250 μm, or about 220 μm to about 250 μm or about 230 μm, such as about 225 μm.

[0095] The process according to the first aspect of the present invention provides, as the product of process step c) described above, a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporin A) in liposome-solubilized form. Preferably, a therapeutically effective amount of the inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporin A) is contained within a liposome-forming structure formed by a membrane-forming substance selected from the group of phospholipids and a solubility-enhancing substance selected from the group of non-ionic surfactants, as outlined above.

[0096] In certain embodiments, the inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporin A) is at least partially incorporated into (or intercalated between) the bilayer membrane of the liposome-forming structure. As used herein, the term "incorporated," particularly with respect to the liposomal compound CsA, means that the CsA is located in or intercalated between the lipophilic interior of the lipid bilayer membrane rather than on the hydrophilic exterior of the lipid bilayer membrane (while the term "surface" refers to both surfaces, i.e., more particularly, the interior or exterior surface of the bilayer membrane forming the liposome-forming structure).

[0097] In preferred embodiments, the inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporine A) is primarily incorporated into (or intercalated between) the bilayer membrane of the liposome-forming structure. In exemplary embodiments, at least about 90%, or even at least about 95%, or even at least about 97.5% of the total amount of the inhalable immunosuppressive macrocyclic active ingredient (particularly CsA) is contained within the bilayer membrane of the liposome-forming structure formed according to the process of the present invention. In further exemplary embodiments, at least about 90%, or about 95% to about 97.5%, or about 99%, or about 99.5%, or even 99.9% of the total amount of the inhalable immunosuppressive macrocyclic active ingredient (particularly CsA) is incorporated into the bilayer membrane of the liposome-forming structure formed by the process of the present invention.

[0098] In a second aspect, the present invention provides a process for the preparation of a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, the lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form, This process comprises the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilised form in an aqueous liquid carrier according to the process of the first aspect of the invention as described above, d) removing the aqueous liquid carrier at least partially under lyophilization conditions to form a lyophilized pharmaceutical composition.

[0099] In other words, the present invention provides a process for the preparation of a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, the lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporin A) in liposome-solubilized form, the process comprising: a) -inhalable immunosuppressive macrocyclic active ingredients (especially CsA); - membrane-forming substances selected from the group of phospholipids; - solubility enhancers selected from the group of nonionic surfactants; optionally one or more excipients; and - providing a mixture comprising an aqueous liquid carrier; b) dispersing the mixture provided in step a) to form an intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; b1) optionally filtering the intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier as formed in step b); c) homogenizing the intermediate aqueous dispersion as formed in step b) or step b1) to form a dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form; c1) optionally sterilizing the dispersion containing the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form; and d) removing the aqueous liquid carrier at least partially under lyophilization conditions to form a lyophilized pharmaceutical composition.

[0100] All features, embodiments, starting materials, processing conditions and combinations thereof described above in relation to the process of the first aspect of the invention also apply to the process according to the second aspect of the invention.

[0101] In certain embodiments of this second aspect of the present invention, the dispersion according to process step b) may also be carried out using the immersion type disperser and in-line disperser described above, in which case the immersion type disperser and in-line disperser are preferably used in series.

[0102] The process of this second aspect of the invention, in addition to the process steps of the process of the first aspect of the invention, comprises the additional step d) in which the aqueous liquid carrier is at least partially removed under lyophilization conditions to form a lyophilized pharmaceutical composition.

[0103] Lyophilization according to process step d) can be carried out according to standard techniques known to those skilled in the art, for example, by using a LyoStar MNL-055-A / LSACC3E lyophilizer or a GEA Lyovac® GT 400-D. Lyophilization to form the lyophilized pharmaceutical composition of this aspect of the invention can be carried out in a continuous manner, for example, at a constant pressure and temperature, or preferably in stages. In this case, each stage of the lyophilization protocol or process can be carried out at a specific pressure, temperature, and for a specified duration. In exemplary embodiments, the lyophilization process or cycle can include up to 20 successive steps, or from about 2 to about 15 successive steps, preferably from about 5 to about 15 successive steps. Each step can be carried out, for example, at a temperature within a range of about 40°C to about −60°C, preferably about 20°C to −50°C, either at a constant temperature or at a temperature that can be increased or decreased at a specific gradient. Furthermore, each freeze-drying step may be carried out under reduced pressure, such as sub-ambient pressure ranging from about 0.005 mbar to about 800 mbar, preferably from 0.009 mbar to about 0.500 mbar, or 0.400 mbar or about 0.300 mbar.

[0104] It should be noted that in addition to the lyophilization described above, part of the aqueous liquid carrier removed according to process step d) can also be removed according to other techniques known to those skilled in the art, such as, for example, distillation under reduced pressure, in particular before lyophilization. Furthermore, it may be advantageous to prepare aliquots of smaller volumes of dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form containing a predetermined amount of the active ingredient, as subjected to process step c).

[0105] In this connection, it is noted that the freeze-drying according to the additional process step d) may in certain embodiments be carried out in the presence of a disaccharide selected from the group consisting of sucrose, lactose and trehalose, in which case at least one disaccharide is present in an amount of at least 40% by weight relative to the total weight of the freeze-dried composition, while said disaccharide is not added to the mixture obtained from process step c) but to the initial mixture provided in process step a).

[0106] In a third aspect, the present invention provides a lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient (particularly cyclosporin A) in liposomally solubilized form for reconstitution in an aqueous liquid carrier, obtained or obtainable by a process according to the second aspect of the invention. In a particular embodiment, such a lyophilized pharmaceutical composition comprises: - inhalable immunosuppressive macrocyclic active ingredients (preferably CsA); - membrane-forming substances selected from the group of phospholipids; a solubility enhancer selected from the group of nonionic surfactants; and - optionally one or more excipients.

[0107] In certain embodiments, depending on the amounts of the above-mentioned components used, the lyophilized pharmaceutical composition that can be prepared according to the process of the second aspect of the present invention may contain the inhalable immunosuppressive macrocyclic active ingredient (particularly CsA) in an amount ranging from about 2 to about 4% by weight, preferably from about 2.2 to about 3.4% by weight, or even more preferably from about 2.4 to about 3.4% by weight, or from about 2.4 to about 3.0% by weight, or from about 2.5% to about 2.9% by weight, or from about 2.6% to about 2.8% by weight, or from about 2.65% to about 2.75% by weight, in each case based on the weight of the lyophilized composition.

[0108] In a further particular embodiment, the content of membrane-forming substances selected from the group of phospholipids (preferably Lipoid S100) in the lyophilized composition may be from about 10 or 15% to about 30% by weight, preferably from about 20 to about 30% by weight, and even more preferably from about 23 to about 27% by weight, based on the total weight of the lyophilized composition.

[0109] In even more specific embodiments, the content of the solubility enhancer selected from the group of nonionic surfactants may be preferably selected in the range of about 0.01 to about 5 wt. %, or about 0.1 to about 4 wt. %, or about 0.5 to about 3.5 wt. %, or about 1 to about 3 wt. %, preferably about 1.5 to about 2.5 wt. %, or about 1.6 to about 2.3 wt. %, or about 1.7 to about 2.1 wt. %, or about 1.8 to about 2.0 wt. %, in each case based on the total weight of the lyophilized composition.

[0110] The lyophilized pharmaceutical composition of this third aspect of the invention, obtainable by the process according to the second aspect of the invention, may or may not further comprise residual moisture after lyophilization. This residual moisture may be bound to the surface of the membrane-forming material or may be contained within the lumen of potentially hollow liposome-forming structures as described above. In preferred embodiments, the amount of residual moisture contained by the lyophilized composition ranges from at most about 5% by weight, or at most about 3% by weight, or preferably at most about 2% by weight, based on the total weight of the lyophilized pharmaceutical composition.

[0111] In a further particular embodiment, the lyophilized pharmaceutical composition according to the third aspect of the invention comprises: a) a liposome-forming structure, i. a therapeutically effective amount of cyclosporine A (CsA); ii. a membrane-forming substance selected from the group of phospholipids; and iii. a liposome-forming structure comprising a solubility enhancer selected from the group of non-ionic surfactants; b) at least one disaccharide selected from the group consisting of sucrose, lactose and trehalose, present in an amount of at least 40% by weight based on the total weight of the lyophilized composition.

[0112] It is noted that in relation to this third aspect of the invention, all features, embodiments, components, process parameters and combinations thereof described above in relation to the first and second aspects of the invention also apply to this third aspect of the invention and all further aspects of the invention.

[0113] In some embodiments, the at least one disaccharide is present in an amount of at least about 40% by weight to at most about 95% by weight, or at most about 90% by weight, or at most about 85% by weight, or at most about 80% by weight, all based on the total weight of the lyophilized composition. In further specific embodiments, the lyophilizate according to this third aspect of the invention preferably comprises sucrose and / or lactose, particularly sucrose, in an amount selected from the range of about 50% by weight to about 80% by weight or about 75% by weight, based on or based on the total weight of the lyophilized composition. In further preferred embodiments, the lyophilized pharmaceutical composition according to this aspect comprises at least one disaccharide, preferably sucrose, trehalose, and / or lactose, particularly sucrose, in an amount selected from the range of about 60% by weight to about 75% by weight, and even more preferably from about 65% by weight to about 70% by weight, based on the total weight of the lyophilized composition.

[0114] Lyophilized pharmaceutical compositions comprising an inhalable immunosuppressive macrocyclic active ingredient (especially CsA) in a liposome-solubilized form for reconstitution in an aqueous liquid carrier preferably contain a therapeutically effective amount of the inhalable immunosuppressive macrocyclic active ingredient (especially CsA) and are particularly useful as drugs for pulmonary application by inhalation.

[0115] Thus, in a fourth aspect, the present invention provides a lyophilized pharmaceutical composition obtained or obtainable by the process of the second aspect of the invention for use as a medicament for pulmonary application, in particular for pulmonary application by inhalation.

[0116] In exemplary embodiments, such lyophilized pharmaceutical compositions according to the third and fourth aspects of the invention comprise the following components: Inhalable immunosuppressive active ingredients: 1~7% by weight Disaccharide: 40~80% by weight Phospholipids: 10-40% by weight Nonionic surfactant: 0.01 to 10% by weight Buffer: 1~6% by weight 0.05 to 0.5% by weight of a chelating agent, (the sum of the components adds up to 100% by weight of the lyophilized composition), or consists essentially of, or consists of, together with any further excipients, and preferably comprises (each based on the total weight of the lyophilized pharmaceutical composition).

[0117] In further exemplary embodiments, such lyophilized pharmaceutical compositions according to the third and fourth aspects of the invention comprise the following components: Cyclosporin A: 2 to 4% by weight Disaccharide: 40~80% by weight Phospholipids: 10-40% by weight Nonionic surfactant: 0.01 to 10% by weight Buffer: 1~6% by weight 0.05 to 0.5% by weight of a chelating agent, (The sum of the components adds up to 100% by weight of the lyophilized composition), or consist essentially of, or consist of, together with optional further excipients, and preferably comprise (each based on the total weight of the lyophilized pharmaceutical composition). Note that the values and ranges set forth above are calculated based on a lyophilized, completely anhydrous composition. However, for practical reasons, the lyophilized composition, in addition to the components listed above, may or may not contain residual moisture in an amount ranging from about 0 to about 5% by weight, based on the weight of the lyophilized pharmaceutical composition.

[0118] In a further exemplary embodiment, such a lyophilized pharmaceutical composition comprises: Cyclosporin A: 2 to 4% by weight Disaccharide: 50~75% by weight Phospholipids: 15-40% by weight Nonionic surfactant: 0.1 to 4% by weight Buffer: 2~6% by weight 0.05 to 0.5% by weight of a chelating agent, (The sum of the components adds up to 100% by weight of the lyophilized composition, and in addition to the components listed above, the lyophilized composition may or may not contain residual moisture in an amount ranging from about 0 to about 2% by weight, based on the weight of the lyophilized pharmaceutical composition), or consists essentially of, or consists of, these components, together with further optional excipients, and preferably includes these components (each based on the total weight of the lyophilized pharmaceutical composition).

[0119] In a preferred exemplary embodiment, the lyophilized pharmaceutical composition comprises: Cyclosporin A: 2.5 to 3% by weight Disaccharide: 60~75% by weight Phospholipids: 20-30% by weight Nonionic surfactant: 1 to 3% by weight Buffer: 3~5% by weight 0.05 to 0.2% by weight of a chelating agent, (The sum of the components adds up to 100% by weight of the lyophilized composition, and in addition to the components listed above, the lyophilized composition may or may not contain residual moisture in an amount ranging from about 0 to about 2% by weight, based on the weight of the lyophilized pharmaceutical composition), or consists essentially of, or consists of, these components, together with further optional excipients, and preferably includes these components (each based on the total weight of the lyophilized pharmaceutical composition).

[0120] The lyophilized pharmaceutical compositions according to the third and fourth aspects of the present invention may be reconstituted (redispersed) in an aqueous liquid carrier, preferably a sterile aqueous liquid carrier, to form a colloidal solution or dispersion. In a preferred embodiment, pulmonary administration of the lyophilized pharmaceutical composition is carried out after said reconstitution in an aqueous liquid carrier to form a colloidal solution or dispersion.

[0121] As described above, compositions containing an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form, which can be prepared by the process of the present invention in the form of a dispersion or lyophilizate, can be used as drugs for the prevention and treatment of autoimmune diseases, skin diseases, sensory organ (eye, nose, ear) transplantation or related diseases, fatigue, and pulmonary diseases such as asthma, chronic obstructive bronchitis, parenchymal, fibrotic, and interstitial lung diseases or inflammation, and lung cancer, preferably for the prevention and treatment of acute or chronic transplant rejection and diseases resulting from acute or chronic transplant rejection, such as obliterative bronchiolitis, particularly after lung, heart, bone marrow, or stem cell transplantation, particularly preferably after lung transplantation, especially after lyophilization and reconstitution in an aqueous liquid carrier as described above. When additive or synergistic effects can be achieved with cyclosporin A due to its efflux pump inhibitory effect, it may also be used to improve the effectiveness of other drugs, especially cytostatic drugs. Nasal, oral, ocular, mucosal, parenteral or topical application of the compositions according to the invention may be advantageous in individual cases. Administration may be effected by application, instillation or spraying onto or into the body, which has proven to be particularly well tolerated in initial human trials.

[0122] Preferably, however, the pharmaceutical compositions which may be prepared according to the present invention, in particular in lyophilized and / or reconstituted form, are useful 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 of acute and chronic organ transplant rejection after lung transplantation and diseases resulting from acute and chronic organ transplant rejection after lung transplantation, such as bronchiolitis obliterans.

[0123] As mentioned above, the pharmaceutical compositions that can be prepared according to the process of the present invention, as described in detail above in relation to the first and second aspects of the present invention, are useful as medicaments for pulmonary administration. Pulmonary administration may be carried out after reconstitution, or more particularly, after redispersion of a lyophilized pharmaceutical composition, which can be prepared as described above in relation to the second aspect of the present invention, in an aqueous liquid carrier, preferably a sterile aqueous liquid carrier, to form a colloidal solution or dispersion, preferably a colloidal dispersion.

[0124] In a preferred embodiment, pulmonary administration of the lyophilized pharmaceutical composition for use as described above is carried out by inhalation. In a further preferred embodiment, pulmonary administration is carried out after converting a pharmaceutical composition containing an inhalable immunosuppressive macrocyclic active ingredient (especially CsA) in liposome-solubilized form into an aerosol, for example by nebulization or aerosolization. After reconstitution, or more particularly dispersion, in an aqueous liquid carrier, the pharmaceutical composition obtainable by the process of the present invention may advantageously be administered by aerosolizing a solution, colloidal formulation, or suspension, such as a composition of the present invention containing a liposome-solubilized inhalable immunosuppressive macrocyclic active ingredient (especially CsA), using a nebulizer capable of converting the solution into very fine droplets that can reach the periphery of the lungs. In particular, a jet nebulizer, ultrasonic nebulizer, piezoelectric nebulizer, electrohydrodynamic nebulizer, membrane nebulizer, electronic membrane nebulizer, or electronic vibrating membrane nebulizer may be used. Examples of suitable nebulizers include the 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) families of devices.

[0125] Preferably, however, a piezoelectric nebulizer, an electrohydrodynamic nebulizer, a membrane nebulizer, an electronic membrane nebulizer, or an electronic vibrating membrane nebulizer may be used, where 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) families of devices.

[0126] In a preferred embodiment, pulmonary administration of a pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient (especially CsA) in reconstituted form for use according to this aspect of the invention is carried out using an ultrasonic nebulizer or an electronic vibrating membrane nebulizer, preferably an electronic vibrating membrane nebulizer such as, for example, an eFlow®, AeroNeb Pro type or -Go or I-Neb type device.

[0127] In a further preferred embodiment, to target the above-described drug CsA (particularly the drug CsA in liposomal solubilized form) to the lower respiratory tract, compositions for use according to this aspect of the invention are aerosolized with an electronic vibrating membrane nebulizer. In a particularly preferred embodiment, reconstituted lyophilized pharmaceutical compositions for use according to the invention are aerosolized using an eFlow® nebulizer (PARI Pharma GmbH).

[0128] The eFlow® nebulizer, equipped with a perforated vibrating membrane that produces an aerosol with low ballistic propulsion and a high percentage of droplets in the respirable size range (typically less than 5 μm), nebulizes liquid formulations, such as pharmaceutical compositions that may be prepared by the process of the present invention, in reconstituted form. Compared to conventional nebulizers, such as jet nebulizers, the eFlow® is designed for more rapid and efficient drug therapy due to its higher nebulization rate, lower drug loss, and a higher percentage of drug available as dose administered (DD) and respirable dose (RD).

[0129] As described above, pharmaceutical compositions containing an inhalable immunosuppressive macrocyclic active ingredient (especially CsA) that can be prepared according to the process of the present invention can be dispersed (reconstituted) in an aqueous liquid carrier, such as water or saline, preferably saline (aqueous sodium chloride solution at a concentration of 0.25% w / v), to provide an opalescent dispersion or solution. In a further embodiment, such liposome dispersions are essentially free of particles visible to the naked eye. The liposomes contained by the dispersions preferably have a mean diameter, more particularly, a z-average diameter of at most about 100 nm, as measured by photon correlation spectroscopy using a Malvern ZetaSizer. Preferably, the liposome dispersion contains liposomes with a z-average diameter, as measured by photon correlation spectroscopy (Malvern ZetaSizer), in the range of about 40 nm to about 100 nm, and even more preferably in the range of about 40 nm to about 70 nm.

[0130] In further particular embodiments, the liposome dispersion has a polydispersity index (PI) as measured by photon correlation spectroscopy of at most about 0.50, preferably at most about 0.4, and even more preferably in the range of about 0.1 to about 0.3.

[0131] In further embodiments, the liposome dispersions described above may have an osmolality in the range of about 300 to about 550 mOsmol / kg, preferably in the range of about 430 to about 550 mOsmol / kg or about 370 to about 470 mOsmol / kg. Preferably, the pH value of the liposome dispersions according to this aspect of the invention is in the range of about 6.0 to 7.0, preferably in the range of about 6.2 to about 6.8. In further embodiments, after a 1:10 dilution, the liposome dispersions according to this aspect of the invention have a turbidity of at most 200 NTC (Nephelometric Turbidity Units), preferably in the range of about 55 to about 90 NTU.

[0132] Surprisingly, it has been found that liposome dispersions of pharmaceutical compositions comprising an inhalable immunosuppressive macrocyclic active ingredient (particularly CsA), obtainable by the process of the second aspect of the invention, and which can be prepared by reconstituting a lyophilized pharmaceutical composition, in particular a lyophilized pharmaceutical composition of the third aspect of the invention, comprising a disaccharide selected from the group consisting of sucrose, lactose, and trehalose in an aqueous liquid carrier, contain liposomes of similar or slightly larger size compared to the liposomes in the corresponding dispersion before lyophilization, as described below. Thus, the process of the invention provides, after lyophilization and reconstitution in an aqueous liquid carrier, aqueous liposome dispersions comprising liposomes with a median diameter, measured as the z-average diameter, as measured by photon correlation spectroscopy (Malvern ZetaSizer). This median size has a median size equal to or up to 20% larger than the z-average diameter of the liposomes used to prepare the lyophilized pharmaceutical composition of the invention before lyophilization, preferably up to only 10% larger, and preferably equal to or up to 20% larger than the liposomes formed by the process according to the second aspect of the invention before lyophilization.

[0133] It has further surprisingly been found that the process according to the first aspect of the invention allows the preparation of dispersions comprising an inhalable immunomacrocyclic active ingredient in liposome-solubilized form (particularly cyclosporine A) having a precise content of active ingredient that correlates with the amount of active ingredient as incorporated into the process, particularly when an intermediate filtration step is included. Thus, in certain embodiments, the content of inhalable macrocyclic active ingredient in liposome-solubilized form (particularly cyclosporine A) contained by a dispersion prepared according to the process of the first aspect of the invention comprises at least about 95%, or at least about 97%, or at least about 98%, for example about 98% or about 99% to about 100%, or about 98% to about 99.95%, or about 98.5% to about 99.9% of the amount of inhalable macrocyclic active ingredient (particularly cyclosporine A) provided in the initial mixture according to step a) of the process of the first aspect of the invention.

[0134] The following is a list of numbered embodiments included in the present invention:

[0135] 1. A process for the preparation of a dispersion containing an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier, the process comprising: a) -inhalable immunosuppressive macrocyclic active ingredients; - membrane-forming substances selected from the group of phospholipids; - solubility enhancers selected from the group of nonionic surfactants; optionally one or more excipients; and - providing a mixture comprising an aqueous liquid carrier; b) dispersing the mixture provided in step a) to form an intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier; and c) homogenizing the intermediate aqueous dispersion as formed in step b) to form a dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form.

[0136] 2. The process according to item 1, wherein the dispersion according to step b) is carried out using a rotor-stator type disperser.

[0137] 3. The process according to item 1 or 2, wherein the dispersion according to step b) is carried out at a shear rate (shear rate) of at least 22,000 1 / s.

[0138] 4. The process according to any one of items 1 to 3, wherein the dispersion according to step b) is carried out using an immersion type disperser.

[0139] 5. The process according to any one of items 1 to 4, wherein the dispersion according to step b) is carried out at a shear rate (shear rate) selected within the range of about 25,000 1 / s to about 40,000 1 / s.

[0140] 6. The process according to any one of items 1 to 5, wherein the dispersion according to step b) is carried out at a shear frequency of at least 42,000 1 / s.

[0141] 7. The process according to any one of items 1 to 6, wherein the dispersion according to step b) is carried out at a shear frequency selected within the range of about 50,000 1 / s to about 80,000 1 / s.

[0142] 8. The process according to any one of items 1 to 7, wherein the dispersion according to step b) is carried out at a shear rate selected within the range of about 28,000 1 / s to about 37,000 1 / s and a shear frequency selected within the range of about 50,000 1 / s to about 80,000 1 / s.

[0143] 9. The process of any one of items 2 to 8, wherein the rotor has a diameter in the range of about 60 mm to about 140 mm.

[0144] 10. The process of any one of items 2 to 9, wherein the rotor has a number of teeth selected within the range of about 10 teeth to about 40 teeth.

[0145] 11. The process of any one of items 2 to 10, wherein the stator has a number of teeth selected within the range of about 10 teeth to about 40 teeth.

[0146] 12. The process of any one of items 2 to 11, wherein the rotor and / or stator have multiple rows of teeth.

[0147] 13. The process according to any one of items 2 to 12, wherein the rotor and / or stator have 2 to 8 rows of teeth, or 3 to 6 rows of teeth.

[0148] 14. The process according to any one of items 2 to 13, wherein the dispersion according to step b) is carried out in a vessel having a diameter in the range of about 700 mm to about 1,000 mm.

[0149] 15. The process according to any one of items 2 to 14, wherein the dispersion according to step b) is carried out at a rotation speed in the range of about 2,000 to about 6,000 rpm.

[0150] 16. The process according to any one of items 2 to 15, wherein the dispersion according to step b) is carried out at a rotation speed in the range of about 3,000 to about 5,500 rpm.

[0151] 17. The process according to any one of items 1 to 3, wherein the dispersing according to step b) is carried out using an in-line disperser.

[0152] 18. The process according to item 17, wherein the dispersion according to step b) is carried out at a shear rate (shear rate) selected within the range of about 45,000 1 / s to about 90,000 1 / s.

[0153] 19. The process according to item 16 or 17, wherein the dispersion according to step b) is carried out at a shear frequency selected within the range of about 100,000 1 / s to about 200,000 1 / s.

[0154] 20. The process according to any one of items 17 to 19, wherein the dispersion according to step b) is carried out at a shear frequency selected in the range of about 120,000 1 / s to about 180,000 1 / s.

[0155] 21. The process according to any one of items 17 to 20, wherein the dispersion according to step b) is carried out at a shear rate selected in the range of about 55,000 1 / s to about 85,000 1 / s and a shear frequency selected in the range of about 130,000 1 / s to about 170,000 1 / s.

[0156] 22. The process of any one of items 17 to 21, wherein the rotor has a diameter in the range of about 100 mm to about 140 mm.

[0157] 23. The process according to any one of items 17 to 22, wherein the rotor has a number of teeth selected within the range of about 30 teeth to about 50 teeth.

[0158] 24. The process of any one of items 17 to 23, wherein the stator has a number of teeth selected within the range of about 30 teeth to about 80 teeth.

[0159] 25. The process of any one of items 17 to 24, wherein the rotor and / or stator have multiple rows of teeth.

[0160] 26. The process of any one of items 17 to 25, wherein the rotor and / or stator have 3 to 12 rows, or 6 to 10 rows, of interlaced teeth.

[0161] 27. The process according to any one of items 17 to 26, wherein the dispersion according to step b) is carried out at a rotation speed in the range of about 2,000 to about 6,000 rpm.

[0162] 28. The process according to any one of items 17 to 27, wherein the dispersion according to step b) is carried out at a rotation speed in the range of about 3,500 to about 4,500 rpm.

[0163] 29. The process according to any one of items 2 to 28, wherein the dispersion according to step b) is carried out at a peripheral speed of the rotor of at least 10 m / s.

[0164] 30. The process according to any one of items 2 to 29, wherein the dispersion according to step b) is carried out at a peripheral speed of the rotor selected in the range of about 15 m / s to about 40 m / s.

[0165] 31. The process according to any one of items 1 to 30, wherein the dispersing according to step b) is carried out using a disperser equipped with a motor having a power of at least 2 kW.

[0166] 32. The process according to any one of items 1 to 31, wherein the dispersion according to step b) is carried out using a disperser equipped with a motor having a power selected within the range of about 2 kW to about 10 kW.

[0167] 33. The process according to any one of items 1 to 32, wherein the dispersion according to step b) is carried out using an immersion type disperser and an in-line disperser.

[0168] 34. The process according to item 33, wherein an immersion disperser and an in-line disperser are used in series.

[0169] 35. The process according to item 33 or 34, wherein dispersion is initiated using an immersion disperser followed by an in-line disperser.

[0170] 36. The process according to any one of items 1 to 15, wherein the dispersion according to step b) is carried out for a period of at least about 3 hours.

[0171] 37. The process according to any one of items 1 to 36, wherein the dispersion according to step b) is carried out for a period ranging from about 3 hours to about 8 hours.

[0172] 38. The process according to any one of items 1 to 37, wherein the dispersion according to step b) is carried out for a period ranging from about 3 hours to about 5 hours.

[0173] 39. The process according to any one of items 33 to 38, wherein the dispersion according to step b) is carried out using an immersion disperser for a period of up to about 1 hour, followed by dispersion using an in-line disperser for a period of about 1 hour to about 4 hours.

[0174] 40. The process according to any one of items 1 to 39, wherein the dispersion according to step b) is carried out at a temperature (of the mixture) selected within the range of about 15°C to about 30°C.

[0175] 41. The process according to any one of items 1 to 40, wherein the dispersion according to step b) is carried out at ambient pressure.

[0176] 42. The process further comprises: b1) filtering the intermediate aqueous dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in an aqueous liquid carrier formed in step b) prior to homogenizing the obtained filtered intermediate aqueous dispersion according to step c).

[0177] 43. The process according to item 41, wherein the filtration according to step b1) is carried out using a filter having an average pore width in the range of about 200 μm to about 250 μm.

[0178] 44. The process according to any one of items 1 to 43, wherein the inhalable immunosuppressive macrocyclic active ingredient is cyclosporin A (CsA) or tacrolimus.

[0179] 45. The process according to any one of items 1 to 44, wherein the inhalable immunosuppressive macrocyclic active ingredient is cyclosporin A (CsA).

[0180] 46. The process according to any one of items 1 to 45, wherein the mixture provided in step a) comprises an inhalable immunosuppressive macrocyclic active ingredient, in particular cyclosporin A (CsA), at a concentration selected within the range of about 1 g / L to about 7 g / L, or about 2 g / L to about 6 g / L, or about 3 g / L to about 5 g / L, for example 4 g / L.

[0181] 47. The process according to any one of items 1 to 46, wherein the membrane-forming substance selected from the group of phospholipids is a mixture of natural phospholipids.

[0182] 48. The process according to any one of items 1 to 47, wherein the membrane-forming substance selected from the group of phospholipids is a lecithin containing unsaturated fatty acid residues.

[0183] 49. The process according to any one of items 1 to 48, wherein the 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 comparable lecithins.

[0184] 50. The process according to any one of items 1 to 49, wherein the mixture provided in step a) comprises a membrane-forming substance selected from the group of phospholipids at a concentration selected within the range of about 20 g / L to about 60 g / L, or about 30 g / L to about 50 g / L, or about 30 g / L to about 40 g / L.

[0185] 51. The process according to any one of items 1 to 50, wherein the solubility enhancer selected from the group of nonionic surfactants is selected from the group of polysorbates.

[0186] 52. The process according to any one of items 1 to 51, wherein the solubility enhancer selected from the group of non-ionic surfactants is polysorbate 80.

[0187] 53. The process according to any one of items 1 to 52, wherein the mixture provided in step a) comprises a solubility enhancer selected from the group of nonionic surfactants at a concentration selected within the range of about 1 g / L to about 5 g / L, or about 2 g / L to about 4 g / L, or about 2.5 g / L to about 3.5 g / L.

[0188] 54. The process according to any one of items 1 to 53, wherein the weight ratio of phospholipid to polysorbate is selected in the range of 15:1 to 9:1, preferably about 14:1 to about 12:1, for example about 13:1.

[0189] 55. The process according to any one of items 1 to 54, wherein the weight ratio of the (total amount of) phospholipid to non-ionic surfactant, on the one hand, and the weight ratio of the phospholipid to the inhalable immunosuppressive macrocyclic active ingredient (in particular CsA), on the other hand, is selected in the range of about 5:1 to about 20:1, preferably about 8:1 to about 12:1, more preferably about 9:1.

[0190] 56. The process according to any one of items 1 to 55, wherein the weight ratio of phospholipid (lecithin), non-ionic surfactant and inhalable immunosuppressive macrocyclic active ingredient (especially CsA) is about 15:1:1.5 to 5:0.3:0.5, preferably about 9:0.7:1.

[0191] 57. The process according to any one of items 1 to 56, wherein the inhalable immunosuppressive macrocyclic active ingredient (in particular CsA) is at least partially incorporated into (or intercalated between) the bilayer membrane of the liposome-forming structure.

[0192] 58. The process according to any one of items 1 to 57, wherein the inhalable immunosuppressive macrocyclic active ingredient (e.g., CsA) is incorporated predominantly (e.g., at least about 90%, or even at least about 95% to about 97.5%) in the bilayer membrane of the liposome-forming structure.

[0193] 59. The process according to any one of items 1 to 58, wherein at least about 90%, or about 95% to about 97.5%, or about 99%, or about 99.5%, or even 99.9% of the total amount of the inhalable immunosuppressive macrocyclic active ingredient (especially CsA) is incorporated into the bilayer membrane of the liposome-forming structure formed in the process of the invention.

[0194] 60. The process according to any one of items 1 to 59, wherein the mixture provided by step a) comprises at least one excipient selected from the group consisting of sugars, buffers and chelating agents.

[0195] 61. The process according to any one of items 1 to 60, wherein the mixture provided by step a) comprises at least one disaccharide as an excipient.

[0196] 62. The process according to any one of items 1 to 61, wherein the mixture provided in step a) comprises at least one disaccharide selected from the group consisting of sucrose, lactose and trehalose as an excipient.

[0197] 63. The process according to any one of items 1 to 62, wherein the mixture provided in step a) comprises at least one disaccharide selected from the group consisting of sucrose, lactose, and trehalose as an excipient at a concentration selected within the range of 60 g / L to about 140 g / L, or about 80 g / L to about 120 g / L, or about 90 g / L to about 110 g / L.

[0198] 64. The process according to any one of items 1 to 63, wherein the mixture provided according to step a) comprises sucrose.

[0199] 65. The process according to any one of items 1 to 64, wherein the aqueous liquid carrier comprises aqueous sodium chloride solution (saline) or sterile water.

[0200] 66. The process according to any one of items 1 to 65, wherein the step of homogenizing the intermediate aqueous dispersion formed in step b) to form a dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form comprises homogenization at high pressure.

[0201] 67. The process according to item 66, wherein the high pressure homogenization is carried out at a pressure selected within the range of about 100 to about 1,000 bar.

[0202] 68. The process according to item 66 or 67, wherein the homogenization at high pressure according to step c) is carried out using a piston-gap homogenizer.

[0203] 69. A process for the preparation of a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, the lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form; 69. The process comprises preparing a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier according to the process of any one of items 1 to 68, d) removing the aqueous liquid carrier at least partially under lyophilization conditions to form a lyophilized pharmaceutical composition.

[0204] 70. A lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form, -inhalable immunosuppressive macrocyclic active ingredients; - membrane-forming substances selected from the group of phospholipids; a solubility enhancer selected from the group of nonionic surfactants; and 70. The process according to item 69, optionally comprising one or more excipients.

[0205] 71. A lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form for reconstitution in an aqueous liquid carrier, which composition is obtained or obtainable by the process according to item 69 or 70.

[0206] 72. a) a liposome-forming structure, -Therapeutically effective doses of cyclosporine A (CsA); membrane-forming substances selected from the group of phospholipids; and - solubility enhancers selected from the group of nonionic surfactants; and b) at least one disaccharide selected from the group consisting of sucrose, lactose, and trehalose; 72. The lyophilized pharmaceutical composition according to item 71, comprising at least one disaccharide present in an amount of at least 40% by weight relative to the total weight of the lyophilized composition.

[0207] 73. A lyophilized pharmaceutical composition according to item 71 or 72 for use as a drug for pulmonary application.

[0208] 74. The lyophilized pharmaceutical composition for use according to item 73, wherein the pulmonary application is carried out after reconstitution (dispersion) of the lyophilized pharmaceutical composition according to item 71 or 72 in an aqueous liquid carrier to form a colloidal solution or dispersion.

[0209] 75. A lyophilized pharmaceutical composition for use according to item 73 or 74, wherein pulmonary application is carried out after converting the composition into an aerosol, for example by nebulization.

[0210] 76. A lyophilized pharmaceutical composition for use according to any one of items 73 to 75, wherein pulmonary application is carried out by inhalation.

[0211] 77. The lyophilized pharmaceutical composition for use according to any one of items 73 to 76, wherein the pulmonary application is carried out by means of an ultrasonic or electronic vibrating membrane nebulizer, preferably a vibrating membrane nebulizer, such as, for example, an eFlow® type, an AeroNeb Pro type or a -Go or I-Neb type device.

[0212] 78. A lyophilized pharmaceutical composition for use according to any one of items 73 to 77 for the prevention and treatment of asthma, refractory asthma, chronic obstructive bronchitis, parenchymal, fibrotic and interstitial lung diseases and inflammation, preferably for the prevention and treatment of acute and chronic organ transplant rejection after lung transplantation, and diseases resulting from bronchiolitis obliterans, etc.

[0213] The following examples serve to illustrate the invention but are not intended to limit it in any way. [Example]

[0214] Example 1: Preparation of a dispersion containing cyclosporin A in liposomal solubilized form 1.1 Step a: Preparation of the initial component mixture 1.1.1 Approximately 70% (approximately 104 L) of water for injection was filled into a preparation vessel. It was degassed by introducing nitrogen gas and adjusted to a temperature of 40-45°C. 18.0 kg of sucrose, 450.0 g of sodium dihydrogen phosphate dihydrate, 612.0 g of disodium hydrogen phosphate decahydrate, and 36.0 g of edetate disodium were added together, using approximately 5% (8.0 L) of water for injection for rinsing. The mixture was stirred until a visually clear solution was obtained.

[0215] 1.1.2 The solution was cooled to 5-25°C, and 6480.0 g of soy lecithin, Lipoid S100, was added and stirred until a homogeneous mixture was obtained. 504.0 g of polysorbate 80HP (Tween 80) was then added with gentle stirring to avoid foaming, and the container containing the polysorbate was rinsed with approximately 100 mL of water for injection. 720.0 g of cyclosporine and approximately 5% (8 L) of water for injection were then added.

[0216] 1.2 Step b: Dispersion of the initial component mixture 1.2.1 The component mixture prepared according to step 1.1 above was then transferred to a 400 L vessel and dispersed for 8 hours at a rotation speed of 4,800 rpm using an Ultra Turrax® UTE 115-P (IKA, Germany) high-shear immersion disperser equipped with a cylindrical stator having an inner diameter of 102.5 mm and 30 teeth, and a rotor (TP4, IKA, Germany) having an outer 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, generating a shear rate of 33,828 1 / s and a shear frequency of 64,800 1 / s. The resulting dispersion was then stirred for 3 hours, after which 10 L (each) of water for injection was added in two portions to reduce foaming.

[0217] 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 stainless steel protective filter (Rigimesh® filter, PALL) with a pore size of 225 μm, and then subjected to high-pressure homogenization at a pressure of 100 bar (first stage) and 1,000 bar (second stage), respectively, at a temperature of up to 25° C. The high-pressure homogenization was repeated 9 times (cycles). After the 8th homogenization, approximately 8 L of water for injection was added.

[0218] 1.4 Step d: Bioburden Reduction 1.4.1 The resulting homogenized dispersion was then filtered through a bioburden reduction filter (Fluorodyne® EX; PALL) with a pore size of 0.2 μm and transferred to a filling / storage tank.

[0219] 1.5. Product Characterization 1.5.1 The resulting homogenized dispersion had a cyclosporine A content of 100% of the total amount of cyclosporine A added in step 1.1.2.

[0220] Example 2: Preparation of a dispersion containing cyclosporin A in liposomal solubilized form 2.1 Step a: Preparation of the initial component mixture 2.1.1 The preparation of the initial component mixture as outlined in section 1.1 above was repeated exactly.

[0221] 2.2 Step b: Dispersion of the initial component mixture 2.2.1 The component mixture prepared according to step 2.1 above was then transferred to a 400 L vessel and dispersed for 80 minutes at 3,000 rpm using an Ultra Turrax® UTE 115-P (IKA, Germany) high-shear immersion disperser equipped with a cylindrical stator having an inner diameter of 102.5 mm and 30 teeth, and a rotor (TP4, IKA, Germany) having an outer 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, generating a shear rate of 21,153 1 / s and a shear frequency of 40,500 1 / s. The resulting dispersion was then stirred for 3 hours, after which 10 L (each) of water for injection was added in two portions to reduce foaming.

[0222] 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 protective filter with a pore size of 40 μm (40 μm HDC II filter (all polypropylene), PALL), and then subjected to high-pressure homogenization at a temperature of 25° C. and a pressure of 100 bar (first stage) and 1,000 bar (second stage), respectively. The high-pressure homogenization was repeated 9 times (cycles). After the 8th homogenization, approximately 8 L of water for injection was added.

[0223] 2.4 Step d: Bioburden Reduction 2.4.1 The resulting homogenized dispersion was then filtered through a bioburden reduction filter (Fluorodyne® EX; PALL) with a pore size of 0.2 μm and transferred to a filling / storage tank.

[0224] 2.5 Product Characterization 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.

[0225] Example 3: Preparation of a dispersion containing cyclosporin A in liposomal solubilized form using an in-line disperser 3.1 Step a: Preparation of the initial component mixture 3.1.1 The preparation of the initial component mixture as outlined in section 1.1 above was repeated exactly.

[0226] 3.2 Step b: Dispersion of the initial component mixture 3.2.1 The component mixture prepared according to step 3.1 above was then transferred to a 400 L vessel and dispersed at a rotation speed of 4,000 rpm for 4 hours using an Ultra Turrax® UTL 1000 / 10 (IKA, Germany) high-shear in-line disperser equipped with a dispersing tool (8SF, IKA, Germany) having a stator with an inner diameter of 120.1 mm, 54 teeth, and a tooth gap of 1.6 mm, and a rotor with an outer diameter of 119.4 mm and a tooth gap of 2.0 mm, with a motor power of 7.5 kW, generating a shear rate of 71,413 1 / s and a shear frequency of 151,200 1 / s. The resulting dispersion was then stirred for 3 hours, after which 10 L (each) of water for injection was added in two portions to reduce foaming.

[0227] 3.3 Step c: Homogenization of the intermediate aqueous dispersion 3.3.1 Homogenization of the intermediate aqueous dispersion undergone in step 3.2.1 was repeated exactly as described in section 1.3.1 above.

[0228] 3.4 Step d: Bioburden Reduction 3.4.1 The resulting homogenized dispersion was then filtered through a bioburden reduction filter (Fluorodyne® EX; PALL) with a pore size of 0.2 μm and transferred to a filling / storage tank.

[0229] 3.5. Product Characterization 3.5.1 The resulting homogenized dispersion had a cyclosporine A content of 100% of the total amount of cyclosporine A added in step 3.1.

[0230] Example 4: Preparation of dispersions containing liposomally solubilized cyclosporine A using an immersion disperser and an in-line disperser 4.1 Step a: Preparation of the initial component mixture 4.1.1 Exactly repeat the preparation of the initial component mixture as outlined in section 1.1 above.

[0231] 4.2 Step b: Dispersion of the initial component mixture 4.2.1 The component mixture as prepared according to step 4.1 above is then transferred to a vessel having a capacity of 400 L and dispersed for 30 minutes at a rotation speed of 4,800 rpm using an Ultra Turrax® UTE 115-P (IKA, Germany) high shear immersion disperser equipped with a cylindrical stator having an inner diameter of 102.5 mm and 30 teeth, and a rotor (TP4, IKA, Germany) having an outer 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 producing a shear rate of 33,828 1 / s and a shear frequency of 64,800 1 / s. The resulting mixture was then dispersed at a rotation speed of 4,000 rpm for 4 hours using an Ultra Turrax® UTL 1000 / 10 (IKA, Germany) high-shear in-line disperser equipped with a dispersing tool (8SF, IKA, Germany) having a stator with an inner diameter of 120.1 mm, 54 teeth, and a tooth gap of 1.6 mm, and a rotor with an outer diameter of 119.4 mm and a tooth gap of 2.0 mm, with a motor power of 7.5 kW, generating a shear rate of 71,413 1 / s and a shear frequency of 151,200 1 / s. The resulting dispersion was then stirred for 3 hours, after which 10 L (each) of water for injection was added in two portions to reduce foam generation.

[0232] 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 stainless steel protective filter (Rigimesh® filter, PALL) with a pore size of 225 μm and then subjected to high-pressure homogenization at a pressure of 100 bar (first stage) and 1,000 bar (second stage) respectively at a temperature of up to 25° C. The high-pressure homogenization is repeated 8 times (cycles). After the sixth homogenization, approximately 8 L of water for injection is added.

[0233] 4.4 Step d: Bioburden Reduction 4.4.1 The resulting homogenized dispersion is then filtered through a bioburden reduction filter (Fluorodyne® EX; PALL) with a pore size of 0.2 μm and transferred to a filling / storage tank.

[0234] 4.5. Product Characterization 4.5.1 The resulting homogenized dispersion has a cyclosporine A content of 100% of the total amount of cyclosporine A added in step 4.1.

[0235] Example 5: Aseptic filling, freeze-drying and packaging 5.1 Glass vials with a fill volume of 10 mL were sterilized in a dry heat sterilization tunnel, allowed to cool, and aseptically sterilized using two sterilizing filters with a pore size of 0.2 μm between the fill / storage tank and the filling needle, before being filled with 1.35 mL (5 mL dose) aliquots of the dispersion prepared according to Example 1 described above. The vials were then partially closed with sterilized lyophilization stoppers, placed in a lyophilizer (GEA Lyovac FCM) and lyophilized according to a 72 hour lyophilization cycle.

[0236] 5.2 After lyophilization was completed, the vials were automatically fully stoppered in the lyophilization chamber. The vials were removed and closed with flip tear-off caps. Each vial contained approximately 190 mg of a nearly white, homogeneous, porous lyophilized cake containing 5 mg of liposomally solubilized cyclosporine A, with a maximum of 2% (w / w) residual moisture and a shelf life of 3 years.

[0237] 5.3 The composition of the lyophilized formulation prepared as described above is summarized in Table 1 below:

[0238] [Table 1]

[0239] Example 6: Reconstitution of a lyophilized composition containing cyclosporin A to obtain a colloidal solution of liposomally solubilized cyclosporin A for nebulization and inhalation 6.1 To an aliquot of 186.1 mg of lyophilized cake containing 5 mg of cyclosporine A and prepared according to Example 5 above, 1.20 mL of sterile aqueous sodium chloride solution (concentration: 0.25% (w / v)) was added to obtain 1.35 mL of an opalescent aqueous solution of liposomal cyclosporine A for inhalation purposes, with a CsA concentration of 4 mg / mL. The deliverable volume of the device is 1.25 mL (5 mg L-CsA).

[0240] 6.2 To prepare the corresponding colloidal solution containing 10 mg of liposomally solubilized cyclosporine A, an aliquot of 372.3 mg of the lyophilized cake prepared according to Example 1 above was dissolved in 2.40 mL of sterile aqueous sodium chloride solution (concentration: 0.25% (w / v)) to obtain 2.65 mL of an opalescent aqueous solution of liposomal cyclosporine A for inhalation purposes with a CsA concentration of 4 mg / mL. The deliverable volume for the device is 2.50 mL (10 mg L-CsA).

[0241] 6.3 The composition of the reconstituted formulation prepared as described above is summarized in Table 2 below:

[0242] [Table 2]

[0243] Example 7: Preparation of a lyophilized composition containing CsA in liposome-solubilized form and its liposome solution reconstituted in the presence of lactose or trehalose 7.1 Lyophilized compositions containing CsA in liposome-solubilized form were prepared in the presence of trehalose and lactose monohydrate as disaccharides according to the protocols of Examples 1 and 5 above. Both disaccharides were used in amounts necessary to obtain 7.5% (w / v) and 10% (w / v) of each sugar in the final reconstituted liposome solution. Furthermore, in addition to the compositions summarized in Table 2 above, corresponding liposome solutions were prepared with sucrose contents of 5.0% (w / v) and 7.5% (w / v). In all cases, opalescent colloidal solutions were obtained with dispersities (PI) and liposome diameters (measured as z-average diameter (ZA)) as summarized in Table 3 below.

[0244] [Table 3]

[0245] Example 8: Comparison of the characteristics of aqueous liposomal dispersions containing CsA in liposomally solubilized form before lyophilization and after reconstitution of the lyophilizate 8.1 Aqueous dispersions of liposomally solubilized CsA containing 10% (w / v) sucrose were prepared as described in Example 1. Similarly, aqueous dispersions of liposomally solubilized CsA containing 10% (w / v) lactose were prepared. Additionally, aqueous dispersions containing 10% (w / v) sucrose were lyophilized and reconstituted using water for injection as described in Example 5. Key characteristics of the resulting dispersions are summarized in Table 4 below.

[0246] [Table 4]

[0247] Example 9: Stability of lyophilized compositions containing liposomally solubilized CsA, stability comparison 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 according to Examples 1 and 5 above. The lyophilized composition, in the form of a nearly white, homogeneous, porous lyophilized cake, was aliquoted into 6R glass vials, sealed, and stored at 25°C and 60% relative humidity (RH) for a period of 36 months. Aliquots of this material were reconstituted with saline (0.25% (w / v)) to yield 1.25 mL volumes of reconstituted solution before and after the storage period. The mean liposome size (Z-average), molecular weight dispersity, and cyclosporine A content were determined after 3, 6, 9, 12, 18, 24, and 36 months.

[0248] 9.1.2 All parameters were found to be within the acceptable ranges before and after the above storage periods. More specifically, the polydispersity index (PI) was 0.50 or less before and after each storage period. Furthermore, the mean liposome diameter (Z-average) was within the specified range of 40-100 nm before and after each storage period. Furthermore, the CsA content of the reconstituted solution was within the acceptable range of 95.0-105.0%, within the acceptable range.

[0249] 9.1.3 The long-term stability study was repeated as described above at a temperature of 30°C and a humidity of 65% relative humidity (RH). All test parameters described above were found to be within their acceptance criteria (as above) before and after 3, 6, 9, and 12 months of storage.

[0250] 9.1.4 The long-term stability study described above was repeated using a lyophilized pharmaceutical composition comprising cyclosporine A (5 mg) prepared according to Example 1 described above, except in this case the lyophilized composition had a sucrose content necessary to obtain a liquid composition having a sucrose content of 7.5% by weight, relative to the total amount of liquid composition after reconstitution.

[0251] 9.1.5 Again, all parameters were found to be within the respective acceptance criteria before and after the storage periods. More specifically, the polydispersity index (PI) was 0.50 or less before and after each storage period. Furthermore, the mean liposome diameter (Z-average) was within the specified range of 40-100 nm before and after each storage period. Furthermore, the CsA content of the reconstituted solution was within the acceptance criteria, ranging from 95.0 to 105.0%.

[0252] 9.1.6 The experiments described under Sections 9.1.1 to 9.1.3 above were repeated using lyophilized pharmaceutical compositions containing 10 mg of cyclosporine A prepared according to Examples 1 and 5 above. Again, all parameters were found to be within the respective acceptance criteria before and after the storage periods. More specifically, the polydispersity index (PI) was 0.50 or less before and after each storage period. Furthermore, the mean liposome diameter (Z-average) was within the specified range of 40 to 100 nm before and after each storage period. Furthermore, the CsA content of the reconstituted solution was within the acceptance criteria, ranging from 95.0 to 105.0%.

[0253] Example 10: Nebulization experiments and aerosol characterization 10.1 2.5 mL (corresponding to 10 mg of CsA) of colloidal solution as prepared in Examples 1, 5 and 6 (successively) was aerosolized according to European Pharmacopoeia 7.3;2.9.44 using a specially adapted PARI eFlow 30 XL electronic vibrating membrane nebulizer with a mixing chamber and breathing in / out valves at a flow rate of 15 L / min.

[0254] 10.2 The droplet size distribution of the generated aerosol was characterized by laser diffraction using a Malvern MasterSizer X. The mass median particle size determined was 3.3 μm (standard deviation (SD) 0.1) with a geometric standard deviation of 1.5. The respirable particle fraction (RF) being less than 5 μm was 65.3% (SD 2.8) and the respirable particle fraction with particles less than 3.3 μm was 37.7% (SD 2.2).

[0255] 10.3 In an inhalation study (adult; flow rate 15 mL / min), a total of 9897 μg of cyclosporine A in the reconstituted liquid formulation described in Example 1 above was loaded into an electronic vibrating membrane nebulizer (PARI eFlow 30XL) and administered. The delivered dose (DD) of cyclosporine A was 7339 μg (SD: 471). The respirable dose (RD) below 5 μm was 6534 μg (66.0%; SD 4.3%); the RD below 3.3 μm was 4461 μg (45.1%, SD 3.2%), and the respirable dose (RD) below 2 μm was 1080 μg (10.9%; SD 0.9%).

[0256] Example 11: Preparation of a dispersion containing tacrolimus in liposomal solubilized form 11.1 Step a: Preparation of the Initial Component Mixture 11.1.1 Fill a preparation vessel with approximately 70% (approximately 104 L) of water for injection. Degas it by introducing nitrogen gas and adjust the temperature to 40-45°C. Add 18.0 kg of sucrose, 450.0 g of sodium dihydrogen phosphate dihydrate, 612.0 g of disodium hydrogen phosphate decahydrate, and 36.0 g of edetate disodium, together with approximately 5% (8.0 L) of water for injection for rinsing. Stir the mixture until a visually clear solution is obtained.

[0257] 11.1.2 Cool the solution to 5-25°C and add 6480.0 g of soy lecithin, Lipoid S100, and stir until a homogeneous mixture is obtained. Then, add 504.0 g of polysorbate 80HP (Tween 80) with gentle stirring to avoid foaming, and rinse the polysorbate container with approximately 100 mL of water for injection. After this, add 720.0 g of tacrolimus and approximately 5% (8 L) of water for injection.

[0258] 11.2 Step b: Dispersion of the initial component mixture 11.2.1 The component mixture as prepared according to step 11.1 above is then transferred to a 400 L vessel and dispersed for 8 hours at a rotation speed of 4,800 rpm using an Ultra Turrax® UTE 115-P (IKA, Germany) high-shear immersion disperser equipped with a cylindrical stator having an inner diameter of 102.5 mm and 30 teeth, and a rotor (TP4, IKA, Germany) having an outer 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 producing a shear rate of 33,828 1 / s and a shear frequency of 64,800 1 / s, until a homogeneous dispersion is formed. The resulting dispersion is then stirred for 3 hours, after which 10 L (each) of water for injection is added in two portions to reduce foam generation.

[0259] 11.3 Step c: Homogenization of the intermediate aqueous dispersion 11.3.1 The resulting dispersion is then transferred to a GEA high-pressure homogenizer through a stainless steel protective filter (Rigimesh® filter, PALL) with a pore size of 225 μm and then subjected to high-pressure homogenization at a pressure of 100 bar (first stage) and 1,000 bar (second stage), respectively, at a temperature of up to 25° C. The high-pressure homogenization is repeated 8 times (cycles). After the sixth homogenization, approximately 8 L of water for injection is added.

[0260] 11.4 Step d: Bioburden Reduction 11.4.1 The resulting homogenized dispersion is then filtered through a bioburden reduction filter (Fluorodyne® EX; PALL) with a 0.2 μm pore size and transferred to a filling / storage tank.

[0261] 11.5. Product Characterization 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

1. 1. A process for the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier, said process comprising: a) - inhalable immunosuppressive macrocyclic active ingredients; - membrane-forming substances selected from the group of phospholipids; - a solubility enhancer selected from the group of non-ionic surfactants; optionally one or more excipients; and the aqueous liquid carrier; providing a mixture comprising: b) dispersing the mixture provided in step a) to form an intermediate aqueous dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in the aqueous liquid carrier; and c) homogenizing the intermediate aqueous dispersion formed in step b) to form the dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form; A process wherein the dispersion according to step b) is carried out at a shear rate of at least 22,000 1 / s.

2. 2. The process according to claim 1, wherein the dispersing according to step b) is carried out using a rotor-stator type disperser.

3. 2. The process according to claim 1, wherein the dispersion according to step b) is carried out using an immersion type disperser.

4. 4. The process according to claim 1, wherein the dispersion according to step b) is carried out at a shear rate selected in the range of about 25,000 1 / s to about 40,000 1 / s.

5. 5. The process according to claim 1, wherein the dispersing according to step b) is carried out at a shear frequency selected in the range of about 50,000 1 / s to about 80,000 1 / s.

6. 6. The process according to any one of claims 1 to 5, wherein the dispersing according to step b) is carried out at a shear rate selected in the range of about 28,000 1 / s to about 37,000 1 / s and at a shear frequency selected in the range of about 50,000 1 / s to about 80,000 1 / s.

7. The process of any one of claims 2 to 6, wherein the rotor has a diameter in the range of about 60 mm to about 140 mm.

8. The process according to any one of claims 2 to 7, wherein the dispersing according to step b) is carried out at a rotation speed ranging from about 2,000 to about 6,000 rpm.

9. The process according to any one of claims 1 to 8, wherein the dispersing according to step b) is carried out using an in-line disperser.

10. 10. The process of claim 9, wherein the dispersion according to step b) is carried out at a shear rate selected within the range of about 45,000 1 / s to about 90,000 1 / s.

11. 11. The process according to claim 9 or 10, wherein the dispersing according to step b) is carried out at a shear frequency selected in the range of about 100,000 1 / s to about 200,000 1 / s.

12. 12. The process according to any one of claims 9 to 11, wherein the dispersing according to step b) is carried out at a shear frequency selected in the range of about 120,000 1 / s to about 180,000 1 / s.

13. 13. The process according to any one of claims 9 to 12, wherein the dispersing according to step b) is carried out at a shear rate selected in the range of about 55,000 1 / s to about 85,000 1 / s and at a shear frequency selected in the range of about 130,000 1 / s to about 170,000 1 / s.

14. The process of any one of claims 9 to 13, wherein the rotor has a diameter in the range of about 100 mm to about 140 mm.

15. The process according to any one of claims 9 to 14, wherein the dispersing according to step b) is carried out at a rotation speed ranging from about 3,500 to about 4,500 rpm.

16. The process according to any one of claims 1 to 15, wherein the dispersing according to step b) is carried out using an immersion disperser and an in-line disperser.

17. 17. The process of claim 16, wherein the immersion disperser and the in-line disperser are used in series.

18. 18. The process of claim 16 or 17, wherein the dispersion is initiated using an immersion type disperser followed by an in-line disperser.

19. The process according to any one of claims 2 to 18, wherein the dispersion according to step b) is carried out at a peripheral speed of the rotor selected in the range of about 15 m / s to about 40 m / s.

20. The process according to any one of claims 1 to 19, wherein the dispersing according to step b) is carried out for a period ranging from about 1 hour to about 8 hours.

21. The process according to any one of claims 1 to 20, wherein the dispersing according to step b) is carried out for a period ranging from about 3 hours to about 8 hours.

22. 22. The process according to any one of claims 1 to 21, wherein the dispersing according to step b) is carried out using an immersion type disperser for a period of up to about 1 hour, followed by dispersing using an in-line disperser for a period of about 1 hour to about 4 hours.

23. 23. The process of any one of claims 1 to 22, wherein the intermediate aqueous dispersion comprising the inhalable immunosuppressive macrocyclic active ingredient in the aqueous liquid carrier formed in step b) is filtered prior to homogenization according to step c).

24. 24. The process of claim 23, wherein the filtration is carried out using a filter having an average pore width in the range of about 200 μm to about 250 μm.

25. The process further comprises the steps 25. The process according to any one of claims 1 to 24, comprising a step c1) of sterilizing the homogenized dispersion obtained from step c) comprising the inhalable immunosuppressive macrocyclic active ingredient, in particular cyclosporin A, in liposome-solubilized form.

26. 26. The process of claim 25, wherein the sterilization is performed by sterile filtration.

27. 27. The process of any one of claims 1 to 26, wherein the inhalable immunosuppressive macrocyclic active ingredient is cyclosporin A (CsA) or tacrolimus.

28. 28. The process of any one of claims 1 to 27, wherein the inhalable immunosuppressive macrocyclic active ingredient is cyclosporin A (CsA).

29. 29. The process according to any one of claims 1 to 28, wherein the 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 comparable lecithins.

30. 30. The process of any one of claims 1 to 29, wherein the solubility enhancer selected from the group of non-ionic surfactants is selected from the group of polysorbates.

31. 31. The process according to any one of claims 1 to 30, wherein the mixture provided in step a) comprises as an excipient at least one disaccharide selected from the group consisting of sucrose, lactose and trehalose.

32. 32. The process of any one of claims 1 to 31, wherein the mixture provided by step a) comprises at least one disaccharide selected from the group consisting of sucrose, lactose, and trehalose as an excipient at a concentration selected within the range of from 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.

33. 1. A process for the preparation of a lyophilized pharmaceutical composition for reconstitution in an aqueous liquid carrier, said lyophilized pharmaceutical composition comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form; 33. The process according to claim 1, comprising the preparation of a dispersion comprising an inhalable immunosuppressive macrocyclic active ingredient in liposome-solubilized form in an aqueous liquid carrier, d) removing said aqueous liquid carrier at least partially under lyophilization conditions to form said lyophilized pharmaceutical composition.

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