JP2008050320A - INTERFERON-beta-CONTAINING MEDICINAL COMPOSITION - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an IFN-β medicinal composition suitable for being used in uses such as treatment, other researches or the like by maintaining its biological activity after preserving for a long period at a low concentration of the IFN-β, without using human serum albumin. <P>SOLUTION: This liquid medicinal composition is provided by containing the IFN-β with citric acid, a phosphoric acid salt and a polyoxyethylenepolyoxypropylene glycol as additives in a pharmacologically acceptable medium, and without containing human serum albumin. Also, the freeze-dried medicinal composition is obtained by freeze-drying the liquid composition having the equal ratios of each of the additives to the IFN-β. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition of interferon-β that is stable over a long period of time and does not contain human serum albumin as a stabilizer. This composition is characterized in that even if it does not contain human serum albumin, a low concentration of interferon-β maintains biological activity over a long period of time and is stable.

  Interferon (IFN) is a protein with a wide range of biological activities such as antiviral activity, antitumor activity, and immunoregulatory activity. It can be used for the treatment of various malignant neoplasms and viral infections. It is used to treat diseases (hepatitis B, hepatitis C), multiple sclerosis, etc. IFN is produced by mammals in response to various inducers such as viruses, polypeptides, and mitogens. In most cases, IFN shows a better response to tissues and cells of the animal species from which IFN was produced than to tissues and cells of other animal species (species specificity). Therefore, human IFN is used for the treatment of human diseases.

  There are at least three different types of human IFN, which are produced from leukocytes such as monocytes and macrophages, fibroblasts, or lymphocytes, and are called IFN-α, IFN-β, and IFN-γ. ing.

  Natural human IFN-β is produced by super-induction with poly-IC in human fibroblasts, and is industrially produced by isolation and purification from the culture supernatant using chromatographic techniques. be able to. Proteins or polypeptides having the same properties as natural IFN-β should be produced industrially by expressing them in E. coli, animal cells, insect cells, or yeast using recombinant DNA technology. Can do.

The concentration unit of IFN-β is usually represented by the biological activity of IFN-β. This is measured using the antiviral activity as an index, expressed in international units (IU) calculated based on IFN-β standard, and sometimes expressed as 1 × 10 ⁶ IU = 1MIU .

  The most commonly used method of administering IFN-β is intravenous, intramuscular, or subcutaneous injection. As a therapeutically effective dose of natural IFN-β, 1 to 6 MIU per day is most commonly used for human administration.

  In order to use IFN-β for treatment, it is essential to develop a preparation that maintains storage stability over a long period of time. However, IFN-β is very unstable, and IFN-β purified so that it can be used for treatment in particular is susceptible to various inactivation reactions during the storage period. For example, the physical destabilizing element includes formation of insoluble aggregates, and the chemical destabilizing element includes hydrolysis, imide formation, amide decomposition, and the like. These changes in IFN-β during storage period result in loss or reduction of its biological activity, resulting in a loss of therapeutic effect. In addition, aggregated or degraded IFN-β may be immunogenic and may cause side effects.

  Stabilization of proteins in pharmaceutical compositions is still an area that requires trial and error. Human serum albumin (HSA), buffers, sugars, surfactants, amino acids, polyethylene glycols, polymers, etc. are commonly used as additives to increase protein stability. Depending on the type, methods that can stabilize other types of proteins may not always stabilize IFN-β.

Natural IFN-β generally has higher specific activity (biological activity per weight: IU / g) than IFN-β produced using recombinant DNA technology. For example, it contains existing natural IFN-β. In the preparation (Feron Toray Co., Ltd.), the specific activity of IFN-β is 3 × 10 ⁸ IU / mg, and as IFN-β, an effective therapeutic effect can be obtained with a very small dose of 3-20 μg. . Since it becomes difficult to maintain stability at a lower concentration of protein, it is very difficult to develop a stable IFN-β preparation for use in therapy.

  Conventionally, various pharmaceutical compositions that keep IFN-β stable have been developed, and human serum albumin has been used as a stabilizer. However, since human serum albumin is a component derived from human blood, there is a risk of contamination with viruses such as human immunodeficiency virus (HIV) and human hepatitis C virus (HCV). Exists. Therefore, development of a safe and stable IFN-β preparation that does not contain biological components such as human serum albumin is desired.

  In recent years, various compositions have been developed as pharmaceutical compositions containing no human serum albumin, and the following reports have been made.

  For example, there is disclosed a stable composition of IFN-β that does not contain human serum albumin and contains sugar and / or sugar alcohol, amino acid, and if necessary, acid and base, and a surfactant (Patent Document 1). However, in the Examples of the present application, the liquid pharmaceutical composition containing sugar and / or sugar alcohol, amino acid and buffer solution includes those whose biological activity decreases when stored at 4 ° C for 1 week. In addition, polysorbate 80 described as being suitable for IFN-β as a surfactant to be additionally added in Patent Document 1 reduces the activity of IFN-β in Examples of the present application. It is shown that.

  For example, an IFN-β-containing liquid composition or lyophilized composition that increases the solubility of IFN-β by maintaining the ionic strength at 60 mM or less and the pH at 3.0 to 5.0 and is stable without human serum albumin. It is disclosed (Patent Document 2). In Patent Document 2 above, storage stability under high protein concentration conditions is determined based on sedimentation coefficient (aggregate formation), UV absorption or IFN-β content by reverse phase HPLC (IFN-β residual rate). It is not shown whether it retains biological activity. In the Examples of the present application, the concentration of natural type IFN-β used for normal treatment (generally about 20 μg / mL or less) decreases the biological activity of the composition described in Patent Document 2 when stored at 4 ° C. for 1 week. It has been shown that the resulting composition is included. Further, it is considered that the composition described in Patent Document 2 has a low pH and may cause problems in living body administration. In the present invention, it is possible to provide a pharmaceutical composition that is stable at a pH closer to a living body.

  For example, a composition of recombinant IFN-β dissolved in an insoluble carrier medium containing a nonionic polymer surfactant as a stabilizer is disclosed (Patent Document 3). This composition is lyophilized and stored and then reconstituted by dissolving in water at the time of use. In addition, this composition was stored in a liquid state, and the change in the amount of protein before and after centrifugation was evaluated by UV absorption (aggregate formation), and further biological activity was evaluated. After storage at 4 ° C for 1 week, Although there is no change in UV absorption, the biological activity is halved and it is not stable enough for therapeutic use. Therefore, it is considered inappropriate to evaluate whether it has biological activity in measurements such as UV absorption. In addition, human serum albumin is mentioned as an additional additive to the composition.

  For example, a liquid composition of IFN-β containing 0.3 to 5% by weight of an acidic amino acid, arginine or glycine as a stabilizer is described (Patent Document 4). The concentration of IFN-β used is 12 MIU / mL (60 μg / mL), which may be too high for use in therapy. For example, when 1MIU, which is often used for treatment, is administered, the dose is about 83 μL, and accurate administration is difficult. Further, in the Examples of the present application, when the IFN-β concentration was 1 MIU / mL with the additive composition described as being most stable in 4 in the above-mentioned patent document, a significant decrease in activity was observed after storage at 37 ° C. for 1 week, Since it was shown that a composition in which biological activity decreases when stored at 37 ° C. for 8 weeks even at an IFN-β concentration of 6 MIU / mL, at a low concentration suitable for treatment, Stabilization by technology is considered insufficient.

  For example, a stable IFN-β liquid medicine whose biological activity of IFN-β is 80% or more after storage for 3 months at 25 ° C. in a solution containing a pH 5-8 buffer without human serum albumin A composition is described (Patent Document 5). The composition of Patent Document 5 is characterized by not containing a surfactant, but in the Examples of the present application, it was shown that the biological activity decreased in the preparation operation when no surfactant was added. It has been shown that the addition of can provide a liquid pharmaceutical composition that is more stable in the preparation operation. Moreover, the concentration of IFN-β substantially used in Patent Document 5 is 6 MIU / mL or more, and the concentration may be too high when used for treatment.

  Thus, a composition that does not contain human serum albumin and maintains the biological activity of IFN-β for a long period of time at a low concentration suitable for treatment has not been developed yet.

International Publication WO2004 / 028557 Special Table 2004-529917 Japanese Unexamined Patent Publication No. 63-179833 Special table 2001-519770 Special table 2001-517635

  The present invention has been made to solve such problems, and maintains the biological activity after long-term storage for low concentrations of IFN-β without using human serum albumin, for treatment or other research. It is an object of the present invention to provide an IFN-β pharmaceutical composition suitable for use in such applications.

  By combining citric acid, phosphate, and polyoxyethylene polyoxypropylene glycol, the present inventors can maintain IFN-β biological activity for a long period of time without human serum albumin. The lyophilized pharmaceutical composition and liquid pharmaceutical composition were found, and the invention shown below was completed.

  That is, the present invention provides a liquid pharmaceutical composition containing IFN-β, citric acid, phosphate and polyoxyethylene polyoxypropylene glycol as additives in a pharmacologically acceptable medium and free of human serum albumin. Offer things. The present invention also provides a lyophilized pharmaceutical composition obtained by lyophilizing a liquid composition in which the ratio of each additive to IFN-β is equal to the liquid pharmaceutical composition of the present invention. Furthermore, the present invention provides an IFN-β administration kit comprising the lyophilized pharmaceutical composition of the present invention and a lysis solution.

  INDUSTRIAL APPLICABILITY According to the present invention, there is provided a pharmaceutical composition that maintains the biological activity of IFN-β at a low concentration suitable for use in treatment for a long period without using human serum albumin as a stabilizer.

  The pharmaceutical composition of the present invention includes a liquid pharmaceutical composition and a lyophilized pharmaceutical composition. The “liquid pharmaceutical composition” refers to a pharmaceutical composition in a solution state that can be directly applied to a living body or can be applied to other uses such as research. A “lyophilized pharmaceutical composition” is obtained by lyophilizing a liquid composition. `` Freeze drying '' means freezing a liquid composition, sublimating ice from the frozen liquid composition to remove moisture and drying, and freeze dryers that perform freeze drying are commercially available, It can be easily operated by those skilled in the art. The “dissolved solution” used for dissolving the lyophilized pharmaceutical composition is a liquid used to make the lyophilized pharmaceutical composition into a form that can be applied to a living body or used for other purposes such as research. It is added to the dry pharmaceutical composition and dissolves it into a solution (reconstituted). The IFN-β pharmaceutical composition of the present invention maintains the biological activity of IFN-β when stored for a long period of time in the form of either a liquid pharmaceutical composition or a lyophilized pharmaceutical composition. Accordingly, the liquid pharmaceutical composition can be used as it is, and in the lyophilized pharmaceutical composition, it can be used after being dissolved in an appropriate solution at the time of administration.

The present invention relates to natural IFN-β (see Japanese Patent Publication No. 1-12760), IFN-β produced using recombinant DNA technology (see US Pat. No. 4966843, US Pat. No. 5,376,567), and chemical synthesis or modification. (See European Patent Application No. 185459B1, U.S. Pat. No. 4518584, U.S. Pat. No. 4,858,585, U.S. Pat. No. 4,743,462), but particularly preferred for natural IFN-β. . The IFN-β of the present invention can be derived from any animal species including, but not limited to, those derived from birds, dogs, cats, cows, pigs, horses and humans. Human IFN-β is preferable. The concentration unit of IFN-β is usually represented by the biological activity of IFN-β. This is measured using antiviral activity as an index, expressed in International units (IU) calculated from IFN-β standards, and expressed as 1 × 10 ⁶ IU = 1MIU in the product There is something. The IFN-β pharmaceutical composition of the present invention can be applied to a low concentration of IFN-β suitable for use in therapy, preferably 0.5 MIU / mL or more, more preferably 0.5 to 12 MIU / mL, more preferably 0.5 to 6 MIU / mL, most preferably 1-6 MIU / mL. Specific activity of IFN-β (biological activity per weight: IU / g) varies depending on its production method, isolation / purification method, type of IFN-β (natural type, recombinant type, etc.) In the present invention, natural type IFN-β is used, and its specific activity corresponds to about 3 × 10 ⁸ IU / mg.

  The additive in the pharmaceutical composition of the present invention refers to components contained in the composition in addition to IFN-β. For example, buffer (pH adjuster), adsorption inhibitor, stabilizer, surfactant, solubilizer (solubilizer), excipient, isotonic agent, soothing agent, preservative, antioxidant, etc. Is mentioned.

  The pH of the pharmaceutical composition of the present invention is preferably from 3.0 to 6.5, more preferably from 4.5 to 6.5, more preferably from 5.0 to 6.0, and most preferably 5.5, which is a physiologically relevant neutral to weakly acidic range. . The pH of the pharmaceutical composition is more preferably pH 4.5 or more, which is said to be a pH that does not cause pain stimulation upon intramuscular injection or subcutaneous injection.

  The pharmaceutical composition of the present invention comprises citric acid and phosphate as a buffering agent that maintains the pH without compromising the stability of IFN-β. The term “citric acid” is used to include not only citric acid but also hydrates and anhydrides of citric acid, and specific examples include citric acid monohydrate and anhydrous citric acid, Citric acid monohydrate is preferred. The term “phosphate” is used to include not only orthophosphate but also monohydrogen phosphate and dihydrogen phosphate. Examples of the phosphate include sodium salt, potassium salt and calcium salt of phosphoric acid, with sodium salt being preferred. Examples of the sodium salt of phosphoric acid include disodium hydrogen phosphate and sodium dihydrogen phosphate, with disodium hydrogen phosphate being most preferred.

  The citric acid and phosphate used in the pharmaceutical composition of the present invention are used after being dissolved at a ratio capable of maintaining a desired pH, and the osmotic pressure ratio and solubility of the pharmaceutical composition as a whole are not particularly problematic, Although it will not specifically limit if it is a density | concentration which can maintain pH, Preferably it is 5-20 mM citric acid and 5-30 mM phosphate.

  As polyoxyethylene polyoxypropylene glycol contained as an additive in the pharmaceutical composition of the present invention, the average degree of polymerization of polyoxyethylene part (the number of repeating polyoxyethylene units) in polyoxyethylene polyoxypropylene glycol is Those having an average polymerization degree of 3 to 200 and a polyoxypropylene part of 5 to 70 are preferred. Specifically, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (120) polyoxypropylene (40) glycol (also known as Pluronic F87), polyoxyethylene (160) polyoxypropylene (30 ) Glycol (also known as Pluronic F68), Polyoxyethylene (42) Polyoxypropylene (67) Glycol (also known as Pluronic P123), Polyoxyethylene (54) Polyoxypropylene (39) Glycol (also known as Pluronic P85), Poly Oxyethylene (196) polyoxypropylene (67) glycol (also known as Pluronic F127), polyoxyethylene (20) polyoxypropylene (20) glycol (also known as Pluronic L44), polyoxyethylene (200) polyoxypropylene (70 ) Glycol, polyoxyethylene (3 And polyoxypropylene (17) glycol. Most preferred is polyoxyethylene (160) polyoxypropylene (30) glycol. Polyoxyethylene (160) polyoxypropylene (30) glycol is also referred to as Pluronic F68 or Poloxamer 188.

  The dose of polyoxyethylene polyoxypropylene glycol in the pharmaceutical composition of the present invention may be an amount acceptable for administration to a living body. For example, the maximum amount of polyoxyethylene (160) polyoxypropylene (30) glycol that has been approved by the Ministry of Health, Labor and Welfare and can be used for human administration is 333 mg for oral administration, 10 mg for intravenous injection, and muscle 10 mg for internal injection, 200 mg / g for general external preparations, and 300 mg for rectal vaginal urethral application (Pharmaceutical Additives Encyclopedia 2000, edited by Japan Pharmaceutical Additives Association, Yakuji Nippo) confirmed safety in the living body The dosage of polyoxyethylene polyoxypropylene glycol in the pharmaceutical composition of the present invention is not limited to this as long as it is an amount that can be used for a short term or a long term without causing harm to the living body. The concentration of the polyoxyethylene polyoxypropylene glycol of the present invention in the liquid pharmaceutical composition is preferably 0.01 to 2 mg / mL, more preferably 0.1 to 0.5 mg / mL.

  The pharmaceutical composition of the present invention may contain one or more amino acids or salts thereof. The amino acid can be used as a purpose of improving stability, a solubilizing agent, an excipient or an isotonic agent. As long as the form of the amino acid is confirmed to be safe for the living body and can be used in the short term or long term without causing harm to the living body, it can be used as a salt form in addition to the free form. Examples of the salt form of amino acid include hydrochloride. The amount of amino acid added may be an amount that is acceptable for administration to a living body and may be an amount that can be dissolved. In the present invention, the amino acids are preferably glycine, histidine and arginine, preferably including at least one amino acid, and most preferably including arginine.

  The amino acid concentration in the liquid pharmaceutical composition of the present invention (when multiple types are included, the sum thereof) is preferably 23 to 200 mM. Arginine hydrochloride is preferably used as the form of arginine, and the concentration of arginine in the liquid pharmaceutical composition is preferably 144 mM or less (30 mg / mL or less as arginine hydrochloride), more preferably 23 to 144 mM (5 to 5 as arginine hydrochloride). 30 mg / mL), more preferably 47-96 mM (10-20 mg / mL as arginine hydrochloride), most preferably 95 mM (20 mg / mL as arginine hydrochloride).

  The pharmaceutical composition of the present invention may contain mannitol and / or lactose. Mannitol and / or lactose can be used for the purpose of improving stability, a solubilizing agent, an excipient or an isotonic agent. Particularly in lyophilized pharmaceutical compositions, it is desirable to add mannitol and / or lactose for cake formation. The amount of mannitol and / or lactose added may be an amount that is acceptable for administration to a living body and may be an amount that can be dissolved. When mannitol is added, its concentration in the liquid pharmaceutical composition is preferably 20 mg / mL or less, more preferably 1.25 to 20 mg / mL, and even more preferably 1.25 to 5 mg / mL. When lactose is added, its concentration in the liquid pharmaceutical composition is preferably 1-20 mg / mL.

  The pharmaceutical composition of the present invention may contain sodium chloride. Sodium chloride can be used as an isotonic agent. The amount of sodium chloride added may be an amount that is acceptable for administration to a living body and may be an amount that can be dissolved. The concentration of sodium chloride in the liquid pharmaceutical composition is preferably 9 mg / mL or less, more preferably 0.6 to 6 mg / mL.

  The pharmaceutical composition of the present invention may further contain other additives than those described above, which are pharmaceutically acceptable. For example, amino acids other than the above-mentioned three types, sugars or sugar alcohols other than the above-described mannitol and lactose, and inorganic salts can be mentioned. An amino acid refers to an organic compound having both an amino group and a carboxyl group, but also includes imino acids such as proline and hydroxyproline. Examples of amino acids include neutral amino acids, acidic amino acids, and basic amino acids. Specifically, as neutral amino acids, alanine, valine, norvaline, leucine, norleucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, phenylalanine, tyrosine, tryptophan, diiodotyrosine, sulamine, hydroxyproline, Examples include thyroxine, cysteine, and α-aminobutyric acid. Examples of acidic amino acids include aspartic acid and glutamic acid. An amino acid can also be used as its salt form, for example, hydrochloride of an amino acid. The sugar or sugar alcohol includes monosaccharides, disaccharides, polysaccharides or water-soluble glucans, and can be used as long as it can be administered to a living body. Specific examples of sugars other than the above include glucose, galactose, fructose, mannose, sorbose, xylose, isomaltose, maltose, sucrose, cellobiose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and the like. . The sugar alcohol is preferably a trihydric or higher sugar alcohol, and specific examples include glycerin, erythritol, arabitol, xylitol, sorbitol, inositol, dulcitol, raffinose, maltitol, lactitol, palatinit, trehalose and the like. Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. In the case of injections, preservatives such as phenol and cresol, sulfites, sodium pyrosulfite, ascorbic acid, ethylenediaminetetraacetic acid (EDTA), antioxidants such as thioglycolic acid, and soothing agents such as benzyl alcohol and chlorobutanol Other acceptable additives may be added. When it is used for oral administration, nasal administration, pulmonary administration, etc., an additive acceptable in each administration route may be added.

  The liquid pharmaceutical composition of the present invention contains the above-mentioned components in a pharmacologically acceptable medium, and is preferably in the form of a solution. Water is preferred as the pharmacologically acceptable medium.

  The lyophilized pharmaceutical composition of the present invention can be obtained by lyophilizing a liquid composition in which the ratio of each additive to IFN-β is equal to that of the liquid pharmaceutical composition. This means that, in addition to the liquid pharmaceutical composition, the liquid composition subjected to lyophilization further includes a diluted solution and a concentrated solution thereof. Of course, the lyophilized pharmaceutical composition of the present invention can also be obtained by lyophilizing the liquid pharmaceutical composition as it is.

  The freeze-dried pharmaceutical composition of the present invention can be dissolved in a solution containing polyoxyethylene sorbitan fatty acid ester. It is preferable that the solution used here contains polyoxyethylene sorbitan fatty acid ester. Specific examples of polyoxyethylene sorbitan fatty acid esters include polysorbate 20 (trade name “Tween 20”), polysorbate 40 (trade name “Tween 40”), polysorbate 60 (trade name “Tween 60”), polysorbate 65 (trade name “Tween 65”). ) And polysorbate 80 (trade name “Tween80”). Polysorbate 20 and polysorbate 80 are preferable, and polysorbate 20 is more preferable. The amount of the polyoxyethylene sorbitan fatty acid ester contained in the solution may be an amount that is acceptable for administration to a living body. For example, the maximum amount of polysorbate 20 that has been approved by the Ministry of Health, Labor and Welfare so far and can be used for one administration to humans is 0.76 mg for oral administration, 0.6 mg for intravenous injection, 66 mg for general external preparations, and external for dental use The maximum amount of polysorbate 80 that can be used per administration is 300 mg for oral administration, 500 mg for intravenous injection, 100.2 mg for intramuscular injection, and 100 mg / g for general topical preparations. For oral and oral use, it is 37mg / g (Pharmaceutical Additives Encyclopedia 2000, edited by Japan Pharmaceutical Additives Association, Yakuji Nippo), but safety to the living body has been confirmed without causing any harm to the living body. The amount is not limited to this as long as it can be used in a short or long term. The amount of polysorbate 20 contained in the solution is preferably 0.01 to 0.5 mg, most preferably 0.5 mg, and the amount of polysorbate 80 is preferably 0.01 to 50 mg. The polysorbate 20 and the polysorbate 80 may be used alone, but may be mixed and used as necessary.

  Further, the solution may contain an isotonic agent such as an inorganic salt for isotonicity. Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, magnesium chloride and the like, with sodium chloride being preferred. The amount of the salt added is preferably such that the liquid pharmaceutical composition after the lyophilized pharmaceutical composition is dissolved in a dissolving solution is isotonic with respect to blood.

  Examples of other isotonic agents added to the solution include sugars, sugar alcohols, and amino acids. Specific examples of sugar or sugar alcohol include glucose, galactose, fructose, mannose, sorbose, xylose, isomaltose, maltose, lactose, sucrose, cellobiose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, glycerin Erythritol, arabitol, xylitol, sorbitol, mannitol, inositol, dulcitol, raffinose, maltitol, lactitol, palatinit, trehalose and the like. Amino acids can be used for the purpose of increasing solubility in addition to isotonicity. Any amino acid can be used as long as it is acceptable for administration to a living body, and examples thereof include neutral amino acids, acidic amino acids, and basic amino acids. Specifically, glycine, alanine, valine, norvaline, leucine, norleucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, phenylalanine, tyrosine, tryptophan, diiodotyrosine, sulamine, hydroxyproline, thyroxine, cysteine , Α-aminobutyric acid, aspartic acid, glutamic acid, lysine, arginine, histidine, hydroxylysine, ornithine, γ-aminobutyric acid, ε-aminocaproic acid and the like. An amino acid can also be used as its salt form, for example, hydrochloride of an amino acid. These isotonic agents may be added in such an amount that the pharmaceutical composition after the lyophilized pharmaceutical composition is dissolved in a solution to be isotonic with respect to blood. The solution may further contain a pharmaceutically acceptable additive. In the case of injections, preservatives such as phenol and cresol, sulfites, sodium pyrosulfite, ascorbic acid, ethylenediaminetetraacetic acid (EDTA), antioxidants such as thioglycolic acid, soothing agents such as benzyl alcohol and chlorobutanol, Other acceptable additives may be added. When it is used for oral administration, nasal administration, pulmonary administration, etc., an additive acceptable in each administration route may be added.

  The lyophilized pharmaceutical composition of the present invention can be provided as a kit. As a preferred specific example of the kit here, the freeze-dried pharmaceutical composition of the present invention is sealed in one chamber of a container composed of a plurality of chambers separated by a partition configured to be easily opened, and the other 1 The solution of the present invention is sealed in a form in which the solution of the present invention is sealed in a chamber, the partition is opened during use, and both are mixed and dissolved, or the freeze-dried pharmaceutical composition of the present invention is sealed in a vial or the like However, the present invention is not limited to this as long as there is no problem in use.

  Regarding the lyophilized pharmaceutical composition, the pharmaceutical composition of the present invention is dissolved in a solution containing polyoxyethylene sorbitan fatty acid ester, and the liquid pharmaceutical composition is directly administered to a living body. Typical routes of administration include oral, nasal, pulmonary, and parenteral (transdermal, intravenous, intramuscular, subcutaneous, intraarterial, intraperitoneal, intrathecal, and intratumoral) Including peripheral injection and injection). Preferred is parenteral administration, and more preferred is a method by intravenous administration, intramuscular administration, or subcutaneous administration.

  Moreover, the pharmaceutical composition of the present invention can be applied to research use as a reagent in addition to therapeutic use. In particular, it is useful for use in analytical methods that have been difficult to analyze for IFN-β alone due to the coexistence of human serum albumin, such as HPLC analysis, UV measurement, and protein quantification.

As the container containing the lyophilized pharmaceutical composition, lysate or liquid pharmaceutical composition of the present invention, a container whose surface is coated with a substance that suppresses adsorption of IFN-β to the container is preferably used. For example, coating with silicon, polypropylene, or polytetrafluoroethylene may be mentioned, and a silicon-coated container is preferable. Examples of the shape of the container include a vial or a syringe.

The IFN-β liquid pharmaceutical composition and lyophilized pharmaceutical composition of the present invention are stable after long-term storage and stable during the preparation operation. In the lyophilized pharmaceutical composition, IFN-β is stable even in the lyophilization process. Retained. “Stable” means that the biological activity of IFN-β is maintained and the therapeutic efficacy of IFN-β is not compromised. “Preservation” means that the prepared liquid pharmaceutical composition is not used immediately after the preparation, and the lyophilized pharmaceutical composition is not dissolved but is held in a lyophilized form (dissolved later). The liquid state). The “preparation operation” means an operation essential for producing the pharmaceutical composition of the present invention, such as a mixing operation of IFN-β and other additives, a transfer operation to a container, and a short-time standing.

  The stable IFN-β liquid pharmaceutical composition of the present invention after long-term storage maintains a biological activity of at least 70% of the initial biological activity after storage for 4 weeks at 37 ° C, and for 8 weeks at 4 ° C. Maintains at least 80% of the biological activity of the initial biological activity after storage. The stable IFN-β frozen pharmaceutical composition of the present invention maintains a biological activity of at least 70% of the initial biological activity when dissolved in a lysis solution after storage at 37 ° C. for 4 weeks. Maintains at least 80% of the biological activity of the initial biological activity when dissolved in a lysate after storage at 8 ° C for 8 weeks. As used herein, “initial biological activity” refers to the biological activity of a liquid pharmaceutical composition immediately after the preparation of the liquid pharmaceutical composition (before the start of storage) in evaluating the stability of the liquid pharmaceutical composition after long-term storage. In the evaluation of the stability of the freeze-dried pharmaceutical composition after long-term storage, the IFN-β liquid composition used for the preparation of the freeze-dried pharmaceutical composition or the freeze-dried pharmaceutical composition immediately after freeze-drying (before the start of storage) Represents biological activity. The stable IFN-β pharmaceutical composition of the present invention maintains a biological activity of 90% or more of the amount of IFN-β used for preparation (IFN-β preparation amount) in the preparation operation. In addition, the freeze-dried pharmaceutical composition needs to retain biological activity in the freeze-drying step included in the production process and the step of dissolving the freeze-dried pharmaceutical composition. The lyophilized pharmaceutical composition of the present invention maintains a biological activity of 80% or more of the biological activity of the IFN-β liquid composition subjected to lyophilized pharmaceutical composition preparation in the lyophilization process and dissolution. .

  The stability of IFN-β can be evaluated by measuring the residual ratio of biological activity after preparation or storage. The biological activity of IFN-β can be measured by a standard method (Cytopathic effect inhibition: CPE method) for evaluating suppression of the cytopathic effect of the virus. Details are described in Armstrong, JA: Cytopathic effect inhibition assay for interferon: microculture plate assay, in: Methods in Enzymology, vol. 78 S. Pestka, (ed) .pp. 381-387, Academic Press (New York), 1981. Has been. As an alternative to this method, an enzyme immunoassay (EIA) method using an anti-human IFN-β antibody can also be used. Yamazaki, S. et al .: Highly sensitive enzyme immunoassay of human interferon-beta 1., J. Immunoassay, 10: 57-73, 1989 is a method that well reproduces the results of CPE method. . Other methods for measuring the residual ratio of IFN-β after storage include the measurement of IFN-β content using UV measurement or reversed-phase HPLC. IFN-β that does not have is also measured as content. Since it is biological activity, not IFN-β content, that is important when IFN-β is used in therapy, these methods are inappropriate for assessing the stability of IFN-β. The long-term stability of IFN-β is preferably evaluated by CPE method or EIA method after storage at 4 ° C or 37 ° C. The temperature condition of 37 ° C. is used for accelerating the decomposition reaction, and is a method generally used by those skilled in the art.

  The present invention will be further described in the following examples, but is not limited thereto. In the following Examples and Comparative Examples, natural human IFN-β derived from fibroblasts was used as IFN-β. Further, pure water was used as a medium for the liquid composition. The additives used in Examples and Comparative Examples are shown in Table 1 below.

  Cytopathic effect inhibition: CPE method and enzyme immunoassay (EIA) method used for the evaluation of stability were performed as follows.

Cytopathic effect inhibition (CPE method) was performed using human amniotic cells (FL cells) and Sindbis virus. 3-4 × 10 ⁴ FL cells were seeded in wells of a 96-well microplate for cell culture (culture medium: MEM supplemented with 5% heat-inactivated FBS, 10 mM HEPES) and cultured overnight (37 ^° C., 5% CO ₂ ). A measurement sample containing IFN-β was appropriately diluted (2-fold serial dilution) in a culture medium, added to FL cells, and further cultured overnight (37 ^° C., 5% CO ₂ ). As a control, human IFN-β standard (Japanese reference from National Institute of Infectious Diseases) was added. After washing and removing the culture solution, Sindbis virus was added to each well and cultured overnight (37 ^° C.). After incubation, the virus solution was discarded, and the plate was immersed in a crystal violet solution containing formalin for about 15 minutes, and the living cells were fixedly stained. Thereafter, the living cells were quantified by measuring the absorbance at a wavelength of 595 nm with a spectrophotometer. The survival rate of cells treated with virus without IFN-β treatment was defined as 0%, and the survival rate of cells treated with IFN-β treatment and non-virus treatment was defined as 100%. Calculated by converting the biological activity of each measurement sample to international units based on the 50% endpoint value of human IFN-β standard, with the point at which the survival rate becomes 50% (50% endpoint) .

  For the enzyme immunoassay (EIA) method, an IFN-β ELISA kit (manufactured by Kamakura Technoscience) was used, a calibration curve was prepared according to the instructions attached to the kit, and the biological activity of each sample was calculated from the calibration curve.

Examples 1-4, Comparative Examples 1-9
IFN-β stability in pharmaceutical composition preparation operations Prepare IFN-β composition containing the ingredients shown in Table 2 below, dispense 0.5 mL each into a 2 mL vial, and store at 4 ° C for the period indicated in the table did. IFN-β biological activity after storage was measured by EIA method. The results are expressed as the residual rate (%) of biological activity with the IFN-β charge as 100%. In Table 2 below, “24 hr” indicates 24 hours after preparation, and “72 hr” indicates 72 hours after preparation.

  Composition 1 (Comparative Example 1) is an additive composition of Ferron (Toray Industries, Inc.), an existing HSA-containing natural IFN-β pharmaceutical composition, and Composition 2 (Comparative Example 2) is Ferron (Toray Industries, Inc.) A composition obtained by removing HSA from the additive composition (containing 1 mg of lactose per IFN-β 1 MIU). Compositions 7 and 8 (Comparative Examples 6 and 7) are compositions containing no surfactant as described in JP-T-2001-517635.

  No surfactant, composition containing only citric acid and phosphate (Composition 3 (Comparative Example 3)), no surfactant, only citric acid, phosphate and amino acid In the composition (Compositions 7 to 10 (Comparative Examples 6 to 9)), the biological activity decreased to 90% or less after standing for 72 hours. On the other hand, in the composition containing polyoxyethylene (160) polyoxypropylene (30) glycol as a surfactant, the biological activity was maintained at 90% or more (Composition 4, 11-13 (Example 1)). ~Four)). In the composition containing other surfactants, the biological activity decreased to 90% or less (Compositions 5 and 6 (Comparative Examples 4 and 5)).

Examples 5-10, Comparative Examples 10-19
IFN-β stability after long-term storage of liquid pharmaceutical compositions (1) -Additive selection-
An IFN-β composition containing the components shown in Table 3 below was prepared, dispensed in 0.5 mL aliquots into 2 mL vials that were siliconized, and stored at a temperature of 4 ° C. or 37 ° C. for the period indicated in the table. IFN-β biological activity after storage was measured by CPE method. The results are expressed as the residual rate (%) of biological activity with the initial biological activity of each liquid pharmaceutical composition immediately after preparation as 100%. In Table 3 below, “1W” represents storage for 1 week, “4W” represents storage for 4 weeks, and “8W” represents storage for 8 weeks.

  Composition 14 (Comparative Example 10) is a low ionic strength, pH 5.0 composition described in JP-T-2004-529917. Compositions 15, 16, 17, and 21 (Comparative Examples 11, 12, 13, and 15) are additive compositions described in JP-T-2001-519700. Composition 20 (Comparative Example 14) is a composition containing a sugar and / or a sugar alcohol and an amino acid described in WO2004 / 028557. Composition 23 (Comparative Example 16) is an additive composition of AVONEX (BIOGEN), which is a genetically modified IFN-β pharmaceutical composition. Composition 24 (Comparative Example 17) is a composition obtained by removing human serum albumin from the additive composition of Ferron (Toray Industries, Inc.), which is an existing human serum albumin-containing natural type IFN-β pharmaceutical composition (IFN- Contains 1 mg lactose per 1 MIU).

  In IFN-β 1MIU / mL, a composition (composition 18, 19 (Examples 5 and 6)) to which citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol was added, low Composition with ionic strength and pH 5.0 (Composition 14 (Comparative Example 10)), Composition described in JP-T-2001-519700 (Compositions 15 and 16 (Comparative Examples 11 and 12)) and citric acid as a buffering agent / A stable composition was obtained as compared with the composition using sodium citrate (composition 17 (Comparative Example 13)). When compositions 18 and 19 (Examples 5 and 6) were stored at 4 ° C. for 8 weeks, the residual ratio of biological activity was 80% or more.

  Phosphoric acid in a composition (composition 22 (Example 7)) containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride at IFN-β 6MIU / mL Long-term stability compared to a composition containing a buffer solution, arginine and sucrose (Composition 20 (Comparative Example 14)) and a composition containing a citrate buffer solution, arginine and sucrose (Composition 21 (Comparative Example 15)) Improvement in sex was confirmed. The composition 22 (Example 7) showed a biological activity remaining rate of 70% or more even after storage at 37 ° C. for 8 weeks.

  IFN-β 10MIU / mL, citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol added composition (Compositions 26-28 (Examples 8-10)), buffered Improvement in long-term stability was confirmed as compared with the composition containing no agent (Composition 25 (Comparative Example 18)) and AVONEX formulation (Composition 23 (Comparative Example 16)). Compositions 26 to 28 (Examples 8 to 10) showed a biological activity remaining ratio of 70% or more even after storage at 37 ° C. for 8 weeks.

  The composition using only polysorbate 20 (Composition 29) (Comparative Example 19) did not improve stability.

Examples 11-15
IFN-β stability after long-term storage of liquid pharmaceutical composition (2) -Effect of pH-
An IFN-β composition containing the components shown in Table 4 below was prepared, dispensed in 0.5 mL portions into 2 mL vials that were siliconized, and stored at a temperature of 37 ° C. for the period indicated in the table. IFN-β biological activity after storage was measured by CPE method. The results are expressed as the residual rate (%) of biological activity with the biological activity of each liquid pharmaceutical composition immediately after preparation as 100%. In Table 4 below, “4W” represents storage for 4 weeks, and “8W” represents storage for 8 weeks.

  In compositions containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride, mannitol, pH 4.5-6.5, after storage at 37 ° C for 8 weeks, The survival rate of biological activity was over 70%. (Compositions 30-34 (Examples 11-15)).

Examples 16-19
IFN-β stability of liquid pharmaceutical compositions after long-term storage (3) -Influence of IFN-β concentration-
An IFN-β composition containing the components shown in Table 5 below was prepared, dispensed in 0.5 mL portions into 2 mL vials that were silicized, and stored at 4 ° C. or 37 ° C. for the period shown in Table 5. IFN-β biological activity after storage was measured by CPE method. The results are expressed as the residual rate (%) of biological activity with the biological activity of each liquid pharmaceutical composition immediately after preparation as 100%. In Table 5 below, “4W” indicates storage for 4 weeks, “8W” indicates storage for 8 weeks, and “17W” indicates storage for 17 weeks.

  In a composition containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride, mannitol, the biological activity of IFN-β at the start of storage is 1 MIU / mL Even after long-term storage at a low concentration of 4 ° C. for 17 weeks, the survival rate of biological activity was 80% or more (Compositions 35 to 38 (Examples 16 to 19)).

Examples 20-24, Comparative Examples 20-24
IFN-β stability during preparation of lyophilized pharmaceutical composition (1)
An IFN-β composition containing the components shown in Table 6 below was prepared, dispensed 1 mL each into a 2 mL glass vial that had been siliconized, and lyophilized with a rubber stopper. IFN-β liquid composition before lyophilization and a solution in which a lyophilized pharmaceutical composition is dissolved immediately after lyophilization (EIA buffer (0.1% (w / v) BSA, 0.05% (v / v IFN-β biological activity of Tween20, 0.1M phosphate buffer (pH 7.0) dissolved in 1 mL) was measured by EIA method. The results are expressed as the residual rate (%) of the biological activity after lyophilization with the biological activity of the IFN-β liquid composition used for preparing the lyophilized pharmaceutical composition as 100%.

  Composition 39 (Comparative Example 20) is an additive composition of Ferron (Toray Industries, Inc.), which is an existing natural IFN-β pharmaceutical composition containing human serum albumin. Composition 40 (Comparative Example 21) was obtained by removing human serum albumin from the additive composition of Ferron (Toray Industries, Inc.), which is a conventional human serum albumin-containing natural IFN-β pharmaceutical composition. Composition 41 (Comparative Example 22) is a composition containing citric acid and phosphate and no surfactant.

  Compositions containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol (Compositions 42, 47 (Examples 20, 24)) and citric acid, phosphate, polyoxyethylene ( 160) The composition containing polyoxypropylene (30) glycol and amino acid (Compositions 44 to 46 (Examples 21 to 23)) exhibited a biological activity remaining rate of 80% or more upon lyophilization. Other surfactants did not improve IFN-β stability (Compositions 43 and 48 (Comparative Examples 23 and 24)).

Examples 25-31, Comparative Example 25
IFN-β stability during preparation of freeze-dried pharmaceutical compositions (2)
An IFN-β composition containing the components shown in Table 7 below was prepared, and 1 mL each was dispensed into a siliconized 5 mL glass vial, stoppered with rubber, and lyophilized. IFN-β biology of IFN-β liquid composition used for preparation of lyophilized pharmaceutical composition and lyophilized pharmaceutical composition dissolved in 2 mL of lysing solution (0.25 mg / mL Tween20, 4.6 mg / mL NaCl) immediately after lyophilization Activity was measured by CPE method. The results are expressed as the residual rate (%) of the biological activity after lyophilization with the biological activity of the IFN-β liquid composition used for preparing the lyophilized pharmaceutical composition as 100%.

  In a composition containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride is added at 5 to 30 mg / mL, and the residual rate of biological activity upon lyophilization is 80% The above was shown (Compositions 49 to 52 (Examples 25 to 28)). In the composition containing citric acid, phosphate, and arginine hydrochloride, the residual rate of biological activity during lyophilization was 80% or more by adding polyoxyethylene (160) polyoxypropylene (30) glycol. (Compositions 54-56 (Examples 29-31)).

Examples 32-42, Comparative Examples 26-30
IFN-β stability after long-term storage of freeze-dried pharmaceutical composition (1)
An IFN-β composition containing the components shown in Table 8 below was prepared, dispensed in an amount of 1 mL each into a siliconized 2 mL glass vial, stoppered with rubber, and lyophilized. The lyophilized pharmaceutical composition was stored at 37 ° C. for 4 or 8 weeks. IFN-β liquid composition used for lyophilized pharmaceutical composition preparation and lyophilized pharmaceutical composition after storage dissolved in EIA buffer (dissolved in EIA buffer for measurement by both EIA method and CPE method) -β biological activity was measured by CPE method and EIA method. The results show the measurement results by the CPE method, and the survival rate of the biological activity after lyophilization with the biological activity of the IFN-β liquid composition used for the preparation of the lyophilized pharmaceutical composition as 100% ( %). In Table 8 below, “4W” represents storage for 4 weeks, and “8W” represents storage for 8 weeks. The result measured by the EIA method also showed the same value as the CPE method.

  Composition 57 (Comparative Example 26) is an additive composition of Ferron (Toray Industries, Inc.), which is an existing natural serum IFN-β pharmaceutical composition containing human serum albumin. Composition 58 (Comparative Example 27) is obtained by removing human serum albumin from the additive composition of Ferron (Toray Industries, Inc.), which is an existing natural serum IFN-β pharmaceutical composition containing human serum albumin. Composition 59 (Comparative Example 28) is a composition containing citric acid and phosphate and no surfactant.

  In lyophilized pharmaceutical compositions (Compositions 60, 61, 63-69, 71, 72 (Examples 32-42)) containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol Even after storage for 8 weeks, the survival rate of biological activity was over 70%. Other surfactants that have been shown to be useful in other known documents (for example, JP-A-61-277633) did not improve IFN-β stability (Compositions 62 and 70 (comparative examples)). 29, 30)). 8 weeks for compositions containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride, mannitol, even at IFN-β concentrations as low as 0.5 MIU / mL Even after storage, the residual rate of biological activity was 100% (Composition 72 (Example 42)).

Examples 43-53
IFN-β stability after long-term storage of lyophilized pharmaceutical composition (2) Effect of various additive concentrations
An IFN-β composition containing the components shown in Table 9 below was prepared, dispensed 1 mL each into a siliconized 5 mL glass vial, stoppered with rubber, and lyophilized. The lyophilized pharmaceutical composition was stored at 37 ° C. for 4 weeks. The lyophilized pharmaceutical composition immediately after lyophilization and the lyophilized pharmaceutical composition after storage are each dissolved in 2 mL of a lysis solution (0.25 mg / mL Tween20, 4.6 mg / mL NaCl) to increase the IFN-β biological activity to CPE. Measured by the method. The results are expressed as the residual rate (%) of the biological activity after storage with the biological activity of the freeze-dried pharmaceutical composition immediately after lyophilization as 100%. “4W” in Table 9 below represents storage for 4 weeks.

  In a composition containing citric acid, phosphate, arginine hydrochloride, mannitol, sodium chloride, the polyoxyethylene (160) polyoxypropylene (30) glycol content is 0.01-1mg / mL and stored at 37 ° C for 4 weeks Later, the survival rate of biological activity was 70% or more (Compositions 73 to 76 (Examples 43 to 46)). In a composition containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, mannitol, sodium chloride, stored at 37 ° C for 4 weeks at an arginine hydrochloride content of 10-30 mg / mL In addition, the survival rate of biological activity was 70% or more (Compositions 77 to 79 (Examples 47 to 49)). A composition containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride, sodium chloride, mannitol content of 5 mg / mL or less at 37 ° C, after storage for 4 weeks The survival rate of the biological activity was 70% or more (Compositions 80 to 83 (Examples 50 to 53)).

Examples 54-61
IFN-β stability after long-term storage of lyophilized pharmaceutical composition (3) Effect of pH
An IFN-β composition containing the components shown in Table 10 below was prepared, dispensed 1 mL each into a 2 mL glass vial that had been siliconized, and lyophilized with a rubber stopper. The lyophilized pharmaceutical composition was stored at 37 ° C. for 4 weeks. The lyophilized pharmaceutical composition immediately after lyophilization and the lyophilized pharmaceutical composition after storage are each dissolved in 2 mL of a lysis solution (0.25 mg / mL Tween20, 4.6 mg / mL NaCl) to increase the IFN-β biological activity to CPE. Measured by the method. The results are expressed as the residual rate (%) of the biological activity after storage with the biological activity immediately after lyophilization as 100%. “4W” in Table 10 below represents storage for 4 weeks.

  A composition containing polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride, mannitol, sodium chloride, adjusted to pH 37-6.5 ° C, adjusted with citric acid and phosphate, 4 Even after weekly storage, the residual ratio of biological activity was 70% or more (Compositions 84 to 91 (Examples 54 to 61)).

Examples 62-64
IFN-β stability after long-term storage of freeze-dried pharmaceutical composition (4) Effect of IFN-β concentration
An IFN-β composition containing the components shown in Table 11 below was prepared, dispensed 1 mL each into a 2 mL glass vial that had been silicized, and lyophilized with a rubber stopper. The lyophilized pharmaceutical composition was stored at 37 ° C. for 4 weeks. The lyophilized pharmaceutical composition immediately after lyophilization and the lyophilized pharmaceutical composition after storage are each dissolved in 2 mL of a lysis solution (0.25 mg / mL Tween20, 4.6 mg / mL NaCl) to increase the IFN-β biological activity to CPE. Measured by the method. The results are expressed as the residual rate (%) of the biological activity after storage with the biological activity immediately after lyophilization as 100%. “4W” in Table 11 below represents storage for 4 weeks.

  A composition containing citric acid, phosphate, polyoxyethylene (160) polyoxypropylene (30) glycol, arginine hydrochloride, mannitol, sodium chloride, with an IFN-β concentration in the range of 1-6 MIU / mL 37 Even after storage at 4 ° C. for 4 weeks, the residual ratio of biological activity was 70% or more (Compositions 92 to 94 (Examples 62 to 64)).

Example 65
Selection of lyophilized pharmaceutical composition solution (1)
An IFN-β composition containing the components shown in Table 12 below was prepared, dispensed 1 mL each into a 2 mL glass vial that had been siliconized, lyophilized with a rubber stopper. Immediately after lyophilization, the lyophilized pharmaceutical composition was dissolved in 1 mL of each lysis solution described in Table 12, and the biological activity of IFN-β was measured by the CPE method. The results are expressed as the residual rate (%) of the biological activity of the lyophilized composition lysate with the biological activity of the liquid composition subjected to lyophilization as 100%.

組成物：1MIU/mL IFN-β、9mMクエン酸、22mMリン酸水素2Na、0.5mg/mL PLU F68、
10mg/mL Arg、2.5mg/mL Man、0.6mg/mL NaCl （pH5.5）

Composition: 1 MIU / mL IFN-β, 9 mM citric acid, 22 mM hydrogen phosphate 2Na, 0.5 mg / mL PLU F68,
10mg / mL Arg, 2.5mg / mL Man, 0.6mg / mL NaCl (pH5.5)

  By using a solution containing the polyoxyethylene sorbitan fatty acid ester (solutions 2 to 5), the IFN-β biological activity remaining rate of the freeze-dried pharmaceutical composition was 80% or more. On the other hand, a solution containing only physiological saline (solution 1) or other nonionic surfactant polyoxyethylene (160) polyoxypropylene (30) glycol (solutions 6 and 7) was used. In this case, the residual rate of biological activity was around 50%, and the activity in the freeze-dried pharmaceutical composition could not be sufficiently maintained.

Example 67
Selection of lyophilized pharmaceutical composition solution (2)
An IFN-β composition containing the components shown in the footnotes in Table 13 below was prepared, dispensed in 1 mL portions into 2 mL glass vials that had been siliconized, stoppered with rubber, and lyophilized. Immediately after lyophilization, the lyophilized pharmaceutical composition was dissolved in 1 mL of each lysis solution described in Table 13, and the IFN-β biological activity was measured by the CPE method. The results are expressed as the residual rate (%) of the biological activity of the lyophilized composition lysate with the biological activity of the liquid composition subjected to lyophilization as 100%.

組成物：6MIU/mL IFN-β、9mMクエン酸、22mMリン酸水素2Na、0.5mg/mL PLU F68、
10mg/mL Arg、2.5mg/mL Man、0.6mg/mLNaCl （pH5.5）

Composition: 6 MIU / mL IFN-β, 9 mM citric acid, 22 mM hydrogen phosphate 2Na, 0.5 mg / mL PLU F68,
10 mg / mL Arg, 2.5 mg / mL Man, 0.6 mg / mL NaCl (pH 5.5)

  When using a lysate containing 0.01 to 0.5 m / mLg of Tween 20 (lysate 8 to 11) and a lysate containing 0.01 to 50 mg / mL of Tween 80 (solution 12 to 16), the residual rate of biological activity is More than 80%.

INDUSTRIAL APPLICABILITY According to the present invention, a safe pharmaceutical composition free from human serum albumin can be provided, which maintains the biological activity of a low concentration of IFN-β suitable for use in therapy for a long period of time. This pharmaceutical composition can be used as a freeze-dried pharmaceutical composition or a liquid pharmaceutical composition for therapeutic use or research use.

Claims (22)

  A liquid pharmaceutical composition comprising interferon-β and citric acid, phosphate and polyoxyethylene polyoxypropylene glycol as additives in a pharmacologically acceptable medium and free of human serum albumin.   2. The liquid pharmaceutical composition according to claim 1, wherein the concentration of interferon-β is 0.5 to 12 MIU / mL.   The liquid pharmaceutical composition according to claim 1 or 2, wherein the concentration of the citric acid is 5 to 20 mM.   The liquid pharmaceutical composition according to any one of claims 1 to 3, wherein a concentration of the phosphate is 5 to 30 mM.   The liquid pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration of the polyoxyethylene polyoxypropylene glycol is 0.01 to 2 mg / mL.   The liquid pharmaceutical composition according to any one of claims 1 to 5, which has a pH of 3.0 to 6.5.   The average polymerization degree of the polyoxyethylene part in the polyoxyethylene polyoxypropylene glycol is 3 to 200, and the average polymerization degree of the polyoxypropylene part is 5 to 70. 7. Liquid pharmaceutical composition.   The liquid pharmaceutical composition according to claim 7, wherein the polyoxyethylene polyoxypropylene glycol is polyoxyethylene (160) polyoxypropylene (30) glycol.   The liquid pharmaceutical composition according to any one of claims 1 to 8, further comprising at least one amino acid selected from the group consisting of arginine, histidine and glycine as an additive.   The liquid pharmaceutical composition according to claim 9, containing 23 to 200 mM of the amino acid.   The liquid pharmaceutical composition according to claim 9 or 10, comprising arginine as the amino acid.   The liquid pharmaceutical composition according to claim 11, comprising 23 to 144 mM of arginine as the amino acid.   The liquid pharmaceutical composition according to any one of claims 1 to 12, further comprising mannitol and / or lactose as an additive.   14. A liquid pharmaceutical composition according to claim 13 containing mannitol.   The liquid pharmaceutical composition according to claim 14, wherein the concentration of mannitol is 1.25 to 20 mg / mL.   A lyophilized pharmaceutical composition obtained by lyophilizing a liquid composition in which the ratio of each additive to interferon-β is equal to the liquid pharmaceutical composition according to any one of claims 1 to 15.   The freeze-dried pharmaceutical composition according to claim 16, wherein the liquid pharmaceutical composition according to any one of claims 1 to 15 is freeze-dried.   An interferon-β administration kit comprising the freeze-dried pharmaceutical composition according to claim 16 or 17 and a lysis solution.   The kit according to claim 18, wherein the solution contains polyoxyethylene sorbitan fatty acid ester.   21. The kit according to claim 19 or 20, comprising one or two polyoxyethylene sorbitan fatty acid esters selected from polysorbate 20 or polysorbate 80 in the solution.   21. The kit according to claim 20, wherein 0.01 to 0.5 mg of polysorbate 20 is contained in the solution. The kit according to claim 21, wherein the dissolution solution contains 0.01-50 mg of polysorbate 80.