Dry powder preparation of interferon beta and method for producing the same

Abstract

The present invention relates to a dry powder formulation of interferon beta and a method for producing the same, and more particularly to a dry powder formulation of interferon beta in the form of spherical particles with high morphological perfection, which is produced by spray drying a liquid composition containing interferon beta, and a method for producing the same.

Description

[Technical Field]

[0001] This application claims priority from Korean Patent Application No. 10-2023-0010191, filed on January 26, 2023, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a dry powder formulation of interferon beta and a method for producing the same, and more particularly to a dry powder formulation of interferon beta in the form of spherical particles with high morphological perfection, which is produced by spray drying a liquid composition containing interferon beta, and a method for producing the same. [Background technology]

[0003] Interferons (IFNs) are a type of cytokine that exhibit antiviral activity, suppress cell proliferation, and regulate innate immune responses. IFNs are classified into IFN-α, IFN-β, and IFN-γ depending on their cellular origin (leukocytes, fibroblasts, and T cells). Interferon-β (IFN-β) is a globular protein with five α-helices, 22 kD in size, which becomes 18 kD when the glycosylation chains are removed.

[0004] Research into the clinical application of IFN-β is actively underway, and it is particularly attracting attention as a treatment for multiple sclerosis, alleviating or alleviating the symptoms. IFN-β also has a variety of other immunological activities, including antiviral activity, inhibition of cell proliferation, enhancing lymphocyte cytotoxicity, immunomodulatory activity, target cell differentiation induction or suppression, macrophage activation, increasing cytokine production, increasing the effectiveness of cytotoxic T cells, and increasing natural killing cells, and has been reported to be effective in treating cancer, autoimmune disorders and viral infections, HIV-related diseases, hepatitis C, and rheumatoid arthritis.

[0005] Human IFN-β is also a glycoprotein, and the glycosylation of proteins plays an important role in their activity, so the activity of glycoproteins can be increased by glycosylation. In other words, protein glycosylation can affect many biochemical properties, such as stability, solubility, intracellular trafficking activity, pharmacokinetics, and antigenicity.

[0006] IFN is typically formulated as an isotonic aqueous solution for parenteral administration. In recent years, clinicians have sought alternative routes of IFN administration that are more suitable for long-term use in patients. Specifically, aerosol formulations of IFN have been prepared for pulmonary delivery as described in WO 91 / 16038. The formulations are dispersed by evaporation of a liquid propellant. This patent describes the addition of a surfactant or its analogues to improve the dispersibility of human IFN from a fluorocarbon delivery system.

[0007] Methods and compositions for producing solid polypeptide microparticles as pharmaceutical aerosol formulations are described in WO 91 / 16038, where IFN-β was prepared in the form of a dry powder by freeze-drying an aqueous solution of IFN and spray-milling after freeze-drying.

[0008] However, this dry powder form has the problem that the particles are not perfectly spherical and are not suitable for effective pulmonary delivery, and it has the limitation of significantly reducing the activity of IFN compared to the liquid formulation before dry powdering. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO91 / 16038 Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, the inventors have conducted extensive research to develop a dry powder formulation with high morphological perfection and interferon activity. As a result, they have found that a liquid interferon beta composition containing acetate buffer, arginine, poloxamer 188, methionine and a hydrophobic amino acid can be spray-dried to produce a formulation with excellent morphology, thereby completing the present invention.

[0011] It is therefore an object of the present invention to provide an interferon beta dry powder formulation produced by spray drying a liquid composition comprising: (a) Interferon beta or its variants; (b) acetate buffer at concentrations between 5 and 100 mM; (c) Arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at concentrations of 0.1–10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5–30% (w / v) hydrophobic amino acids.

[0012] Another object of the present invention is to provide an inhalable capsule filled with a dry powder formulation.

[0013] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, comprising the dry powder formulation.

[0014] Furthermore, another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, which comprises a dry powder formulation.

[0015] Furthermore, another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, which consists essentially of the dry powder formulation.

[0016] Another object of the present invention is to provide a method for producing a dry powder of interferon beta, comprising the step of spray-drying a liquid composition comprising: (a) Interferon beta or its variants; (b) acetate buffer at concentrations between 5 and 100 mM; (c) Arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at concentrations of 0.1–10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5–30% (w / v) hydrophobic amino acids. [Means for solving the problem]

[0017] In order to achieve the above object of the present invention, the present invention provides a dry powder formulation of interferon beta prepared by spray drying a liquid composition comprising: (a) Interferon beta or its variants; (b) acetate buffer at concentrations between 5 and 100 mM; (c) Arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at concentrations of 0.1–10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5–30% (w / v) hydrophobic amino acids.

[0018] In order to achieve another object of the present invention, the present invention provides an inhalation capsule filled with a dry powder formulation.

[0019] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, comprising a dry powder formulation.

[0020] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, comprising a dry powder formulation.

[0021] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infection, HIV infection, hepatitis C, and rheumatoid arthritis, consisting essentially of the dry powder formulation.

[0022] In order to achieve another object of the present invention, the present invention provides a method for producing a dry powder of interferon beta, comprising the step of spray-drying a liquid composition comprising the following at an inlet air temperature of 90-130°C and an outlet air temperature of 40-80°C: (a) Interferon beta or its variants; (b) acetate buffer at concentrations between 5 and 100 mM; (c) Arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at concentrations of 0.1–10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5–30% (w / v) hydrophobic amino acids.

[0023] The present invention will be described in detail below.

[0024] The present invention provides an interferon-beta dry powder formulation produced by spray drying a liquid composition comprising: (a) Interferon beta or its variants; (b) acetate buffer at concentrations between 5 and 100 mM; (c) Arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at concentrations of 0.1–10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5–30% (w / v) hydrophobic amino acids.

[0025] One of the most fundamental challenges in producing protein-based formulations is determining the desired therapeutic protein concentration in solution. This is because protein stability is not only dependent on protein concentration but also on the solution's pH, temperature, ionic strength, and additive concentrations. Therefore, protein-based formulations must be suitable for stabilizing therapeutic proteins and avoid problems such as aggregation, precipitation, or fragmentation. When producing protein-based dry powder formulations, the protein dispersed in solution is powdered using methods such as freeze-drying and spray-drying, so a composition that maintains the protein's stability in solution is essential.

[0026] Furthermore, when a protein-based dry powder formulation is to be used for inhalation administration, it is preferable to prepare a dry powder formulation of particles with high morphological perfection, i.e., nearly spherical morphological characteristics, so that they are uniformly absorbed into the nasal mucosa or lungs.

[0027] Interferon is meant to include a class of naturally occurring or recombinantly prepared low-molecular-weight proteins and glycoproteins (sometimes called cytokines) with molecular weights of approximately 15,000 to 27,000 daltons and interferon activity. Generally, such activity is exerted by binding to specific membrane receptors on the cell surface. Once bound, interferons initiate a complex series of interferon-dependent intracellular processes. Interferons are useful in treating a variety of human diseases, ranging from cancer to immune system suppression. Natural interferons are produced and secreted by cells in response to viral infections, as well as synthetic and biological derivatives. Some interferons are modified forms of natural substances and are produced using recombinant DNA technology. Interferons are sometimes abbreviated as "IFN" and will be used in this specification. Examples of interferons include recombinant IFN-α-2A (Roferon® A, Roche Laboratories), recombinant IFN-α-2B (Intron® A, Shering), human leukocyte-derived IFN-α-N3 (Alferon® N, Purdue Frederick), IFN-gamma 1B (Actimmune®, Genentech), recombinant IFN-beta (Betaseron®, Chiron, Berlex), and natural IFN-beta (Feron®, Toray, Japan). As used herein, the term "interferon-beta (IFN-β)" refers to fibroblast interferon of human origin obtained by isolation from biological fluids or by recombinant DNA technology from eukaryotic or prokaryotic host cells, as well as its salts, functional derivatives, variants, analogs, and active fractions.

[0028] Preferably, the IFN-β of the present invention refers to human IFN-β.

[0029] A preferred IFN-β may refer to IFN-β-1a, which may include an N-linked carbohydrate chain at any one or more asparagine residues of the amino acid residues of the protein.

[0030] In a preferred embodiment of the present invention, the IFN-β mutant may be a human IFN-β mutant in which arginine at position 27 is replaced with threonine (R27T). The R27T mutant is a recombinant human IFN-β mutant (hereinafter referred to as rhIFN-β) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for additional glycosylation at position 25 of IFN-β1a, allowing glycosylation at two positions, the 25th and 80th amino acids.

[0031] In the present invention, the "liquid composition" means that the solvent contains the above (a) to (g). The type of solvent is not particularly limited as long as it is a pharmaceutically acceptable solvent, and is preferably water.

[0032] In the present invention, the term "powder" refers to a formulation consisting of finely dispersed solid particles that are free-flowing and can be easily dispersed by an inhalation device, and then inhaled by a patient, where the particles reach and penetrate the nasal mucosa or lungs. Therefore, the powder is said to be "respirable." The average size of the particles is preferably less than about 10 microns (μm) in diameter and has a relatively uniform ellipsoidal distribution. A diameter of less than about 7.5 μm is more preferred, and a diameter of less than about 5.0 μm is most preferred. The particle size distribution can generally be from about 0.1 μm to about 5 μm, particularly from about 2 μm to about 5 μm.

[0033] In the present invention, the term "dry" means that the composition has a moisture content that allows the particles to be readily dispersed in an inhalation device to form an aerosol, and the moisture content is generally less than about 10% by weight of water, usually less than about 5% by weight, and preferably less than about 4% by weight.

[0034] According to one embodiment of the present invention, a dry powder IFN-β preparation produced by spray-drying a liquid composition containing (a) interferon beta or a variant thereof; (b) acetate buffer solution at a concentration of 5 to 100 mM; (c) arginine at a concentration of 10 to 150 mM; (d) poloxamer 188 at a concentration of 0.1 to 10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5 to 30% (w / v) of a hydrophobic amino acid, exhibits a highly spherical morphology and has been confirmed to exhibit a high recovery rate and stable activity.

[0035] This is because arginine, poloxamer 188, and methionine not only effectively maintain a high level of protein stability of IFN-β in the liquid composition for spray drying, but also enable the protein to be dispersed uniformly without being destroyed during the spray drying process, resulting in the production of particles with a morphologically nearly spherical shape.

[0036] In the present invention, the liquid composition for spray drying contains arginine at a concentration of 10 to 150 mM, poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and methionine at a concentration of 0.5 to 5 mM, and more preferably contains arginine at a concentration of 50 to 100 mg / mL, poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and methionine at a concentration of 0.5 to 2 mM.

[0037] If the arginine, poloxamer 188, and methionine concentrations are outside the above ranges, the size and shape of the particles after spray drying may not be uniform, and the pharmacokinetics as an inhalant may be undesirable.

[0038] In the present invention, the acetate buffer is a solution that has the effect of adjusting or maintaining the pH of the formulation so that it falls within a preferred pH range for the formulation, and may have a concentration of preferably 5 to 100 mM, more preferably 10 to 30 mM.

[0039] In the present invention, the preferred pH range of the acetate buffer is 3.6 to 4.4, which not only maintains the stability of interferon beta in the liquid composition for spray drying and prevents abnormal aggregation reactions, but also maintains protein stability during the spray drying process and allows the powder particles to form a spherical shape.

[0040] According to one embodiment of the present invention, the dry powder produced by spray-drying the IFN-β liquid composition comprising the components of the present invention was rehydrated and the IFN-β content in the dry powder was confirmed. It was confirmed that 100% of the IFN-β before spray-drying was recovered.

[0041] The therapeutically effective amount of IFN-β can vary within a formulation depending on the biological activity of the IFN-β used and the amount required for the unit dosage form. Because IFN-β is highly active, it must be manufactured on a unit basis to facilitate easy handling by formulation equipment and consumers. This generally means that the unit dose is about 0.5 mg to 15 mg, preferably about 2 mg to 10 mg, of all ingredients in the dry powder formulation. Preferably, the amount of IFN-β in the dry powder formulation can vary from about 0.05% (w / w) to about 5.0% (w / w).

[0042] To adjust the amount of IFN-β in the dry powder to the above level, IFN-β may be contained in the liquid composition at a concentration of, but not limited to, 0.01 to 1% (w / v).

[0043] The dry powder formulations of the present invention can be prepared by spray-drying together with a pharmaceutically acceptable carrier. The amount of the pharmaceutically acceptable carrier is that amount necessary to provide the necessary stability, dispersibility, consistency, and packing properties to ensure uniform pulmonary delivery of the composition to a patient in need. Numerically, the amount can be from about 95.0% (w / w) to about 99.95% (w / w), depending on the activity of the IFN-β used. It may be desirable to use about 98% (w / w) to about 99.8% (w / w).

[0044] Carriers can be made by combining one or more pharmaceutical excipients, but are generally substantially free of "penetration enhancers." "Penetration enhancers" are surface-active compounds that facilitate drug penetration through mucous membranes or mucosal barriers and are proposed for use in intranasal, rectal, and vaginal pharmaceutical formulations. Penetration enhancers include bile salts such as taurocholate, glycocholate, and deoxycholate; fujidates such as taurodihydrofujidate; and biologically acceptable surfactants such as Tweens and laureth-9. However, the use of penetration enhancers in pulmonary formulations is generally not preferred because the pulmonary epithelial blood barrier may be adversely affected by the surface-active compounds. The dry powder compositions of the present invention are readily absorbed into the lungs without the need for penetration enhancers.

[0045] Types of pharmaceutical excipients useful as carriers in the present invention include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides, pH adjusting or buffering agents, salts such as sodium chloride, etc. The carriers can be crystalline or amorphous, or can be a mixture of the two.

[0046] Particularly useful bulking agents may include acceptable carbohydrates, polypeptides, amino acids, or combinations thereof.

[0047] Hydrophobic amino acids may be included in the composition of the present invention for steric stability during spray drying and to improve powder dispersibility. These hydrophobic amino acids may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine and tryptophan, and preferably leucine.

[0048] In one embodiment of the present invention, the hydrophobic amino acid may be contained in the liquid composition at 15 to 25% (w / v), preferably 17 to 23% (w / v), and most preferably 19 to 21% (w / v).

[0049] In one embodiment of the present invention, the hydrophobic amino acid may be present in the liquid composition at 15% (w / v), 16% (w / v), 17% (w / v), 18% (w / v), 19% (w / v), 20% (w / v), 21% (w / v), 22% (w / v), 23% (w / v), 24% (w / v), or 25% (w / v).

[0050] If the concentration of the hydrophobic amino acid in the liquid composition is less than 15% (w / v), the yield of the dry powder formulation may decrease, and if it is more than 25% (w / v), the morphological properties of the dry powder particles may be undesirable.

[0051] In another aspect of the present invention, the hydrophobic amino acid may be contained in the liquid composition as leucine at 15 to 25% (w / v), preferably 17 to 23% (w / v), and most preferably 19 to 21% (w / v).

[0052] In another aspect of the invention, the leucine may be present in the liquid composition at 15% (w / v), 16% (w / v), 17% (w / v), 18% (w / v), 19% (w / v), 20% (w / v), 21% (w / v), 22% (w / v), 23% (w / v), 24% (w / v), or 25% (w / v).

[0053] A concentration of leucine in the liquid composition below 15% (w / v) may result in a reduced yield of the precursor powder formulation, while a concentration above 25% (w / v) may result in undesirable morphological properties of the dry powder particles.

[0054] In one aspect of the present invention, the formulation can be characterized in that 90% (w / w) of the dry powder has a particle size of 0.1 μm to 10 μm, preferably 1 μm to 10 μm, even more preferably 3 μm to 8 μm, and most preferably 5 μm to 7 μm.

[0055] In one aspect of the invention, the dry powder formulation may be for administration by inhalation.

[0056] The term "inhalation administration" refers to administration via the oral or nasal cavity due to the structure of the respiratory tract, for example, by spraying or injecting IFN-β or a carrier containing IFN-β in a liquid, aerosol, or gas phase through the oral or nasal cavity so that it contacts respiratory cells.

[0057] In the present invention, the term "respiratory system" refers to the entire organs through which breathing takes place, including the nose, mouth, airways, and lungs. Preferably, the respiratory system in the present invention includes the nasal mucosa, nasopharynx, oropharynx, laryngopharynx, larynx, trachea, bronchi, bronchioles, and lungs.

[0058] The present invention further provides an inhalation capsule filled with the dry powder formulation according to the present invention.

[0059] The inhalation capsule according to the present invention may be a capsule and cartridge made of gelatin or hypromellose or the like used in an inhalation device, or a blister made of a layered aluminum film or the like, filled with the dry powder formulation according to the present invention.

[0060] The capsules may have different internal volumes depending on the size, for example, a No. 0 capsule may have an internal volume of about 0.68 ml, a No. 1 capsule may have an internal volume of about 0.47 ml, a No. 2 capsule may have an internal volume of about 0.37 ml, a No. 3 capsule may have an internal volume of about 0.27 ml, and a No. 4 capsule may have an internal volume of about 0.20 ml. The capsule size can be appropriately selected by a person skilled in the art and manufactured as a capsule.

[0061] The capsule may be transparent in that it allows a patient to visually confirm whether the dry powder formulation has been inhaled into the capsule after inhalation. Furthermore, a transparent capsule allows visual confirmation of a decrease in stability or product defects, such as aggregation or discoloration of the dry powder formulation in the capsule.

[0062] The inhalation capsules can be administered using any known dry powder inhaler. The dry powder inhaler may include a means for rupturing, puncturing, or otherwise opening the capsule to deliver a metered amount of the active ingredient therein to the patient's lungs. The dry powder inhaler may also include an inlet port through which air enters to create an airflow, an outlet port through which the patient inhales and expels the active ingredient, and a sieve to filter out foreign matter. Examples of such devices include, but are not limited to, the commercially available ROTAHALER® from GlaxoSmithKline, HANDIHALER® from Behringer Ingelheim, or AEROLIZER® from Plastiape.

[0063] In the present invention, the capsule for inhalation may be for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, but is not limited thereto.

[0064] The present invention also provides a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infection, HIV infection, hepatitis C, and rheumatoid arthritis, comprising the dry powder formulation.

[0065] In one embodiment of the present invention, the viral infection may be a respiratory viral infection. The respiratory virus may be selected from the group consisting of adenovirus, avian influenza virus, bocavirus, coronavirus, cytomegalovirus, hantavirus, herpes simplex virus, influenza virus, measles virus, metapneumovirus, parainfluenza virus, respiratory syncytial virus, rhinovirus, and varicella-zoster virus. Preferably, in the present invention, the respiratory virus is a coronavirus.

[0066] In the present invention, the coronavirus is i) an alphacoronavirus, such as 229E or NL63 that infect humans, or a non-human-infective porcine epidemic diarrhea virus (PEDV), a non-human-infective transmissible gastroenteritis virus (TGEV), a canine coronavirus (CCoV), a feline coronavirus (FCoV), a Miniopterus bat coronavirus 1, a Miniopterus bat coronavirus HKU8, a Rhinolophus bat coronavirus HKU2, or a Scotophilus bat coronavirus 512; or ii) a human-infective OC43, a HKU1, a SARS-CoV, a MERS-CoV, a SARS-CoV-2, or a non-human-infective porcine hemagglutinating encephalomyelitis virus (PEDV). encephalomyelitis virus (PHEV), bovine coronavirus (BCoV), equine coronavirus (EqCoV), murine coronavirus (MuCoV), Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9 (betacoronaviruses); iii) gammacoronaviruses such as avian coronavirus (Avian coronavirus) and Beluga whale-coronavirus SW1 (which do not infect humans);iv) It may be one of the deltacoronaviruses, namely the Jeju brown-eared brown-eared brown bird (Bulbul)-coronavirus HKU11, the Thrush-coronavirus HKU12, and the Munia-coronavirus HKU13, which do not infect humans;

[0067] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

[0068] The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, a suitable oil, physiological saline, aqueous glucose, glycol, etc. Furthermore, stabilizers and preservatives may be further included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be found in the art.

[0069] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method, for example, orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration, and preferably intranasal administration.

[0070] The pharmaceutical composition of the present invention can be formulated into a preparation for oral or parenteral administration according to the above-mentioned administration route. When formulated, one or more buffering agents (e.g., physiological saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrating agents or surfactants, diluents or excipients can be used.

[0071] Examples of solid preparations for oral administration include tablets, pills, acids, granules, liquids, gels, syrups, slurries, suspensions, and capsules. These solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one or more excipients, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or gelatin. For example, the active ingredient can be blended with a solid excipient, crushed, and processed into a granular mixture after addition of suitable auxiliaries to give tablets or sugar-coated tablets.

[0072] In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, or preservatives in addition to water or liquid paraffin, which are commonly used simple diluents.

[0073] In some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrating agent, and the composition may further contain an anti-coagulating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

[0074] For parenteral administration, the pharmaceutical composition of the present invention can be formulated into the form of an injectable, transdermal, or nasal inhalant with a suitable parenteral carrier according to methods known in the art. Injectables must be sterilized and protected from microbial contamination, such as bacteria and fungi. Suitable carriers for injectables include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferred carriers include Hank's solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or sterile water for injection, and isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injectables from microbial contamination, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal, may be further included. Furthermore, in most cases, the injections may further contain isotonic agents, such as sugars or sodium chloride.

[0075] Transdermal administration includes ointments, creams, lotions, gels, topical liquids, pastes, liniments, air rolls, etc. As used above, "transdermal administration" means that a pharmaceutical composition is applied topically to the skin, and an effective amount of an active ingredient contained in the pharmaceutical composition is delivered to the skin.

[0076] For inhalants, the compositions of the present invention are conveniently delivered in aerosol spray form from pressurized packs or nebulizers using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in inhalers or insufflators can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch. Formulations for parenteral administration are described in guidelines generally known in all fields of pharmaceutical chemistry.

[0077] The pharmaceutical composition of the present invention can provide desirable disease prevention and treatment effects when it contains an effective amount of IFN-β. As used herein, "effective amount" refers to an amount that shows a response greater than or equal to that of a negative control, and preferably refers to an amount sufficient to alleviate the above-mentioned pathological symptoms. The pharmaceutical composition of the present invention can contain 0.01 to 99.99% IFN-β, with the remainder being a pharmaceutically acceptable carrier. The effective amount of IFN-β contained in the pharmaceutical composition of the present invention varies depending on the form in which the composition is commercialized, etc.

[0078] The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The content of the active ingredient in the pharmaceutical composition of the present invention can be varied depending on the severity of the disease. For parenteral administration, IFN-β is administered in an amount of 0.01 to 50 mg, preferably 0.1 to 30 mg, per kg of body weight per day. For oral administration, IFN-β can be administered in an amount of 0.01 to 100 mg, preferably 0.01 to 10 mg, per kg of body weight per day, in one or several divided doses. However, the effective dose of IFN-β for a patient is determined taking into account various factors, such as the administration route and number of treatments, as well as the patient's age, body weight, health condition, sex, severity of the disease, diet, and excretion rate. Taking these factors into consideration, a person skilled in the art would be able to determine an appropriate effective dose of IFN-β for a specific purpose for preventing and treating the aforementioned diseases. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method, as long as it exhibits the effects of the present invention.

[0079] The pharmaceutical composition of the present invention can also be provided as an external preparation containing IFN-β as an active ingredient.

[0080] When the pharmaceutical composition of the present invention is used as an external preparation for the skin, it may further contain any other ingredient commonly used in external preparations for the skin, such as fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents, or lipid vesicles, and other adjuvants commonly used in the field of dermatology. The ingredients may be incorporated in amounts commonly used in the field of dermatology.

[0081] When the pharmaceutical composition of the present invention is provided as an external preparation for skin, it may be in the form of a formulation such as, but not limited to, an ointment, patch, gel, cream, or spray.

[0082] The present invention also provides a method for producing a dry powder of interferon beta, comprising spray drying a liquid composition comprising: (a) Interferon beta or its variants; (b) acetate buffer at concentrations between 5 and 100 mM; (c) Arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at concentrations of 0.1–10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5–30% (w / v) hydrophobic amino acids.

[0083] In the above manufacturing method, the intake temperature is preferably 100 to 120°C, and the exhaust temperature may be 60 to 70°C.

[0084] The present invention also provides use of the dry powder formulation for producing a composition for treating a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infection, HIV infection, hepatitis C, and rheumatoid arthritis.

[0085] The present invention also provides a method for treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, comprising administering an effective amount of a composition containing the dry powder formulation as an active ingredient to an individual in need thereof.

[0086] The "effective amount" of the present invention means an amount that, when administered to an individual, shows an effect of improving, treating, detecting, or diagnosing a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, or suppressing or reducing the disease. The "individual" may be an animal, preferably a mammal, particularly an animal including a human, and may be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the above-mentioned effect.

[0087] The "treatment" of the present invention comprehensively refers to improving a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disease, viral infection, HIV infection, hepatitis C, and rheumatoid arthritis, or symptoms caused by the disease, and includes curing, substantially preventing, or improving the condition of the disease, including, but not limited to, alleviating, curing, or preventing one or most of the symptoms caused by the disease.

[0088] As used herein, the term "comprising" is used interchangeably with "including" or "characterized by" and does not exclude additional components or method steps, etc., not specifically recited in a composition or method according to the present invention. The term "consisting of" also means excluding additional elements, steps, or ingredients, etc., not otherwise recited. The term "essentially consisting of" means that a composition or method may include, in addition to the recited materials or steps, materials or steps that do not substantially affect its basic properties. [Effects of the Invention]

[0089] The IFN-β dry powder formulation according to the present invention is an IFN-β dry powder formulation with excellent morphological integrity and activity, and can effectively deliver IFN-β to the respiratory tract by inhalation administration, making it highly useful for the treatment of respiratory diseases, respiratory viral infections, etc., as well as for the effective delivery of IFN-β to the body. [Brief explanation of the drawings]

[0090] [Figure 1] FIG. 1 shows the results of scanning electron microscopy (SEM) of dry powder particles obtained after spray-drying liquid compositions containing IFN-β (Batch 1: leucine-free liquid composition, Batch 2: 10% (w / v) leucine-containing liquid composition, Batch 3: dry powder formulation of 20% (w / v) leucine-containing liquid composition). [Figure 2] 2 shows the results of ELISA analysis performed on rehydrated IFN-β dry powder formulations according to the present invention to evaluate their antiviral activity. A non-sprayed IFN-β liquid composition was used as a control. [Figure 3] 3 shows the results of a cytopathic effect (CPE) assay performed on a rehydrated IFN-β dry powder formulation according to the present invention to evaluate its antiviral activity. A non-sprayed IFN-β liquid composition was used as a control. DETAILED DESCRIPTION OF THE INVENTION

[0091] The present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. [Example]

[0092] Preparation and particle size analysis of interferon-beta powder (Dry-Powder Inhalation (DPI)) For the preparation of a DPI composition of interferon-beta, powder particle size analysis was performed upon conversion of the interferon-beta composition into a DPI.

[0093] To prepare dry powder particles, a liquid interferon beta composition (1 mg / mL of human interferon beta R27T mutant, 50 mM arginine, 0.5 mg / mL of poloxamer 188, 20% (w / v), and 10% (w / v) or 20% (w / v) of leucine in 20 mM acetate buffer at pH 3.8) was placed in a spray dryer according to the conditions shown in Table 1 to prepare dry powder. The final DPI interferon beta loading was 1 μg per mg of dry powder.

[0094] [Table 1]

[0095] Dry powder compositions were analyzed for particle size distribution using a Malvern Mastersizer MS3000 instrument, and all powder preparations were stored in a Safetech Powder Containment booth under humidity conditions of 30% or less.

[0096] As a result, as shown in Table 2, the recovery rate of the dry powdered interferon-beta composition increased when 20% (w / v) of leucine was added, and the D90 (90% of particle size) was confirmed to be 6 μm or less. As shown in Figure 1, the particle morphology of each batch was confirmed using a scanning electron microscope, and it was confirmed that uniform circular particles were produced.

[0097] [Table 2] [Example]

[0098] Moisture content analysis of interferon-beta powder (Dry-Powder Inhalation (DPI)) products

[0099] To measure the water content of the DPI preparation of interferon-beta prepared in Example 1 above, the water content in the preparation was measured by Karl Fischer titration method.

[0100] All experiments were performed in a facility with humidity below 30% and analyzed using a Metrohm 851 titrando Karl Fischer Coulometer. The analytical conditions are shown in Table 3.

[0101] [Table 3]

[0102] As a result, the moisture content of the powdered interferon-beta formulation was determined to be 3.4%, which is a suitable level for DPI inhalation formulations. Furthermore, measurements were performed at oven temperatures of 150 and 180°C, resulting in moisture values ​​of 3.46% and 3.41%, respectively, as shown in Table 4.

[0103] [Table 4] [Example]

[0104] Recovery Analysis of Interferon-Beta Powder (Dry-Powder Inhalation (DPI)) For the component analysis of the interferon-beta powder composition prepared in Example 1, the total protein amount was measured using a UV spectrophotometer, and the interferon-beta content was measured using SEC-HPLC.

[0105] Interferon-beta powder was rehydrated and analyzed using an Evolution 260 Bio-UV spectrophotometer, and titrated to A280nm minus A320nm wavelength values ​​after dissolving in sterile water for hydration to produce a concentration of 13.5-14.5ug / mL.

[0106] In addition, the amount of rehydrated interferon beta was measured using SEC-HPLC, and the rehydrated material, such as the sample used for UV analysis, was analyzed under the conditions shown in Table 5.

[0107] [Table 5]

[0108] To confirm the amount of interferon-beta actually contained in the interferon-beta powder, the recovery rate was calculated using the values ​​measured by UV and SEC-HPLC using the following formula: -UV protein concentration

[0109]

number

[0110]

number

[0111]

number

[0112] As a result, as shown in Table 6, it was confirmed that 100% of interferon-beta was recovered in the powder.

[0113] [Table 6] [Example]

[0114] Measurement of the activity of interferon-beta powder (Dry-Powder Inhalation (DPI)) To measure the antiviral activity of the dry powder formulation of interferon-beta prepared in Example 1, the powder preparation was rehydrated and subjected to ELISA and CPE analysis. A liquid composition of the same composition that was not spray-dried was used as a control.

[0115] 0.21 g of the interferon-beta dry powder formulation was titrated and converted into a liquid form by hydration with 2.1 mL of sterile water to a concentration of 0.1 mg / mL of interferon-beta. The resulting solution was used in an ELISA assay using a Toray ELISA kit, and the antiviral activity was analyzed by CPE assay.

[0116] For the ELISA assay, the liquid composition and rehydrated dry powder formulation were diluted approximately 260,000-fold with ELISA kit dilution buffer solution and reacted with HRP-conjugate in a 96-well plate coated with anti-human interferon-beta antibody at 25°C for 2 hours with shaking at 400 rpm. After washing three times with 0.5% Tween-20 PBS, TMB solution was added and the reaction was continued at 25°C for another 30 minutes. The reaction was then terminated with stop solution, and the absorbance was measured at 450 nm. The measured absorbance was converted to MIU / mL by substituting it into the standard solution curve.

[0117] The converted MIU / mL was divided by interferon-beta mg / mL to convert it into specific activity (MIU / mg), and the depreciation rate was measured in comparison with the liquid composition before powderization.

[0118] As a result, as shown in Figure 2, it was confirmed that the specific activity of the rehydrated dry powder formulation (Reconstituted ABN101) was reduced by 31.6% compared to the liquid composition before powderization (Control).

[0119] For the intracellular antiviral activity assay, A549 cells were grown in MEM media containing 2% heat-inactivated FBS, 100x sodium pyruvate, and 100x non-essential amino acid, and 2x10 cells were plated in a 96-well plate. 5 Cells were seeded at 1000 cells / well, and the liquid composition or the hydrated dry powder composition was diluted in medium at 50 IU / mL and cultured for 22 hours in a 37°C, 5% CO2 incubator. The liquid composition (Control) was diluted at 1 / 7,100,000, and the rehydrated dry powder (Reconstituted ABN101) was diluted at 1 / 900,000. After 22 hours, the drug was removed, and the cells were infected with EMCV stock at approximately 1,000 TCID50 / mL for 22 hours. After removing EMCV, the cells were treated with WST-8 solution for 2 hours, and cell viability (%) was measured to determine antiviral activity. The conversion method for intracellular activity measurement results is as follows: CPE Assay (IU / mL) = Standard activity in column 1 (IU / mL) x 2 (Nsam-Nstd) ×Dilution factor of sample in 1st column Ac: Mean absorbance of cell control Vc: Mean absorbance of virus control A 50 : (Ac+Vc) / 2 N std : n + (A n -A 50 ) / (A n -A n+1 ) N sam : n + (A n -A 50 ) / (A n -A n+1 ) N: Dilution factor index, A 50 This corresponds to the lowest OD value among the above highest OD values. [Industrial Applicability]

[0120] The IFN-β dry powder formulation of the present invention is an IFN-β dry powder formulation with excellent morphological integrity and activity, and can effectively deliver IFN-β to the respiratory tract by inhalation administration. It is therefore highly useful for the effective delivery of IFN-β to the body, including the treatment of respiratory diseases, respiratory viral infections, etc., and therefore has great industrial applicability.

Claims

1. An interferon-beta dry powder formulation prepared by spray drying a liquid composition comprising: (a) interferon beta or a variant thereof; (b) acetate buffer at a concentration of 5 to 100 mM; (c) arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5-30% (w / v) of a hydrophobic amino acid.

2. The dry powder formulation according to claim 1, wherein the interferon beta mutant is characterized in that the 27th amino acid of human interferon beta, arginine, is replaced with threonine.

3. The dry powder formulation of claim 1, wherein the interferon beta mutant contains an N-linked glycan at the asparagine residue, which is the 25th amino acid of human interferon beta.

4. 2. The dry powder formulation of claim 1, wherein the acetate buffer has a pH in the range of 3.6 to 4.

4.

5. The dry powder formulation according to claim 1, wherein the liquid composition contains interberon beta or a variant thereof at a concentration of 0.01 to 1% (w / v).

6. 2. The dry powder formulation of claim 1, wherein the hydrophobic amino acid is selected from the group consisting of tryptophan, tyrosine, leucine, and phenylalanine.

7. 2. The dry powder formulation of claim 1, wherein the hydrophobic amino acid is contained in the liquid composition at a concentration of 15 to 25% (w / v).

8. 2. The dry powder formulation of claim 1, wherein 90% (w / w) of the dry powder has a particle size between 0.1 μm and 10 μm.

9. 10. The dry powder formulation of claim 1, wherein the formulation is for inhalation administration.

10. An inhalation capsule filled with the dry powder formulation according to any one of claims 1 to 9.

11. A pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infection, hepatitis C, and rheumatoid arthritis, comprising the dry powder formulation of any one of claims 1 to 9.

12. A method for producing a dry powder of interferon beta, comprising spray drying a liquid composition comprising: (a) interferon beta or a variant thereof; (b) acetate buffer at a concentration of 5 to 100 mM; (c) arginine at a concentration of 10 to 150 mM; (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL; (e) methionine at a concentration of 0.5 to 5 mM; and (f) 5-30% (w / v) of a hydrophobic amino acid.

13. Use of the dry powder formulation according to any one of claims 1 to 9 for the manufacture of a composition for treating a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infection, HIV infection, hepatitis C, and rheumatoid arthritis.

14. A method for treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infections, hepatitis C, and rheumatoid arthritis, comprising administering an effective amount of a composition containing the dry powder formulation according to any one of claims 1 to 9 as an active ingredient to an individual in need thereof.

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