Dry powder preparation of interferon beta and preparation method thereof

Abstract

The present invention relates to a dry powder formulation of interferon beta and a method for preparing the same, and more particularly, to a dry powder formulation of interferon beta in the form of spherical particles having high morphological integrity prepared by spray drying a liquid composition containing interferon beta, and a method for preparing the dry powder formulation of interferon beta.

Description

Technical Field

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

[0002] The present disclosure relates to a dry powder formulation of interferon-β and a method for preparing the same, and more particularly, to a dry powder formulation of interferon-β comprising spherical particles having high morphological integrity prepared by spray drying a liquid composition comprising interferon-β and a method for preparing the same. Background Art

[0003] Interferon (IFN) is a cytokine that exhibits antiviral activity, inhibits cell proliferation, and regulates innate immune responses. IFNs are classified into IFN-α, IFN-β, and IFN-γ based on their cellular origin (leukocytes, fibroblasts, T cells). Interferon-β (IFN-β) is a globular protein with five α-helices (α-helices) and a size of 22 kDa, which decreases to 18 kDa when glycans are removed.

[0004] Research into the clinical application of IFN-β is actively underway, and it is receiving particular attention as a therapeutic agent for alleviating, relieving, or treating the symptoms of multiple sclerosis (MS). In addition, it has been reported that due to its various immunological activities, including antiviral activity, inhibition of cell growth, enhancement of lymphocyte cytotoxicity, immunomodulatory activity, induction or inhibition of target cell differentiation, macrophage activation, increased cytokine production, enhancement of cytotoxic T cell efficacy, and increased activity of natural killer cells, it is effective in treating various conditions such as cancer, autoimmune diseases, viral infections, HIV-related diseases, hepatitis C, and rheumatoid arthritis.

[0005] Human IFN-β is also a type of glycoprotein, and the glycan moieties attached to proteins play an important role in their activity. In the case of glycoproteins, the addition of glycans can sometimes increase their activity. Specifically, protein glycosylation is known to affect many biochemical properties, such as stability, solubility, intracellular trafficking activity, pharmacokinetics, and antigenicity.

[0006] IFN is typically formulated as an isotonic solution for parenteral administration. Recently, clinicians have sought alternative routes of IFN administration that are more suitable for long-term use in patients. Specifically, aerosol formulations of IFN for pulmonary delivery have been developed, as described in WO91 / 16038. These formulations are dispersed by evaporation of a liquid propellant. This patent describes improving the dispersibility of human IFN from a freon delivery system by adding a surfactant or its analogue.

[0007] WO 91 / 16038 describes a method and composition for preparing solid polypeptide microparticles as pharmaceutical aerosol formulations, wherein IFN-β is prepared in dry powder form by freeze-drying an IFN solution and then jet-milling the freeze-dried product.

[0008] However, such dry powder forms have the problem of not exhibiting a completely spherical particle shape, making them unsuitable for efficient pulmonary delivery. In addition, there is a limitation that the activity of IFN is significantly reduced compared to liquid preparations before the drying process.

[0009] Technical issues

[0010] Therefore, the present inventors have conducted repeated studies to develop dry powder formulations with high morphological integrity and interferon activity. Therefore, the present inventors have confirmed that spray drying a liquid interferon beta composition comprising acetate buffer, arginine, poloxamer 188, methionine and hydrophobic amino acids can produce an interferon beta dry powder formulation with excellent morphological integrity and activity, thereby completing the present disclosure.

[0011] Therefore, an object of the present disclosure is to provide a dry powder formulation of interferon-β prepared by spray drying a liquid composition, wherein the liquid composition comprises:

[0012] (a) interferon beta or a variant thereof;

[0013] (b) acetate buffer at a concentration of 5 to 100 mM;

[0014] (c) arginine at a concentration of 10-150 mM;

[0015] (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL;

[0016] (e) methionine at a concentration of 0.5-5 mM; and

[0017] (f) 5 to 30% (w / v) hydrophobic amino acids.

[0018] Another object of the present disclosure is to provide an inhalation capsule filled with a dry powder formulation.

[0019] Another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating a disease selected from multiple sclerosis, cancer, autoimmune disease, viral infectious disease, HIV infectious disease, hepatitis C and rheumatoid arthritis, comprising the dry powder preparation.

[0020] In addition, another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating a disease selected from multiple sclerosis, cancer, autoimmune disease, viral infectious disease, HIV infectious disease, hepatitis C and rheumatoid arthritis, which consists of the dry powder preparation.

[0021] In addition, another object of the present disclosure is to provide a pharmaceutical composition consisting essentially of a dry powder preparation for preventing or treating a disease selected from multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C and rheumatoid arthritis.

[0022] Another object of the present disclosure is to provide a method for preparing a dry powder formulation of interferon beta, comprising spray drying a liquid composition at an inlet temperature of 90 to 130° C. and an outlet temperature of 40 to 80° C., wherein the liquid composition comprises:

[0023] (a) interferon beta or a variant thereof;

[0024] (b) acetate buffer at a concentration of 5 to 100 mM;

[0025] (c) arginine at a concentration of 10 to 150 mM;

[0026] (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL;

[0027] (e) methionine at a concentration of 0.5-5 mM; and

[0028] (f) 5 to 30% (w / v) hydrophobic amino acids.

[0029] Technical Solution

[0030] In order to achieve the above-mentioned object of the present disclosure, the present disclosure provides a dry powder formulation of interferon-β prepared by spray-drying a liquid composition, wherein the liquid composition comprises:

[0031] (a) interferon beta or a variant thereof;

[0032] (b) acetate buffer at a concentration of 5 to 100 mM;

[0033] (c) arginine at a concentration of 10 to 150 mM;

[0034] (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL;

[0035] (e) methionine at a concentration of 0.5-5 mM; and

[0036] (f) 5 to 30% (w / v) hydrophobic amino acids.

[0037] To achieve another object of the present invention, the present invention provides an inhalation capsule filled with a dry powder formulation.

[0038] To achieve another object of the present disclosure, the present disclosure provides a pharmaceutical composition for preventing or treating a disease selected from multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C and rheumatoid arthritis, comprising a dry powder preparation.

[0039] In addition, the present disclosure provides a pharmaceutical composition for preventing or treating a disease selected from multiple sclerosis, cancer, autoimmune disease, viral infectious disease, HIV infectious disease, hepatitis C, and rheumatoid arthritis, which consists of a dry powder preparation.

[0040] Furthermore, the present disclosure provides a pharmaceutical composition for preventing or treating a disease selected from multiple sclerosis, cancer, autoimmune disease, viral infectious disease, HIV infectious disease, hepatitis C, and rheumatoid arthritis, which consists essentially of a dry powder preparation.

[0041] In order to achieve another object of the present disclosure, the present disclosure provides a method for preparing a dry powder formulation of interferon beta, the method comprising spray-drying a liquid composition at an inlet temperature of 90 to 130° C. and an outlet temperature of 40 to 80° C., wherein the liquid composition comprises:

[0042] (a) interferon beta or a variant thereof;

[0043] (b) acetate buffer at a concentration of 5 to 100 mM;

[0044] (c) arginine at a concentration of 10 to 150 mM;

[0045] (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL;

[0046] (e) methionine at a concentration of 0.5 to 5 mM; and

[0047] (f) 5 to 30% (w / v) hydrophobic amino acids.

[0048] Hereinafter, the present disclosure will be described in detail.

[0049] The present disclosure provides a dry powder formulation of interferon-β prepared by spray drying a liquid composition, wherein the liquid composition comprises:

[0050] (a) interferon beta or a variant thereof;

[0051] (b) acetate buffer at a concentration of 5 to 100 mM;

[0052] (c) arginine at a concentration of 10 to 150 mM;

[0053] (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL;

[0054] (e) methionine at a concentration of 0.5 to 5 mM; and

[0055] (f) 5 to 30% (w / v) hydrophobic amino acids.

[0056] One of the most fundamental issues in preparing protein-based formulations is achieving the desired concentration of the therapeutic protein in solution, as protein stability depends not only on protein concentration but also on the pH, temperature, ionic strength, and additive concentration of the solution. Therefore, protein-based formulations must be suitable for stabilizing the therapeutic protein and preventing problems such as aggregation, precipitation, or fragmentation. When preparing protein-based dry powder formulations, since the protein dispersed in the solution is powdered by methods such as freeze-drying or spray-drying, it is necessary to have a composition in which the protein remains stably dispersed in the solution.

[0057] Furthermore, when protein-based dry powder formulations are used for inhalation administration, it is desirable to prepare dry powder formulations with high morphological integrity, ie, particles with nearly spherical morphological characteristics so that they can be uniformly absorbed in the nasal mucosa or lungs.

[0058] Interferon refers to a low molecular weight protein and glycoprotein (sometimes referred to as cytokine) with a molecular weight of about 15,000 to 27,000 daltons, which is naturally or recombinantly produced and exhibits interferon activity. Typically, this activity is exerted by binding to specific membrane receptors on the cell surface. Once combined, interferon triggers a complex series of intracellular processes that vary according to the type of interferon. Interferon can be used to treat a variety of human diseases ranging from cancer to immune system suppression. Natural interferon is produced and secreted by cells in response to viral infection or synthetic and biological inducers. Some interferons are modified forms of natural substances and are prepared using recombinant DNA technology. Interferon is sometimes abbreviated as "IFN", and this abbreviation will also be used in this article. Examples of interferons include IFN-alpha-2A recombinant (RoferonR A-Roche Laboratories), IFN-alpha-2B recombinant (IntronR A-Shering), IFN-α-N3 derived from human leukocytes (AlferonR N-PurdueFrederick), IFN-gamma-1B (ActimmuneR-Genentech), IFN-β recombinant (BetaseronR-Chiron, Berlex) and natural IFN-β (FeronR-Toray, Japan). As used herein, the term "interferon-β (IFN-β)" refers to fibroblast interferon of human origin obtained by separation from biological fluids or by DNA recombinant technology from eukaryotic or prokaryotic host cells, as well as salts, functional derivatives, variants, analogs and active fractions thereof.

[0059] Preferably, the IFN-β of the present disclosure represents human IFN-β.

[0060] Preferred IFN-β may refer to IFN-β-1a and may comprise N-linked glycans at one or more asparagine residues of the amino acid residues of the protein.

[0061] In a preferred embodiment of the present disclosure, the IFN-β variant may be a variant in which the 27th amino acid arginine of human IFN-β is substituted with threonine (R27T). The R27T variant is a recombinant human IFN-β variant (hereinafter referred to as rh INF-β) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for additional glycosylation at position 25 of IFN-β1a, and glycans can be bound at amino acids 25 and 80.

[0062] In the present disclosure, the term "liquid composition" means that components (a) to (g) are contained in a solvent. The type of solvent is not particularly limited as long as it is pharmaceutically acceptable, and is preferably water.

[0063] In the present disclosure, the term "powder" refers to a formulation consisting of finely dispersed solid particles that are free-flowing, can be easily dispersed through an inhalation device, and are subsequently inhaled by the patient, where the particles reach and penetrate the nasal mucosa or the lungs. Therefore, powders are referred to as "respirable". The average size of the particles is preferably less than about 10 microns (μm) in diameter with a relatively uniform elliptical distribution. More preferably, the diameter is less than about 7.5 μm, and most preferably, less than about 5.0 μm. The diameter of the particle size distribution is typically from about 0.1 μm to about 5 μm, particularly from about 2 μm to about 5 μm.

[0064] In this disclosure, the term "dry" means that the composition has a moisture content that allows the particles to be easily dispersed in an inhalation device to form an aerosol. The moisture content is typically less than about 10% by weight water, typically less than about 5% by weight, and preferably less than about 4% by weight.

[0065] According to one embodiment of the present disclosure, a dry powder formulation of IFN-β prepared by spray drying a liquid composition comprising (a) interferon β 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 to 30% (w / v) hydrophobic amino acids exhibits a highly morphological spherical shape with integrity, high recovery, and stable activity.

[0066] That is, arginine, poloxamer 188, and methionine not only play a role in maintaining the protein stability of IFN-β at a high level in the liquid composition for spray drying, but also allow the protein to be uniformly dispersed without being destroyed during the spray drying process, thereby enabling the production of particles that exhibit a shape close to spherical in morphology.

[0067] In the present disclosure, the liquid composition for spray drying may comprise 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, arginine at a concentration of 50 to 100 mM, poloxamer 188 at a concentration of 0.1 to 1 mg / mL, and methionine at a concentration of 0.5 to 2 mM.

[0068] If the concentrations of arginine, poloxamer 188, and methionine deviate from the above ranges, the particle size and shape after spray drying may not be uniform, and the pharmacokinetics as an inhalation formulation may be undesirable.

[0069] In the present disclosure, the acetate buffer is a solution having the effect of adjusting or maintaining the pH of the preparation to fall within the desired pH range of the preparation, and preferably has a concentration of 5 to 100 mM, more preferably 10 to 30 mM.

[0070] In the present disclosure, the desired pH range of the acetate buffer may be in the range of 3.6 to 4.4, which is a pH that maintains the stability of interferon β in the liquid composition for spray drying, prevents abnormal aggregation reactions, and maintains protein stability during the spray drying process while allowing the spherical shape of the powder particles to form well.

[0071] According to one embodiment of the present disclosure, when the dry powder prepared by spray drying the IFN-β liquid composition comprising the components according to the present disclosure was rehydrated and the content of IFN-β in the dry powder was examined, it was confirmed that 100% of IFN-β was recovered before spray drying.

[0072] The therapeutically effective amount of IFN-β can vary within the formulation, depending on the biological activity of the IFN-β used and the amount required for the unit dosage form. Since IFN-β is highly active, it should be prepared in unit doses to be easy to handle 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 the total components 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).

[0073] In order to adjust the amount of IFN-β in the dry powder to the above-mentioned level, IFN-β in the liquid composition may be included at a concentration of 0.01 to 1% (w / v), but is not limited thereto.

[0074] According to the dry powder formulation of the present disclosure, it can be spray-dried and made into powder together with a pharmaceutically acceptable carrier. The amount of a pharmaceutically acceptable carrier is the amount required for providing stability, dispersibility, consistency and filling properties, and the stability, dispersibility, consistency and filling properties are to ensure that the composition is uniformly delivered to the lungs of patients in need. In numerical terms, depending on the activity of the IFN-β used, the amount can be about 95.0% (w / w) to about 99.95% (w / w). Preferably, about 98% (w / w) to about 99.8% (w / w) is used.

[0075] The carrier can be composed of a combination of one or more pharmaceutically acceptable excipients, but it generally does not contain a "penetration enhancer". A "penetration enhancer" is a surfactant compound that promotes drug penetration through the mucosa or mucosal wall and is proposed for use in nasal, rectal and vaginal drug formulations. Penetration enhancers include, for example, bile salts such as taurocholate, glycocholate and deoxycholate, fusidates such as taurodihydrofusidate, and biologically acceptable surfactants such as Tweens and laureth-9. However, the use of a penetration enhancer in preparations for the lungs is generally undesirable because the epithelial blood barrier of the lungs may be adversely affected by the surfactant compound. The dry powder composition of the present disclosure is easily absorbed into the lungs without the need for a penetration enhancer.

[0076] In the present disclosure, the types of pharmaceutically acceptable excipients that can be used as carriers include stabilizers such as human serum albumin (HSA), fillers such as carbohydrates, amino acids and polypeptides, pH adjusters or buffers, salts such as sodium chloride and other excipients. The carrier can be in crystalline or amorphous form, or a mixture thereof.

[0077] Particularly useful bulking agents may include acceptable carbohydrates, polypeptides, amino acids, or combinations thereof.

[0078] The hydrophobic amino acids in the compositions of the present disclosure may be included to improve the steric morphological stability during spray drying and to enhance the dispersibility of the powder. These hydrophobic amino acids may be selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine and tryptophan, preferably leucine.

[0079] In one aspect of the present disclosure, the hydrophobic amino acid may be included in the liquid composition at 15 to 25% (w / v), preferably 17 to 23% (w / v), and most preferably 19 to 21% (w / v).

[0080] In one aspect of the present disclosure, the hydrophobic amino acid may be included 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).

[0081] If the concentration of the hydrophobic amino acid in the liquid composition is less than 15% (w / v), the yield of dry powder formulation may decrease, and if it exceeds 25% (w / v), the morphological properties of dry powder particles may be undesirable.

[0082] In another aspect of the present disclosure, the hydrophobic amino acid may be leucine, and may be included in the liquid composition at 15 to 25% (w / v), preferably 17 to 23% (w / v), and most preferably 19 to 21% (w / v).

[0083] In another aspect of the present disclosure, leucine may be included 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).

[0084] If the concentration of leucine in the liquid composition is less than 15% (w / v), the yield of dry powder formulation may decrease, and if it exceeds 25% (w / v), the morphological characteristics of dry powder particles may be undesirable.

[0085] In one aspect of the disclosure, the formulation may 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, more preferably 3 μm to 8 μm, and most preferably 5 μm to 7 μm.

[0086] In one aspect of the present disclosure, the dry powder formulation may be used for administration by inhalation.

[0087] The term "inhalation administration" refers to administration through the oral or nasal cavity due to the structure of the respiratory system, for example, oral or nasal spraying or injection of a carrier containing IFN-β or IFN-β in liquid, aerosol or gaseous state to contact respiratory cells.

[0088] In the present disclosure, the term "respiratory system" refers to the organs involved in breathing as a whole, including every organ from the nose and mouth to the trachea and lungs. Preferably, in the present disclosure, the respiratory system includes the nasal mucosa, nasopharynx, oropharynx, hypopharynx, larynx, trachea, bronchi, bronchioles and lungs.

[0089] The present disclosure also provides an inhalation capsule filled with the dry powder formulation according to the present disclosure.

[0090] The inhalation capsule according to the present disclosure may be a capsule or cartridge (eg, gelatin or hypromellose) or blister (eg, laminated aluminum film) that is used in an inhalation device and is filled with the dry powder formulation according to the present disclosure.

[0091] Depending on the size, the capsule can have various internal capacities, for example, a size 0 capsule can have an internal capacity of about 0.68 mL, a size 1 capsule about 0.47 mL, a size 2 capsule about 0.37 mL, a size 3 capsule about 0.27 mL, and a size 4 capsule about 0.20 mL. The size of the capsule can be appropriately selected and prepared as a capsule by one skilled in the art.

[0092] The capsule can be transparent, as the patient can visually confirm whether the dry powder formulation within the capsule is inhaled after inhalation. In addition, the transparent capsule allows visual confirmation of the stability degradation or product defects, such as aggregation or discoloration, of the dry powder formulation contained inside.

[0093] The inhalation capsule can be administered using any known dry powder inhalation device. The dry powder inhalation device can include a device for rupturing the capsule, piercing the capsule, or otherwise opening the capsule to deliver the active ingredient in the metered capsule to the patient's lungs. In addition, it can also include an air inlet for forming an air flow, an outlet through which the active ingredient is discharged by the patient inhaling with their mouth, and a sieve for filtering foreign matter. Examples of such devices include the Twincer® marketed by GlaxoSmithKline Boehringer Ingelheim's or Plastiape's but are not limited thereto.

[0094] In the present invention, the inhalation capsule can be used for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disease, viral infectious disease, HIV infectious disease, hepatitis C, and rheumatoid arthritis, but is not limited thereto.

[0095] The present disclosure also provides a pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disease, viral infectious disease, HIV infectious disease, hepatitis C, and rheumatoid arthritis, comprising a dry powder formulation.

[0096] In one aspect of the present disclosure, the viral infectious disease can be a respiratory viral infectious disease. The respiratory virus can 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 disclosure, the respiratory virus is a coronavirus.

[0097] In the present disclosure, the coronavirus can be: i) an alpha-coronavirus selected from the group consisting of 229E, NL63, which infect humans, or porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), canine coronavirus (CCoV), feline coronavirus (FCoV), Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Rhinolophus bat coronavirus HKU2, and Scotophilus bat coronavirus 512, which do not infect humans; ii) a beta-coronavirus selected from the group consisting of OC43, HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, which infect humans, or porcine hemagglutinating encephalomyelitis virus (PHEV), bovine coronavirus (BCoV), equine coronavirus (EqCoV), murine coronavirus (MuCoV), Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, and Rousettus bat coronavirus HKU9, which do not infect humans; iii) a gamma-coronavirus selected from the group consisting of avian coronavirus and beluga whale coronavirus SW1, which do not infect humans; or iv) a delta-coronavirus selected from the group consisting of nightingale coronavirus HKU11, thrush coronavirus HKU12, and finch coronavirus HKU13, which do not infect humans.

[0098] The pharmaceutical composition of the present disclosure can further include a pharmaceutically acceptable carrier.

[0099] The pharmaceutically acceptable carrier can further include, for example, a carrier for oral administration or a carrier for parenteral administration. The carrier for oral administration can include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration can include water, a suitable oil, saline, aqueous dextrose, glycol, and the like. Furthermore, a stabilizer and a preservative can be additionally included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl paraben, or propyl paraben, and chlorobutanol. Other pharmaceutically acceptable carriers can be referred to as known in the art.

[0100] The pharmaceutical composition of the present disclosure can be administered to a mammal, including a human, by any method. For example, it can be administered orally or parenterally, and the parenteral administration method can include, but is not limited to, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration, and is preferably intranasal administration.

[0101] The pharmaceutical compositions of the present disclosure can be formulated for oral or parenteral administration according to the above-mentioned routes of administration. When formulated, it can be prepared using one or more buffers (e.g., 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, disintegrants, surfactants, diluents, or excipients.

[0102] Solid formulations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions, capsules, or other solid formulations. And such solid formulations can be prepared by mixing the pharmaceutical composition of the present disclosure with at least one excipient, 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 methyl cellulose, or gelatin. For example, tablets or sugar-coated tablets can be obtained by mixing the active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and processing it into a granular mixture.

[0103] In addition to simple excipients, lubricants such as magnesium stearate and talc are used. Liquid formulations for oral administration include suspensions, solutions, emulsions, or syrups, and in addition to simple diluents such as water or liquid paraffin, various excipients can be included, such as wetting agents, sweeteners, flavoring agents, or preservatives.

[0104] In addition, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate can be added as needed, and anti-caking agents, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives can also be included.

[0105] When parenteral administration, pharmaceutical compositions of the present disclosure can be prepared together with suitable parenteral carriers in the form of injection, transdermal preparation or nasal inhalation according to methods known in the art. In the case of injection, they must be sterile and free from the contamination of microorganisms such as bacteria and fungi. The example of a suitable carrier for injection includes but is not limited to a solvent or a dispersion medium, which comprises water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol), a mixture thereof and / or a vegetable oil. More preferably, a suitable carrier can include an isotonic solution, such as Hank's solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, 10% ethanol, 40% propylene glycol and 5% glucose. In order to protect injection from microbial contamination, various antimicrobial agents and antifungal agents can be included in addition, such as parahydroxybenzoate, chlorobutanol, phenol, sorbic acid and thimerosal. In addition, in most cases, injection can include an isotonic agent in addition, such as sugar or sodium chloride.

[0106] Transdermal preparations include ointments, creams, lotions, gels, external solutions, pastes, liniments and aerosols. Here, "transdermal administration" refers to topical application of a pharmaceutical composition to the skin, allowing an effective amount of the active ingredient contained in the pharmaceutical composition to be delivered to the skin.

[0107] For inhalation formulations, compositions according to the present disclosure can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant (such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in the generally known manuals in pharmaceutical chemistry.

[0108] When an effective amount of IFN-β is included, the pharmaceutical composition of the present disclosure can provide the desired effect for preventing and treating diseases. As used herein, the term "effective amount" refers to an amount that exhibits a response greater than that of a negative control group, and is preferably an amount sufficient to alleviate the aforementioned pathological symptoms. The pharmaceutical composition of the present disclosure may contain IFN-β in an amount of 0.01 to 99.99%, with the remainder being a pharmaceutically acceptable carrier. The effective amount of IFN-β included in the pharmaceutical composition of the present disclosure may vary depending on the form in which the composition is commercialized.

[0109] The total effective amount of the pharmaceutical composition of the present disclosure can be administered to a patient as a single dose or through a fractionated treatment regimen involving multiple doses over an extended period of time. The pharmaceutical composition of the present disclosure can vary the content of the active ingredient according to the severity of the disease. For parenteral administration, IFN-β can be preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per kilogram of body weight per day. For oral administration, IFN-β can be preferably administered in an amount of 0.01 to 100 mg, more preferably 0.01 to 10 mg per kilogram of body weight per day, divided into one or several doses. However, the dose of IFN-β is determined as an effective dose for a patient in consideration of various factors, such as administration route, treatment frequency, age, body weight, health condition, gender, disease severity, diet, and excretion rate of the patient. The appropriate effective dose of IFN-β for specific use in the prevention and treatment of the above-mentioned diseases can be determined by those skilled in the art in consideration of these factors. The pharmaceutical composition according to the present disclosure is not particularly limited in its formulation, administration route, or administration method, as long as it exhibits the effects of the present disclosure.

[0110] The pharmaceutical composition of the present disclosure can also be provided in the form of a topical preparation containing IFN-β as an active ingredient.

[0111] When the pharmaceutical composition of the present disclosure is used as a topical preparation for the skin, it can additionally include an auxiliary agent commonly used in the dermatological field, such as a lipid, an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic emulsifier, a non-ionic emulsifier, a filler, a metal ion chelating agent, a chelating agent, a preservative, a vitamin, a blocking agent, a moisturizer, an essential oil, a dye, a pigment, a hydrophilic activator, a lipophilic activator, a lipid vesicle, or any other component commonly used as a topical preparation for the skin. These components can also be introduced in an amount commonly used in the dermatological field.

[0112] When the pharmaceutical composition of the present disclosure is provided as a topical preparation for the skin, it can be in the form of, but is not limited to, an ointment, a patch, a gel, a cream, or a spray.

[0113] The present disclosure also provides a method for preparing a dry powder formulation of interferon beta, comprising spray drying a liquid composition at an inlet temperature of 90 to 130℃ and an outlet temperature of 40 to 80℃, wherein the liquid composition comprises:

[0114] (a) interferon beta or a variant thereof;

[0115] (b) an acetate buffer having a concentration of 5 to 100 mM;

[0116] (c) arginine having a concentration of 10-150 mM;

[0117] (d) poloxamer 188 at a concentration of 0.1 to 10 mg / mL;

[0118] (e) methionine at a concentration of 0.5-5 mM; and

[0119] (f) 5 to 30% (w / v) of a hydrophobic amino acid.

[0120] In the preparation method, the inlet temperature can be preferably 100 to 120°C, and the outlet temperature can be preferably 60 to 70°C.

[0121] The present disclosure also provides a use of the above-described dry powder formulation for the preparation of a composition for the treatment of a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infectious disease, an HIV infectious disease, hepatitis C, and rheumatoid arthritis.

[0122] The present disclosure also provides a method for treating a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infectious disease, an HIV infectious disease, hepatitis C, and rheumatoid arthritis, the method comprising administering to a subject in need thereof an effective amount of a composition comprising the above-described dry powder formulation as an active ingredient.

[0123] As used herein, the term "effective amount" means an amount that shows an effect of improving, treating, detecting, diagnosing, or inhibiting or alleviating a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infectious disease, an HIV infectious disease, hepatitis C, and rheumatoid arthritis when administered to a subject. The term "subject" can include an animal, preferably a mammal, particularly a human, and can also include a cell, tissue, or organ derived from an animal. The subject can be a patient in need of the above-described effect.

[0124] As used herein, the term "treatment" used in the present disclosure comprehensively means improving a disease selected from the group consisting of multiple sclerosis, cancer, an autoimmune disease, a viral infectious disease, an HIV infectious disease, hepatitis C, and rheumatoid arthritis, or improving symptoms caused by the disease. This can include curing, substantially preventing or improving the condition, and alleviating, curing or preventing one or most of the symptoms caused by the disease, but is not limited thereto.

[0125] As used herein, the term "comprising" as used herein is used in the same sense as "including" or "characterized by," and does not exclude additional components or steps of methods not specifically mentioned in the compositions or methods according to the present disclosure. Furthermore, the term "consisting of means that additional elements, steps, or components not specifically described are excluded. The term "consisting essentially of means that within the scope of the composition or method, it can include substances or steps that do not substantially affect the essential characteristics of the substance or step.

[0126] Beneficial effects

[0127] The dry powder formulation of IFN-β according to the present disclosure is a dry powder formulation of IFN-β having excellent morphological integrity and activity, which can effectively deliver IFN-β to the respiratory tract by inhalation administration. Thus, it can be very usefully used for effective in vivo delivery of IFN-β, including the treatment of respiratory diseases and respiratory viral infections. BRIEF DESCRIPTION OF DRAWINGS

[0128] Figure 1 is a graph of dry powder particles under a scanning electron microscope (SEM) after spray drying a liquid composition comprising IFN-β (Batch 1: dry powder formulation of a liquid composition not containing leucine, Batch 2: dry powder formulation of a liquid composition containing 10% (w / v) leucine, Batch 3: dry powder formulation of a liquid composition containing 20% (w / v) leucine).

[0129] Figure 2 is a result of an ELISA analysis to evaluate the antiviral activity of the dry powder formulation of IFN-β according to the present disclosure by rehydrating the dry powder formulation. A liquid composition of IFN-β not spray-dried was used as a control group.

[0130] Figure 3 is a result of a CPE (cytopathic effect) analysis to evaluate the antiviral activity of the dry powder formulation of IFN-β according to the present disclosure by rehydrating the dry powder formulation. A liquid composition of IFN-β not spray-dried was used as a control group. DETAILED DESCRIPTION

[0132] Hereinafter, the present disclosure will be described in detail with reference to the following examples. However, the following examples are merely illustrative of the present disclosure and the present disclosure is not limited thereto.

[0133] Example 1: Manufacture and particle size analysis of interferon-β dry powder (dry powder inhalation (DPI))

[0134] In order to manufacture DPI (dry powder formulation of interferon-β), particle size analysis was performed during DPI conversion of a liquid composition of interferon-β.

[0135] In order to prepare dry powder particles, a liquid composition of interferon-β (1 mg / mL human interferon-β R27T variant, 50 mM arginine, 0.5 mg / mL poloxamer 188, 1 mM methionine, and 10% (w / v) or 20% (w / v) leucine in 20 mM acetate buffer, pH 3.8) of interferon-β was spray-dried under the conditions shown in Table 1 to produce a dry powder. The purpose was to load 1 μg of final DPI (dry powder formulation of interferon-β) into 1 mg of dry powder.

[0136] [Table 1]

[0137]

[0138]

[0139] The particle size distribution of the dry powder compositions was analyzed using a Malvern Mastersizer MS3000 apparatus and all powder products were stored in Safetech Powder Containment compartments at a humidity of 30% or less.

[0140] Therefore, as shown in Table 2, in the composition containing 20% ​​(w / v) leucine, the recovery rate of the interferon β dry powder preparation was improved, and D90 (90% particle size) was confirmed to be 6 μm or less. Figure 1 As shown, the particle morphology of each batch was confirmed using scanning electron microscopy, and it was observed that spherical particles were uniformly formed.

[0141] [Table 2]

[0142]

[0143] Example 2: Moisture content analysis of dry powder formulations of interferon-β (dry powder inhalation (DPI))

[0144] In order to measure the moisture content of the DPI (dry powder formulation of interferon-β) manufactured in Example 1, the moisture content of the powder was measured using the Karl Fischer titration method.

[0145] All experimental conditions were carried out in a facility with humidity below 30%, and the analysis was performed using a Metrohm 851 Titrando Karl Fischer coulometer. The analysis conditions are shown in Table 3.

[0146] [Table 3]

[0147] parameter set up Nitrogen flow rate 50mL / min (±10mL / min) stirring speed 8 Background drift <10 μg / min Oven temperature 150 or 180°C Sample weight 50mg (±5mg) Extraction time 120 seconds

[0148] As a result, it was found that the moisture content of the dry powder formulation of interferon-β was 3.4%, which was measured to be at an appropriate level for DPI formulations. In addition, when the oven temperature was set at 150°C or 180°C for measurement, the moisture content of the analyzed powder was 3.46% and 3.41%, respectively, as shown in Table 4.

[0149] [Table 4]

[0150]

[0151] Example 3: Recovery Analysis of Interferon-β Dry Powder Formulation (Dry Powder Inhalation (DPI))

[0152] In order to analyze the components of the dry powder formulation of interferon-β produced in Example 1, the total protein amount was measured using a UV spectrophotometer, and the interferon-β content was measured using SEC-HPLC.

[0153] Interferon-β dry powder formulations were rehydrated and analyzed using an Evolution 260 Bio-UV spectrophotometer. For rehydration, the powder was dissolved in sterile water to prepare a concentration of 13.5-14.5 μg / mL, and the value obtained by subtracting the A320 nm wavelength value from the A280 nm wavelength value was measured.

[0154] In addition, to measure the amount of interferon-β contained in the rehydrated powder, analysis was performed using SEC-HPLC under the conditions shown in Table 5, using the same rehydrated sample as used for the UV analysis.

[0155] [Table 5]

[0156] Mobile phase 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 Sample tray temperature 4±2℃ Injection volume 50 μL Pillar TSKgel G2000SWXL 7.8x3000mm, 5μm Column temperature 25±5℃ Detector wavelength 280nm,214nm Runtime 40min flow rate 0.5mL / min

[0157] In order to confirm the actual amount of interferon-β contained in the powder, the recovery rate was calculated according to the following formula using the values ​​measured by UV and SEC-HPLC.

[0158] -UV protein concentration

[0159]

[0160] -% w / w concentration

[0161]

[0162] -% Theoretical content

[0163]

[0164] As a result, as shown in Table 6, it was confirmed that 100% of interferon-β was recovered in the powder.

[0165] [Table 6]

[0166]

[0167] Example 4: Activity Measurement of Dry Powder Formulations of Interferon-β (Dry Powder Inhalation (DPI))

[0168] To measure the antiviral activity of the interferon-β dry powder formulation prepared in Example 1, the powder was rehydrated and subjected to ELISA and CPE analysis. As a control group, a liquid composition of the same formulation that had not been spray-dried was used.

[0169] To rehydrate the dry powder formulation of interferon-β, 0.21 g of dry powder was dissolved in 2.1 mL of sterile water to achieve an interferon-β content of 0.1 mg / mL, thereby converting the powder into a liquid state. This solution was used for ELISA analysis using a Toray ELISA kit and for analysis of antiviral activity using the CPE assay.

[0170] For ELISA analysis, the liquid composition and the rehydrated dry powder preparation were diluted approximately 260,000 times with an ELISA kit dilution buffer solution. The diluted samples were aliquoted into a 96-well plate coated with an anti-human interferon-β antibody. Subsequently, HRP-conjugate was added, and the mixture was reacted at 25°C for 2 hours while shaking at 400 rpm. After washing three times with 0.5% Tween-20 PBS, TMB solution was added and reacted again at 25°C for 30 minutes. The reaction was terminated with a stop solution, and the absorbance was measured at 450 nm. The measured absorbance was substituted into the standard solution curve and converted to MIU / mL.

[0171] The converted MIU / mL was divided by interferon-β mg / mL to calculate the specific activity (MIU / mg), and the reduction rate was measured by comparing it with the liquid composition before powderization.

[0172] Results, such as Figure 2 As shown, the specific activity of the rehydrated dry powder formulation (reconstituted ABN101) was confirmed to have decreased by 31.6% compared to the liquid composition before pulverization (control).

[0173] For intracellular antiviral activity measurement, A549 cells were cultured in MEM medium containing 2% heat-inactivated FBS, 100x sodium pyruvate, and 100x non-essential amino acids. 5 Cells / well were seeded in 96-well plates, and the liquid composition or rehydrated powder composition was diluted to 50 IU / mL in culture medium and incubated at 37°C in a 5% CO2 incubator for 22 hours. The liquid composition before powdering (control) was diluted 7,100,000 times from 1, and the rehydrated dry powder (reconstructed ABN101) was diluted 900,000 times from 1 for treatment. After 22 hours, the drug was removed and the EMCV stock solution was infected at approximately 1,000 TCID50 / mL for 22 hours. Subsequently, EMCV was removed and the WST-8 solution was treated for 2 hours to measure cell viability (%) and determine the antiviral activity. The calculation method for measuring intracellular antiviral activity is as follows.

[0174] CPE determination (IU / mL)

[0175] = Standard activity of sample in row 1 (IU / mL) × 2 (Nsam-Nstd)X dilution fold of the 1st row sample

[0176] Ac: average absorbance of cell control

[0177] Vc: average absorbance of virus control

[0178] A 50 : (Ac+Vc) / 2

[0179] N std : n+(A n -A 50 ) / (A n -A n+1 )

[0180] N sam : n+(A n -A 50 ) / (A n -A n+1 )

[0181] N: the index of dilution time corresponds to the lowest O.D. value among high O.D. values greater than A 50

[0182] Industrial applicability

[0183] The dry powder formulation of IFN-β according to the present disclosure is a dry powder formulation of IFN-β having excellent morphological integrity and activity, and can effectively deliver IFN-β to the respiratory tract by inhalation. Therefore, the formulation can be very usefully applied to effective in vivo delivery of IFN-β for uses including the treatment of respiratory diseases and respiratory viral infections, and thus has significant industrial applicability.​

Claims

1. An interferon beta dry powder preparation prepared by spray drying a liquid composition, wherein the liquid composition comprises: (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 0.5-5 mM; and (f) 5 to 30% (w / v) hydrophobic amino acids.

2. The dry powder formulation according to claim 1, wherein the interferon beta variant is a variant of human interferon beta, wherein the 27th amino acid arginine is substituted by threonine.

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

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

4. 5 . The dry powder formulation according to claim 1 , wherein the interferon beta or variant thereof is contained in the liquid composition at a concentration of 0.01 to 1% (w / v). The dry powder formulation according to claim 1 , wherein the hydrophobic amino acid is selected from tryptophan, tyrosine, leucine and phenylalanine.

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

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

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

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

11. A pharmaceutical composition comprising the dry powder formulation according to any one of claims 1 to 9, for preventing or treating a disease selected from multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C and rheumatoid arthritis.

12. A method for preparing a dry powder formulation of interferon beta, comprising spray drying a liquid composition at an inlet temperature of 90 to 130° C. and an outlet temperature of 40 to 80° C., wherein the liquid composition comprises: (a) interferon beta or a variant thereof; (b) acetate buffer at a concentration of 5 to 100 mM; (c) arginine at a concentration of 10-150 mM; (d) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL; (e) methionine at a concentration of 0.5-5 mM; and (f) 5 to 30% (w / v) hydrophobic amino acids.

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

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

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