Preparations containing HMGB1 partial peptides

Abstract

This pharmaceutical composition, which contains a substance described in any of the following (a) to (c), an organic acid, an organic acid metal salt, and a sugar and / or a sugar alcohol and which has a pH of 3.0-4.5 when dissolved in distilled water for injection, can stabilize the substance described in any of the following (a) to (c): (a) an HMGB1 fragment peptide that contains the amino acid sequence of SEQ ID NO: 1, or an acid addition salt of said peptide; (b) a peptide that contains the amino acid sequence of SEQ ID NO: 1, in which one or more amino acids have been replaced, deleted, inserted, or added, and that has activity to stimulate cell migration, or an acid addition salt of said peptide; and (c) a peptide that contains an amino acid sequence having a sequence identity of approximately 80% or more to the amino acid sequence of SEQ ID NO: 1 and that has activity to stimulate cell migration, or an acid addition salt of said peptide.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for stabilizing a partial peptide of HMGB1 that induces tissue regeneration. [Background technology]

[0002] Currently, regenerative medicine is being actively developed, in which bone marrow mesenchymal stem cells are collected by bone marrow blood collection, expanded in cell culture, and then transplanted into the site of intractable tissue damage or into the peripheral blood circulation to induce regeneration of damaged tissue. Clinical applications of bone marrow mesenchymal stem cell transplantation have already been advanced in regenerative medicine for cerebral infarction, myocardial infarction, and intractable skin ulcers. Furthermore, transplanted bone marrow mesenchymal stem cells have been shown to suppress inflammation and immune responses and fibrous scar formation locally in the body. Clinical trials of bone marrow mesenchymal stem cell transplantation have been initiated as a new treatment for graft-versus-host disease (GVHD), a serious side effect after bone marrow transplantation or transfusion, and for the autoimmune disease scleroderma. However, bone marrow blood containing bone marrow mesenchymal stem cells can only be obtained by an invasive technique involving repeated insertion of a thick needle into the iliac bone. Furthermore, bone marrow mesenchymal stem cells gradually lose their proliferation and pluripotency when subcultured ex vivo. Furthermore, because bone marrow mesenchymal stem cell culture based on high-quality control that guarantees the safety of in vivo transplantation requires specialized culture facilities such as a cell processing center (CPC), it is currently only possible at a very limited number of universities and companies. In other words, in order to deliver regenerative medicine using bone marrow mesenchymal stem cells to the many patients around the world who are suffering from intractable tissue damage, it is an urgent task to develop technology for mesenchymal stem cell regenerative medicine that can be performed at any medical facility.

[0003] HMGB1 (High mobility group box 1) was identified approximately 30 years ago as a non-histone chromatin protein that regulates intranuclear chromatin structure, thereby controlling gene expression and DNA repair. The structure of the HMGB1 protein is primarily composed of two DNA-binding domains: the N-terminal DNA-binding domain is called the A-box, and the C-terminal DNA-binding domain is called the B-box. Previous studies have revealed that the domain within the HMGB1 molecule that binds to TLRs and induces an inflammatory response is located within the B-box.

[0004] It has been found that a fragment peptide of the HMGB1 (High mobility group box 1) protein mobilizes bone marrow mesenchymal stem cells from the bone marrow into the peripheral blood, and that administration of this fragment peptide during the acute phase of myocardial infarction causes bone marrow-derived mesenchymal stem cells to accumulate at and near the site of infarction, resulting in an improvement in cardiac function (Patent Documents 1 and 2).

[0005] Many peptides and proteins are degraded when administered orally and are often administered as injections. Various additives are added to injections to stabilize the active ingredients, but the types of additives used vary significantly depending on the active ingredient (Patent Documents 3 to 5). Various additives that stabilize HMGB1 fragment peptides also needed to be investigated. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2012 / 147470 International Publication Pamphlet [Patent Document 2] WO2014 / 065347 International Publication Pamphlet [Patent Document 3] WO2008 / 102849 International Publication Pamphlet [Patent Document 4] WO2013 / 154045 International Publication Pamphlet [Patent Document 5] JP 2000-169372 A Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present application is to provide a pharmaceutical composition that stabilizes an HMGB1 fragment peptide. [Means for solving the problem]

[0008] The present inventors investigated the physical properties of pharmaceutical composition additives and formulations that stabilize HMGB1 fragment peptides, and found that HMGB1 fragment peptides can be stabilized by a pharmaceutical composition containing an HMGB1 fragment peptide and having a pH of 3 to 4.5 when dissolved in purified water. Specifically, the peptides can be stabilized by including an HMGB1 fragment peptide, an organic acid buffer, a sugar and / or sugar alcohol, and an alkali metal hydroxide.

[0009] That is, the present invention (1) A pharmaceutical composition containing a substance described in any one of the following (a) to (c), wherein the pH of the pharmaceutical composition when dissolved in distilled water for injection is 3.0 to 4.5: (a) an HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or an acid addition salt thereof; (b) a peptide or an acid addition salt thereof, which comprises an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1 and has the activity of stimulating cell migration; and (c) a peptide or an acid addition salt thereof, which comprises an amino acid sequence having about 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and has the activity of stimulating cell migration; (2) The pharmaceutical composition according to (1) above, which contains one or more selected from the group consisting of organic acids, alkali metal salts of organic acids, and alkaline earth metal salts of organic acids. (3) The pharmaceutical composition according to (2) above, which contains an organic acid, and the organic acid is one or more selected from the group consisting of citric acid, acetic acid, and phosphoric acid. (4) The pharmaceutical composition according to (2) above, which contains an organic acid and / or an alkali metal salt of an organic acid, and the organic acid and the alkali metal salt of an organic acid are citric acid and sodium citrate, respectively. (5) The pharmaceutical composition according to any one of (1) to (4) above, which contains a sugar and / or a sugar alcohol. (6) The pharmaceutical composition according to (5) above, which contains a sugar, and the sugar is one or more selected from the group consisting of monosaccharides, disaccharides, and polysaccharides. (7) The pharmaceutical composition according to (5) above, wherein the sugar and / or sugar alcohol is one or more selected from the group consisting of glucose, fructose, sucrose, mannitol, and trehalose. (8) The pharmaceutical composition according to (7) above, wherein the sugar and / or sugar alcohol is sucrose. (9) The pharmaceutical composition according to any one of (5) to (8) above, wherein the amount of sugar and / or sugar alcohol in the pharmaceutical composition is 10 to 300 times the molar amount of the substance according to any one of (a) to (c). (10) The pharmaceutical composition according to (9), wherein the amount of sugar and / or sugar alcohol in the pharmaceutical composition is 50 to 300 times the molar amount of the substance according to any one of (a) to (c). (11) The pharmaceutical composition according to any one of (1) to (10) above, wherein the pH is adjusted using a hydroxide of one or more metals selected from the group consisting of alkali metals, alkaline earth metals, and magnesium. (12) The pharmaceutical composition according to (11) above, wherein the hydroxide is one or more selected from the group consisting of potassium hydroxide, calcium hydroxide, sodium hydroxide, and magnesium hydroxide. (13) The pharmaceutical composition according to (11), wherein the hydroxide is sodium hydroxide. (14) A pharmaceutical composition according to any one of (1) to (13) above, which contains the acid addition salt according to any one of (a) to (c), wherein the acid addition salt is a trifluoroacetic acid salt. (15) The pharmaceutical composition according to any one of (1) to (14) above, which is a lyophilized product. (16) The pharmaceutical composition according to (15), wherein the amount of the substance according to any one of (a) to (c) per 1 g of the lyophilized product is 6.0 to 50.0 mg. (17) An injectable preparation prepared by dissolving the pharmaceutical composition according to any one of (1) to (16) above in distilled water for injection. (18) A method for producing a pharmaceutical composition, comprising the steps of: 1) dissolving an organic acid hydrate, an organic acid metal salt hydrate, and a sugar and / or sugar alcohol in distilled water for injection, and adjusting the pH to 3.0 to 4.0; 2) A step of cooling the liquid produced in step 1) to 18°C ​​or less; 3) A step of dissolving the substance described in any one of (a) to (c) of (1) above in the liquid obtained in step 2); 4) adjusting the pH of the liquid obtained in step 3) to 2.5 to 4.0 with a hydroxide of one or more metals selected from the group consisting of alkali metals, alkaline earth metals, and magnesium; and 5) freeze-drying the liquid obtained in step 4); (19) A method for producing a pharmaceutical composition, comprising the following steps: 1) dissolving citric acid hydrate, sodium citrate hydrate, and sucrose in distilled water for injection and adjusting the pH to 3.0 to 4.0; 2) A step of cooling the liquid produced in step 1) to 18°C ​​or less; 3) A step of dissolving the substance described in any one of (a) to (c) of (1) above in the liquid obtained in step 2); 4) adjusting the pH of the solution obtained in step 3) to 2.9 to 3.5 with sodium hydroxide; and 5) freeze-drying the liquid obtained in step 4); (20) A pharmaceutical composition produced by the production method according to (18) or (19) above. Regarding. [Effects of the Invention]

[0010] The stability of pharmaceutical compositions containing the HMGB1 fragment peptides of the present invention can be increased by adjusting pH and additives. BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The active ingredient of the formulation of the present invention is defined herein as: (a) an HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or an acid addition salt thereof; (b) a peptide or an acid addition salt thereof, which comprises the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, inserted, or added, and which has the activity of stimulating cell migration; or (c) A peptide or an acid addition salt thereof, which comprises an amino acid sequence having about 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and has the activity of stimulating cell migration.

[0012] In this application, the term "pharmaceutical composition" is used interchangeably with "medicament," "drug," or "pharmaceutical composition."

[0013] In the present application, an HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 refers to a peptide consisting of a portion of the HMGB1 protein, and comprising the amino acid sequence set forth in SEQ ID NO: 1. Such peptides can be obtained as recombinants by incorporating DNA encoding the peptide into an appropriate expression system, or they can be artificially synthesized.

[0014] In the present application, examples of HMGB1 proteins include, but are not limited to, proteins comprising the amino acid sequence set forth in SEQ ID NO: 2 and proteins encoded by DNA comprising the base sequence set forth in SEQ ID NO: 3.

[0015] Examples of HMGB1 fragment peptides comprising the amino acid sequence of SEQ ID NO: 1 in the present application include, but are not limited to, the following: 1) An HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and having the activity of stimulating cell migration; 2) an HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and having the activity of stimulating the migration of mesenchymal stem cells; 3) An HMGB1 fragment peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0016] In the present application, cells whose migration is stimulated by HMGB1 fragment peptides include, but are not limited to, bone marrow cells or bone marrow-derived cells (eg, bone marrow stem cells or bone marrow-derived stem cells).

[0017] In this application, "bone marrow cells" refers to cells present in the bone marrow, while "bone marrow-derived cells" refers to "bone marrow cells" mobilized from the bone marrow to the outside of the bone marrow. Furthermore, "bone marrow cells" may include undifferentiated cells such as stem cells and progenitor cells present in the bone marrow.

[0018] In the present application, cells whose migration is stimulated by HMGB1 fragment peptides include, but are not limited to, mesenchymal stem cells. "Mesenchymal stem cells" are cells collected from bone marrow or other tissues (blood, e.g., umbilical cord blood, skin, fat, dental pulp, etc.), can be cultured and proliferated as adherent cells on a culture dish (made of plastic or glass), and have the potential to differentiate into mesenchymal tissues such as bone, cartilage, fat, and muscle. In one embodiment, mesenchymal stem cells also have the potential to differentiate into epithelial tissue and neural tissue. Mesenchymal stem cells in the present application may exist as a heterogeneous cell population containing not only stem cells in the strict sense but also progenitor cells, and may, under culture conditions, contain differentiated cells in addition to stem cells and / or progenitor cells in the strict sense. In one embodiment, mesenchymal stem cells may be composed solely of stem cells in the strict sense, or may be a cell population consisting of multiple types of progenitor cells.

[0019] In the present application, progenitor cells are defined as cells that have the ability to differentiate unidirectionally into specific tissue cells other than blood cells, and include cells that have the ability to differentiate into mesenchymal tissue, epithelial tissue, neural tissue, parenchymal organs, and vascular endothelium.

[0020] In the present application, cells whose migration is stimulated by HMGB1 fragment peptides include, but are not limited to, bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells. "Bone marrow mesenchymal stem cells" are cells present in the bone marrow that are collected from the bone marrow and can be cultured and proliferated as adherent cells on a culture dish (made of plastic or glass). They are characterized by their ability to differentiate into mesenchymal tissues such as bone, cartilage, fat, and muscle, as well as neural tissue and epithelial tissue. In the present application, the term "bone marrow mesenchymal stem cells" is used interchangeably with "bone marrow mesenchymal stromal cells," "bone marrow pluripotent stem cells," and "bone marrow pluripotent stromal cells."

[0021] Furthermore, "bone marrow-derived mesenchymal stem cells" refer to bone marrow mesenchymal stem cells that have been mobilized from the bone marrow to the outside of the bone marrow, and are cells that can be obtained by peripheral blood sampling or by collection from mesenchymal tissues such as fat, epithelial tissues such as skin, or neural tissues such as the brain. In this application, the term "bone marrow-derived mesenchymal stem cells" is used interchangeably with "bone marrow-derived mesenchymal stromal cells," "bone marrow-derived pluripotent stem cells," and "bone marrow-derived pluripotent stromal cells."

[0022] Furthermore, bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells have the ability to differentiate into epithelial tissues, such as keratinocytes that make up the skin, and nervous system tissues that make up the brain, when administered to an injured area of ​​the body, either directly after collection or after first attaching the cells to a culture dish.

[0023] It is preferable that bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells have the ability to differentiate into osteoblasts (which can be identified by, for example, the formation of calcium deposits when differentiation is induced), chondrocytes (which can be identified by positive Alcian blue staining, positive Safranin-O staining, etc.), adipocytes (which can be identified by positive Sudan III staining, etc.), as well as mesenchymal cells such as fibroblasts, smooth muscle cells, skeletal muscle cells, stromal cells, and tendon cells, nerve cells, pigment cells, epidermal cells, hair follicle cells (which express the cytokeratin family, hair keratin family, etc.), epithelial cells (for example, epidermal keratinocytes and intestinal epithelial cells express the cytokeratin family, etc.), endothelial cells, and even parenchymal organ cells such as the liver, kidney, and pancreas, although the differentiated cells are not limited to the above cells.

[0024] Examples of markers for human mesenchymal stem cells include all or some of the following, but are not limited to: PDGFRα positive, PDGFRβ positive, Lin negative, CD45 negative, CD44 positive, CD90 positive, CD29 positive, Flk-1 negative, CD105 positive, CD73 positive, CD90 positive, CD71 positive, Stro-1 positive, CD106 positive, CD166 positive, CD31 negative, CD271 positive, and CD11b negative.

[0025] Examples of mouse mesenchymal stem cell markers used in mice include, but are not limited to, all or some of the following: CD44 positive, PDGFRα positive, PDGFRβ positive, CD45 negative, Lin negative, Sca-1 positive, c-kit negative, CD90 positive, CD105 positive, CD29 positive, Flk-1 negative, CD271 positive, and CD11b negative.

[0026] Examples of markers for rat mesenchymal stem cells used in rats include, but are not limited to, all or some of the following: PDGFRα positive, CD44 positive, CD54 positive, CD73 positive, CD90 positive, CD105 positive, CD29 positive, CD271 positive, CD31 negative, and CD45 negative.

[0027] In the present application, cells whose migration is stimulated by HMGB1 fragment peptides also include, but are not limited to, PDGFRα-positive cells. Examples of PDGFRα-positive cells whose migration is stimulated by HMGB1 fragment peptides include, but are not limited to, PDGFRα-positive mesenchymal stem cells, PDGFRα-positive bone marrow-derived mesenchymal stem cells, and PDGFRα-positive bone marrow-derived cells obtained as adherent cells by culturing mononuclear cells fractionated from blood obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection. Examples of PDGFRα-positive mesenchymal stem cells include cells that are positive for PDGFRα and CD44, cells that are positive for PDGFRα and CD90, cells that are positive for PDGFRα and CD105, and cells that are positive for PDGFRα and CD29. In one embodiment, the PDGFRα-positive mesenchymal stem cells may be CD44-negative cells.

[0028] In the formulations of the present invention, instead of or in addition to the HMGB1 fragment peptide containing the amino acid sequence of SEQ ID NO: 1, a peptide containing an amino acid sequence in which one or more amino acid residues have been modified (substituted, deleted, inserted, or added) in the amino acid sequence of SEQ ID NO: 1 and which has the activity of stimulating cell migration can be used. Examples of such peptides include, but are not limited to, the following: i) A peptide comprising the amino acid sequence of SEQ ID NO: 1 in which one or more (e.g., 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, deleted, inserted, or added, and which has the activity of stimulating cell migration; ii) A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one or more (e.g., 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, deleted, inserted, or added, and which has the activity of stimulating cell migration; iii) a peptide comprising an amino acid sequence having about 80% or more, for example, about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having the activity of stimulating cell migration; iv) A peptide consisting of an amino acid sequence having about 80% or more, for example about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having the activity of stimulating cell migration. Furthermore, examples of cells whose migration is stimulated by these peptides include, but are not limited to, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, PDGFRα-positive cells, PDGFRα-positive mesenchymal stem cells, PDGFRα-positive bone marrow-derived mesenchymal stem cells, and PDGFRα-positive bone marrow-derived cells obtained as adherent cells by cell culture of mononuclear cells in blood obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection.

[0029] The HMGB1 fragment peptide in the formulation of the present invention may be in the form of a salt, but is preferably an acid addition salt. Acid addition salts include salts formed with inorganic acids such as sulfuric acid and hydrochloric acid, or organic acids such as trifluoroacetic acid and p-toluenesulfonic acid. A preferred acid addition salt is trifluoroacetic acid salt.

[0030] An effective amount of the formulation of the present invention containing the peptide of the present application is administered to a subject for the treatment or prevention of the diseases and conditions described herein.

[0031] In particular, the HMGB1 fragment peptide, which is the active ingredient of the formulation of the present invention, is effective against epidermolysis bullosa, cerebral infarction, myocardial infarction, heart disease, old myocardial infarction, spinal cord injury, fibrotic disease, inflammatory bowel disease, psoriasis, and the like.

[0032] As used herein, an effective amount refers to an amount sufficient to treat or prevent a disease or condition as described herein. As used herein, treating includes, but is not limited to, reducing, delaying, preventing, ameliorating, remission, curing, and complete recovery. As used herein, preventing includes, but is not limited to, reducing, delaying, preventing, and the like.

[0033] The subject in the present application is not particularly limited, and includes mammals, birds, fish, etc. Mammals include humans and non-human animals, such as, but not limited to, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, whales, etc. In the present application, the term "subject" is used interchangeably with "patient," "individual," and "animal."

[0034] There are no limitations on the site of administration of the peptides, etc. of the present application, and the peptides, etc. of the present application can exert their effects regardless of the site of administration, such as a site having a structural or functional abnormality in tissue or its vicinity, a site different from these (sites other than these), a site distant from the site having a structural or functional abnormality in tissue, a site distal to the site having a structural or functional abnormality in tissue, or a site distal to and ectopic to the site having a structural or functional abnormality in tissue.

[0035] The formulations of the present invention can be administered orally or parenterally, and parenteral administration includes, but is not limited to, intravascular administration (intra-arterial administration, intravenous administration, etc.), intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, nasal administration, pulmonary administration, transdermal administration, etc. Furthermore, the peptides and the like of the present application can be administered systemically or locally (e.g., subcutaneously, intradermally, to the skin surface, the eyeball or palpebral conjunctiva, the nasal mucosa, the oral and gastrointestinal mucosa, the vaginal or intrauterine mucosa, or an injured site) by injection, for example, intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0036] The formulation of the present invention can be administered by intravenous drip infusion. When administering intravenous drip infusion, the lyophilized formulation in the vial can be redissolved in physiological saline, and the resulting solution can be added to a physiological saline bag as needed to prepare an administration solution. The administration solution has a concentration of 0.1 to 2.5 mg / mL and can be administered intravenously.

[0037] In addition, instead of the peptides of the present application, cells that secrete the peptides of the present application, vectors for gene therapy into which DNA encoding the peptides has been inserted, and pharmaceutical compositions containing these can also be used.

[0038] In addition, the administration method can be selected appropriately depending on the patient's age and symptoms. When administering the peptide of the present application, the dosage can be selected, for example, within the range of 0.0000001 mg to 1000 mg per kg of body weight per administration. When administering cells that secrete the peptide of the present application or a gene therapy vector into which DNA encoding the peptide has been inserted, the amount of the peptide can also be administered so that it falls within the above range. However, the pharmaceutical composition of the present application is not limited to these dosages.

[0039] The content of the HMGB1 fragment peptide or its acid addition salt in the formulation of the present invention is not particularly limited, but may be any content (potency) that allows the HMGB1 fragment peptide or its acid addition salt to be stabilized in the formulation. The content of the HMGB1 fragment peptide or its acid addition salt in the formulation of the present invention is generally 1.0 to 50.0% by weight, preferably 3.0 to 40.0% by weight, more preferably 6.0 to 30.0% by weight, and particularly preferably 6.5 to 28.3% by weight (64.6 to 283.3 mg / g), based on the total weight of the formulation, particularly in the case of a lyophilized formulation. If the amount is less than this range, the formulation may need to be administered in excess, while if the amount is greater, it may not be possible to lyophilize the formulation.

[0040] When the preparation of the present invention is dissolved in distilled water for injection, the pH is 3.0 to 4.5, preferably 3.0 to 4.3, more preferably 3.0 to 4.0, and particularly preferably 3.0 to 3.6. Although the pH is measured using a pH meter or the like, there may be an error of about ±0.05 during measurement.

[0041] The preparation of the present invention may contain various additives.

[0042] The formulation of the present invention may contain a buffering agent, and buffering agents listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia Non-Japanese Pharmaceutical Standards, the Pharmaceutical Additives Standards, or the Official Specification of Food Additives can be used. A buffering agent is a compound added to a solution to maintain the pH of the solution at a specific value even when other acids or bases are added to the solution to a certain extent. In other words, a buffering agent suppresses rapid changes in pH. Examples of buffering agents include amino acids or their salts such as hydrochlorides, sodium salts, and potassium salts; ampholytes or their salts such as sodium salts; salts of weak acids such as sodium salts and potassium salts; combinations of salts of weak acids with strong acids such as weak acids, hydrochloric acid, and sulfuric acid; and trishydroxymethylaminomethane. Examples of amino acids or salts thereof, such as hydrochlorides, sodium salts, and potassium salts, include amino acids such as glycine, alanine, arginine, glutamine, cysteine, serine, threonine, valine, histidine, phenylalanine, methionine, aspartic acid, glutamic acid, ε-aminocaproic acid, lysine, and leucine, and salts thereof, such as hydrochlorides, sodium salts, and potassium salts. Examples of ampholytes or salts thereof, such as sodium salts, include ampholytes such as nicotinamide and aminobenzoic acid, and salts thereof, such as sodium salts. Examples of sodium salts and potassium salts of weak acids include sodium salts and potassium salts of weak acids such as citric acid, phosphoric acid, lactic acid, acetic acid, boric acid, propionic acid, butyric acid, valeric acid, glycolic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, malic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phthalic acid, and tartaric acid. Examples of combinations of weak acids or their salts with strong acids such as hydrochloric acid and sulfuric acid include combinations of salts of weak acids such as citric acid, phosphoric acid, lactic acid, acetic acid, boric acid, propionic acid, butyric acid, valeric acid, glycolic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, malic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phthalic acid, and tartaric acid with strong acids such as weak acids, hydrochloric acid, and sulfuric acid.Preferred are citric acid, sodium citrate, citric acid hydrate, sodium citrate hydrate, sodium dihydrogen citrate, disodium citrate, calcium citrate, acetic acid, sodium acetate, sodium acetate hydrate, phosphoric acid, calcium monohydrogen phosphate, dipotassium phosphate, monopotassium phosphate, disodium phosphate, and monosodium phosphate. More preferred are citric acid, sodium citrate, citric acid hydrate, and sodium citrate hydrate. Particularly preferred are citric acid hydrate and sodium citrate hydrate. Two or more of these buffering agents may be used.

[0043] The content of the buffering agent in the formulation of the present invention is not particularly limited, as long as it is sufficient to stabilize the HMGB1 fragment peptide or its acid addition salt in the formulation. The content of the buffering agent in the formulation of the present invention is generally 0.1 to 10 wt.%, preferably 0.25 to 7.5 wt.%, more preferably 0.5 to 5.0 wt.%, and particularly preferably 0.9 to 3.9 wt.%, of the total weight of the formulation, particularly in the case of a lyophilized formulation. If the content is less or more than this amount, the active ingredient, the HMGB1 fragment peptide, may not be stabilized.

[0044] The formulations of the present invention may contain additives (hereinafter sometimes referred to as "stabilizers" herein) that stabilize the active ingredient, HMGB1 fragment peptide or its acid addition salt, and additives listed in the Japanese Pharmacopoeia, Japanese Pharmacopoeia Non-Japanese Pharmaceutical Standards, Pharmaceutical Additives Standards, Food Additives Official Specification, etc. Examples of additives that stabilize the active ingredient include sugars, sugar alcohols, amino acids, alkali metal chlorides, alkaline earth metal chlorides, etc. Examples of sugars include monosaccharides, disaccharides, and polysaccharides, and more specifically, sucrose (white sugar, refined white sugar), trehalose, glucose, fructose, raffinose, maltose, maltotriose sucrose, and the like. Examples of sugar alcohols include mannitol, sorbitol, erythritol, xylitol, powdered maltose syrup, and maltitol. Examples of amino acids include methionine, histidine, and arginine. Examples of the alkali metal chloride include sodium chloride, lithium chloride, and potassium chloride. The alkaline earth metal chlorides include beryllium chloride and calcium chloride. Preferred are sugars and sugar alcohols, more preferred are monosaccharides, disaccharides, polysaccharides, etc. Also, more preferred are glucose, fructose, sucrose, mannitol, and trehalose, and particularly preferred is sucrose (white sugar, refined white sugar).

[0045] The amount of stabilizer contained in the formulation of the present invention is not particularly limited, as long as it is sufficient to stabilize the HMGB1 fragment peptide or its acid addition salt in the formulation. The amount of stabilizer contained in the formulation of the present invention is generally 30.0 to 99.0 wt.%, preferably 35.0 to 97.5 wt.%, more preferably 40.0 to 95.0 wt.%, and particularly preferably 67.8 to 92.7 wt.% of the total weight of the formulation, particularly in the case of a lyophilized formulation. If the amount is greater or less than this range, the active ingredient, the HMGB1 fragment peptide, may not be stabilized.

[0046] The amount of stabilizer contained in the formulation of the present invention, particularly the amount of sugar and / or sugar alcohol, is 10 to 300 times the molar amount of the HMGB1 fragment peptide (active ingredient), preferably 25 to 300 times the molar amount, and more preferably 50 to 300 times the molar amount. If the amount is more or less than this range, it may not be possible to produce the lyophilized formulation.

[0047] The formulations of the present invention are produced by freeze-drying a liquid containing the active ingredient, an HMGB1 fragment peptide or its acid addition salt, and additives (hereinafter sometimes referred to as the "preparation liquid"). The stability of the HMGB1 fragment peptide in the freeze-dried formulation varies depending on the pH of the preparation liquid. To stabilize the HMGB1 fragment peptide, the pH of the preparation liquid should be 2.5 to 5.0, preferably 2.5 to 4.5, more preferably 2.5 to 4.0, and particularly preferably 2.9 to 3.5. The concentration of the active ingredient in the preparation liquid may be any concentration that allows for freeze-drying, but is typically 1.0 to 100.0 mg / g, preferably 2.5 to 80.0 mg / g, more preferably 5.0 to 60.0 mg / g, and particularly preferably 7.5 to 50.0 mg / g.

[0048] As described above, the pH of the preparation of the present invention must be adjusted, and this can be achieved by using additives capable of adjusting the pH of the preparation. For example, the pH can be adjusted by using alkali metal hydroxides, alkaline earth metal hydroxides, or magnesium hydroxide. Preferably, potassium hydroxide, calcium hydroxide, sodium hydroxide, or magnesium hydroxide can be used, and more preferably, sodium hydroxide can be used.

[0049] The amount of the additive used to adjust the pH of the preparation of the present invention may be any amount that can adjust the optimum pH of the preparation.

[0050] The formulation of the present invention may contain other additives that are generally used for injections, or additives listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia Standards for Pharmaceuticals Other Than the Japanese Pharmacopoeia, the Pharmaceutical Additives Standards, the Official Specification of Food Additives, etc. Examples of additives include suspending agents, surfactants, hydrophilic polymers, emulsifiers, preservatives, soothing agents, isotonic agents, solvents, etc.

[0051] The formulation of the present invention may contain a suspending agent. Any additive capable of suspending an active ingredient in a liquid may be used as the suspending agent. Examples of suspending agents include surfactants, hydrophilic polymers, gum arabic, and tragacanth. Examples of surfactants include stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionate, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, poloxamer, aluminum monostearate, and polysorbate 80. Examples of hydrophilic polymers include polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

[0052] The formulation of the present invention may contain an emulsifier. Any emulsifier capable of emulsifying an active ingredient in a liquid may be used, and emulsifiers listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia's Non-Drug Standards, the Pharmaceutical Additives Standards, or the Official Specification of Food Additives may be used. Examples of emulsifiers include phospholipids, surfactants, colloidal clay, metal hydroxides, natural gums, and gelatin. Examples of phospholipids include natural phospholipids and synthetic phospholipids (anionic phospholipids). Examples of natural phospholipids include egg yolk lecithin, soybean lecithin, and soybean lecithin. Synthetic phospholipids (anionic phospholipids) include dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, oleoyl palmitoyl phosphatidylglycerol, dioctanoyl phosphatidic acid, didecanoyl phosphatidic acid, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, diheptadecanoyl phosphatidic acid, Examples of the surfactant include phatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, arachidonylstearoylphosphatidic acid, dipalmitoylphosphatidylserine, dioleoylphosphatidylserine, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, distearoylphosphatidylinositol, dioleoylphosphatidylinositol, dimyristoylphosphatidylserine, distearoylphosphatidylserine, etc. Examples of the surfactant include nonionic surfactants (nonionic surfactants), anionic surfactants, cationic surfactants, etc.Examples of nonionic surfactants include polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene alkyl ethers, polyoxyethylene alkylaryl ethers, hydrogenated castor oil polyoxyethylene derivatives, polyoxyethylene sorbitan derivatives, polyoxyethylene sorbitol derivatives, polyoxyethylene alkyl ether sulfates, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid, sucrose fatty acid esters, glycerin fatty acid esters, sorbitan fatty acid esters, and propylene glycol fatty acid esters. Examples of anionic surfactants include sodium oleate, sodium stearate, calcium stearate, and sodium lauryl sulfate. Examples of cationic surfactants include benzalkonium chloride. Examples of colloidal clays include bentonite and Veegum. Examples of metal hydroxides include magnesium hydroxide, aluminum hydroxide, potassium hydroxide, and sodium hydroxide. Examples of natural gums include gum arabic, gum tragacanth, and tragacanth.

[0053] The formulation of the present invention may contain a preservative. Examples of preservatives that can be used include those listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia's Non-Japanese Pharmaceutical Standards, the Pharmaceutical Additives Standards, and the Official Specification of Food Additives. Examples of preservatives include parabens, benzoic acids, sorbic acid, potassium sorbate, sodium dehydroacetate, benzyl alcohol, chlorobutanol, cresol, sodium dehydroacetate, and sodium edetate. Examples of parabens include methylparaben, ethylparaben, propylparaben, and butylparaben. Examples of benzoic acids include benzoic acid, sodium benzoate, benzyl benzoate, and parahydroxybenzoic acid esters (isobutylparahydroxybenzoate, isopropylparahydroxybenzoate, ethylparahydroxybenzoate, sodium ethylparahydroxybenzoate, butylparahydroxybenzoate, propylparahydroxybenzoate, sodium propylparahydroxybenzoate, methylparahydroxybenzoate, and sodium methylparahydroxybenzoate).

[0054] The preparation of the present invention may contain a soothing agent. Examples of soothing agents that can be used include those listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia's Non-Japanese Pharmaceutical Standards, the Pharmaceutical Additives Standards, and the Official Specification of Food Additives. Examples of soothing agents include ethyl aminobenzoate, inositol, liquid phenol, meprylcaine hydrochloride, lidocaine hydrochloride, creatinine, chlorobutanol, sodium bicarbonate, clove oil, glucose, procaine hydrochloride, propylene glycol, benzyl alcohol, mepivacaine hydrochloride, lidocaine, magnesium sulfate hydrate, and benzalkonium chloride.

[0055] The formulation of the present invention may contain a preservative. Examples of preservatives that can be used include those listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia's Non-Japanese Pharmaceutical Standards, the Pharmaceutical Additives Standards, and the Official Specification of Food Additives. Examples of preservatives include parahydroxybenzoic acid esters (parabens), alcohol, benzalkonium chloride, benzoic acid, salicylic acid, sorbic acid, chlorhexidine, and hydrogen peroxide. Examples of parahydroxybenzoic acid esters (parabens) include methyl parahydroxybenzoate (methylparaben) and propyl parahydroxybenzoate (propylparaben). Examples of alcohols include benzyl alcohol, phenethyl alcohol, chlorobutanol, and phenol.

[0056] The formulation of the present invention may contain an isotonicity agent. Examples of isotonicity agents that can be used include those listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia's Non-Japanese Pharmaceutical Standards, the Pharmaceutical Additives Standards, or the Official Specification of Food Additives. Examples of isotonicity agents include ionic isotonicity agents and nonionic isotonicity agents. Examples of ionic isotonicity agents include inorganic salts and organic bases. Examples of inorganic salts include disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium bisulfite, sodium sulfite, sodium thiosulfate, magnesium sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, boric acid, and borax. Examples of organic bases include potassium acetate, sodium acetate, sodium bicarbonate, and sodium carbonate. Nonionic tonicity agents include polyhydric alcohols having two or more alcoholic hydroxyl groups in the molecule, amino acids, and sugars. Examples of polyhydric alcohols having two or more alcoholic hydroxyl groups in the molecule include glycerin, propylene glycol, and polyethylene glycol. Examples of amino acids include glycine and alanine. Examples of sugars include glucose, fructose, trehalose, sucrose, xylitol, sorbitol, and mannitol.

[0057] The formulation of the present invention may contain a solvent. Alternatively, the formulation of the present invention may be dissolved or suspended in a solvent. Solvents listed in the Japanese Pharmacopoeia, the Japanese Pharmacopoeia's Non-Japanese Pharmaceutical Standards, the Pharmaceutical Additives Standards, or the Official Specification of Food Additives can be used. Examples of solvents include aqueous solvents, alcohol, propylene glycol, macrogol, sesame oil, and corn oil. Examples of aqueous solvents include distilled water, physiological saline, Ringer's solution, water for injection, and distilled water for injection.

[0058] The dosage form of the preparation of the present invention may be any dosage form as described in each monograph of the Japanese Pharmacopoeia, but is preferably an injection, which may be administered directly into a blood vessel or tissue, or by intravenous drip.

[0059] The method for producing the formulation of the present invention may be any method for producing a pharmaceutical composition that can stabilize the active ingredient, an HMGB1 fragment peptide or an acid addition salt thereof, such as a method for producing a pharmaceutical composition comprising the following steps: 1) a step of dissolving an additive containing an organic acid hydrate, an alkali metal salt hydrate of an organic acid, and a sugar and / or a sugar alcohol in distilled water for injection, and adjusting the pH to 3.0 to 4.0; 2) A step of cooling the liquid produced in step 1) to 18°C ​​or less; 3) dissolving the HMGB1 fragment peptide or its acid addition salt in the liquid obtained in step 2); 4) adjusting the pH of the liquid obtained in step 3) to 2.5 to 4.0 with a hydroxide of one or more metals selected from the group consisting of alkali metals, alkaline earth metals, and magnesium; and 5) A step of freeze-drying the liquid obtained in step 4). Note that a step of adjusting the concentration with distilled water for injection may be included after step 4) and before step 5).

[0060] Another embodiment of the method for producing the formulation of the present invention may be a method for producing a pharmaceutical composition that can stabilize the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, including, for example, the following steps: 1) dissolving additives including citric acid hydrate, sodium citrate hydrate, and sucrose in distilled water for injection and adjusting the pH to 3.0 to 4.0; 2) A step of cooling the liquid produced in step 1) to 18°C ​​or less; 3) dissolving the HMGB1 fragment peptide or a trifluoroacetate thereof in the liquid obtained in step 2); 4) adjusting the pH of the solution obtained in step 3) to 2.9 to 3.5 with sodium hydroxide; and 5) A step of freeze-drying the liquid obtained in step 4). Note that a step of adjusting the concentration with distilled water for injection may be included after step 4) and before step 5).

[0061] The pharmaceutical composition of the present invention contains an HMGB1 fragment peptide or an acid addition salt thereof as an active ingredient, an organic acid, a metal salt of an organic acid, and a sugar and / or sugar alcohol.

[0062] Another embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition containing the active ingredient HMGB1 fragment peptide or its acid addition salt, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of monosaccharides, disaccharides, and polysaccharides.

[0063] Another embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition containing the active ingredient HMGB1 fragment peptide or its acid addition salt, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of glucose, fructose, sucrose, mannitol, and trehalose.

[0064] Another embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, an organic acid, a metal salt of an organic acid, and sucrose.

[0065] Another embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, citric acid, a metal salt of an organic acid, and sucrose.

[0066] Another embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, an organic acid, sodium citrate, and sucrose.

[0067] Another embodiment of the pharmaceutical composition of the present invention is a pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, citric acid, sodium citrate, and sucrose.

[0068] The pharmaceutical composition of the formulation of the present invention contains the active ingredients HMGB1 fragment peptide or its acid addition salt, an organic acid, a metal salt of an organic acid, and a sugar and / or sugar alcohol, and has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0069] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition that contains an HMGB1 fragment peptide or its acid addition salt as an active ingredient, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of monosaccharides, disaccharides, and polysaccharides, and that has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0070] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition that contains an HMGB1 fragment peptide or its acid addition salt as an active ingredient, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of glucose, fructose, sucrose, mannitol, and trehalose, and that has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0071] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition that contains the active ingredient HMGB1 fragment peptide or its trifluoroacetate salt, an organic acid, a metal salt of an organic acid, and sucrose, and has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0072] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition that contains an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, citric acid, a metal salt of an organic acid, and sucrose, and has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0073] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition that contains an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, an organic acid, sodium citrate, and sucrose, and has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0074] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition that contains an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, citric acid, sodium citrate, and sucrose, and has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0075] The formulation of the present invention can be produced by freeze-drying the preparation. Freeze-drying is a drying method in which an aqueous solution or dispersion containing an active ingredient is frozen and water is removed by sublimation. That is, the active ingredient is frozen in its aqueous solution or dispersion state, and then dried by sublimation from the frozen surface under high vacuum.

[0076] The pharmaceutical composition of the present invention is a lyophilized pharmaceutical composition containing the active ingredient HMGB1 fragment peptide or its acid addition salt, an organic acid, a metal salt of an organic acid, and a sugar and / or sugar alcohol.

[0077] Another embodiment of the pharmaceutical composition of the present invention is a freeze-dried pharmaceutical composition containing an HMGB1 fragment peptide or its acid addition salt as an active ingredient, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of monosaccharides, disaccharides, and polysaccharides.

[0078] Another embodiment of the pharmaceutical composition of the present invention is a freeze-dried pharmaceutical composition containing an HMGB1 fragment peptide or its acid addition salt as an active ingredient, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of glucose, fructose, sucrose, mannitol, and trehalose.

[0079] Another embodiment of the pharmaceutical composition of the present invention is a lyophilized pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, an organic acid, a metal salt of an organic acid, and sucrose.

[0080] Another embodiment of the pharmaceutical composition of the present invention is a lyophilized pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, citric acid, a metal salt of an organic acid, and sucrose.

[0081] Another embodiment of the pharmaceutical composition of the present invention is a lyophilized pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, an organic acid, sodium citrate, and sucrose.

[0082] Another embodiment of the pharmaceutical composition of the present invention is a lyophilized pharmaceutical composition containing an HMGB1 fragment peptide or its trifluoroacetate salt as an active ingredient, citric acid, sodium citrate, and sucrose.

[0083] The pharmaceutical composition of the formulation of the present invention is a freeze-dried pharmaceutical composition containing the active ingredients HMGB1 fragment peptide or its acid addition salt, an organic acid, a metal salt of an organic acid, and a sugar and / or sugar alcohol, and has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0084] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a freeze-dried pharmaceutical composition containing an HMGB1 fragment peptide or its acid addition salt as an active ingredient, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of monosaccharides, disaccharides, and polysaccharides, and the pharmaceutical composition has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0085] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a freeze-dried pharmaceutical composition containing an HMGB1 fragment peptide or its acid addition salt as an active ingredient, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of glucose, fructose, sucrose, mannitol, and trehalose, and which has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0086] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a freeze-dried pharmaceutical composition containing the active ingredient HMGB1 fragment peptide or its trifluoroacetate salt, an organic acid, a metal salt of an organic acid, and sucrose, and the pharmaceutical composition has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0087] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a freeze-dried pharmaceutical composition containing the active ingredient HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, a metal salt of an organic acid, and sucrose, and the pharmaceutical composition has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0088] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a freeze-dried pharmaceutical composition containing the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, an organic acid, sodium citrate, and sucrose, and the pharmaceutical composition has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0089] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a freeze-dried pharmaceutical composition containing the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, sodium citrate, and sucrose, and the pharmaceutical composition has a pH of 3.0 to 4.5 when dissolved in distilled water for injection.

[0090] The pharmaceutical composition of the formulation of the present invention is prepared by dissolving the active ingredients, HMGB1 fragment peptide or its acid addition salt, organic acid hydrate, organic acid metal salt hydrate, and sugar and / or sugar alcohol, in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with hydroxide.

[0091] The pharmaceutical composition of the formulation of the present invention is prepared by dissolving the active ingredient, an HMGB1 fragment peptide or its acid addition salt, a hydrate of an organic acid, a hydrate of a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from monosaccharides, disaccharides, and polysaccharides, in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with a hydroxide.

[0092] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its acid addition salt, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of glucose, fructose, sucrose, mannitol, and trehalose, in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with a hydroxide.

[0093] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, an organic acid, a metal salt of an organic acid, and sucrose in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with hydroxide.

[0094] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, a metal salt of an organic acid, and sucrose in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with hydroxide.

[0095] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, an organic acid, sodium citrate, and sucrose in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with hydroxide.

[0096] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, sodium citrate, and sucrose, in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with hydroxide.

[0097] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, sodium citrate, and sucrose, in distilled water for injection, and adjusting the pH to 2.9 to 3.5 with sodium hydroxide.

[0098] The pharmaceutical composition of the formulation of the present invention is produced by dissolving the active ingredients, HMGB1 fragment peptide or its acid addition salt, organic acid hydrate, organic acid metal salt hydrate, and sugar and / or sugar alcohol, in distilled water for injection, adjusting the pH to 2.9 to 3.5 with hydroxide, and lyophilizing.

[0099] The pharmaceutical composition of the formulation of the present invention is produced by dissolving the active ingredient, an HMGB1 fragment peptide or its acid addition salt, a hydrate of an organic acid, a hydrate of a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from monosaccharides, disaccharides, and polysaccharides, in distilled water for injection, adjusting the pH to 2.9 to 3.5 with hydroxide, and lyophilizing the solution.

[0100] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its acid addition salt, an organic acid, a metal salt of an organic acid, and one or more sugars and / or sugar alcohols selected from the group consisting of glucose, fructose, sucrose (refined white sugar), mannitol, and trehalose, in distilled water for injection, adjusting the pH to 2.9 to 3.5 with hydroxide, and lyophilizing the mixture.

[0101] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, an organic acid, a metal salt of an organic acid, and sucrose in distilled water for injection, adjusting the pH to 2.9 to 3.5 with hydroxide, and lyophilizing the mixture.

[0102] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, a metal salt of an organic acid, and sucrose in distilled water for injection, adjusting the pH to 2.9 to 3.5 with hydroxide, and lyophilizing the mixture.

[0103] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, an organic acid, sodium citrate, and sucrose, in distilled water for injection, adjusting the pH to 2.9 to 3.5 with hydroxide, and lyophilizing the resulting solution.

[0104] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, sodium citrate, and sucrose, in distilled water for injection, adjusting the pH to 2.9 to 3.5 with hydroxide, and lyophilizing the resulting solution.

[0105] Another embodiment of the pharmaceutical composition of the formulation of the present invention is a pharmaceutical composition produced by dissolving the active ingredient, an HMGB1 fragment peptide or its trifluoroacetate salt, citric acid, sodium citrate, and sucrose, in distilled water for injection, adjusting the pH to 2.9 to 3.5 with sodium hydroxide, and lyophilizing the resulting solution.

[0106] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0107] The present invention will be described in detail below with reference to Examples, Comparative Examples and Reference Examples, but the present invention is not limited thereto. The preparations in the Examples, Comparative Examples and Reference Examples were produced by the following methods.

[0108] (1) pH of the preparation The formulation of the present invention is produced by freeze-drying a preparation solution. Therefore, the effect of differences in the pH of the preparation solution on the quality of the freeze-dried formulation was evaluated. The active ingredient (hereinafter sometimes referred to as "drug substance") refers to any of the substances listed below in (a) to (c). (a) an HMGB1 fragment peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or an acid addition salt thereof; (b) a peptide or an acid addition salt thereof, which comprises the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, inserted, or added, and which has the activity of stimulating cell migration; and (c) A peptide or an acid addition salt thereof, which comprises an amino acid sequence having about 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and has the activity of stimulating cell migration.

[0109] (Experimental Method) a. Preparation manufacturing method Table 1 shows the formulation. Citric acid hydrate (Merck) and sodium citrate hydrate (Merck) were each measured in predetermined amounts and dissolved in water for injection. These solutions were mixed and adjusted to a predetermined pH to prepare a citrate buffer solution of a predetermined concentration. Predetermined amounts of the drug substance and polysorbate 80 (Merck) were then dissolved in this citrate buffer solution, after which the final pH was adjusted with aqueous sodium hydroxide, the mixture was stirred, and the volume of the solution was adjusted to the final weight with the citrate buffer solution. The prepared solution was filtered through a hydrophilic PVDF filter (manufactured by Merck Millipore), and the filtrate was filled into a 3 mL vial (manufactured by Taisei Kako Co., Ltd.), which was then partially sealed with a rubber stopper. The solution was then freeze-dried using a freeze-dryer under the following conditions. After freeze-drying was completed, the pressure inside the freeze-dryer was restored with nitrogen, and the rubber stopper was fully sealed.

[0110] (Freeze-drying conditions) The freeze-dried product was produced by the following steps: 1) cooling at 5°C, 2) cooling at -5°C for 1 hour, 3) freezing at -40°C for 4 hours, 4) primary drying at -35°C for 24 hours or more under a vacuum pressure of 10 Pa, and 6) secondary drying at 25°C for 5 hours or more under a vacuum pressure of 2 Pa.

[0111] [Table 1] *1 : As a free form *2 : Prepared by mixing 20 mmol / L citric acid hydrate solution and 20 mmol / L sodium citrate hydrate solution to achieve a specified pH.

[0112] b. Measurement method for related substances Related substances were measured by UPLC (model: ACQUITY (Waters)). (1) Preparation of sample dilution solvent For a preparation volume of 500 mL, use Cetyltrimethylammonium Chloride was dissolved in a proportion of 0.16 g to prepare a 0.001 M sample dilution solvent. (2) Preparation of standard solutions The drug substance from the same lot as the sample was dissolved in the sample dilution solvent to prepare a concentration of approximately 0.5 mg / mL. (3) Preparation of sample solution The prepared solution was dissolved in a sample dilution solvent to give a drug substance concentration of 0.5 mg / mL, and used as a sample solution. (4) Preparation of sample solution for freeze-dried preparation One vial of the lyophilized formulation was dissolved in a sample dilution solvent to a drug substance concentration of 0.5 mg / mL to prepare a sample solution. The amount of related substances was calculated using the area percentage method. Test conditions Detector: UV spectrophotometer (measurement wavelength 220 nm) Column: AQUITY UPLC BEH300 C18, 2.1 x 150 mm 1.7 μm (Waters) Column temperature: constant temperature around 70°C Mobile phase A:I: Water / liquid chromatography grade trifluoroacetic acid (1000:1) II: Prepare acetonitrile for liquid chromatography / trifluoroacetic acid for liquid chromatography (1000:1), and mix I / II (95:5). Mobile phase B:I: Water / trifluoroacetic acid for liquid chromatography (1000:1), II: Prepare acetonitrile for liquid chromatography / trifluoroacetic acid for liquid chromatography (1000:1), and mix I / II (75:25). The gradient program for mobile phases A and B is as shown in Table 2.

[0113] [Table 2] Flow rate: 0.2 mL / min Injection volume: 5μL Sample cooler temperature: Constant temperature around 5℃ Area measurement range: 55 minutes after sample injection

[0114] c.pH measurement method While stirring the prepared solution, the pH was measured using a pH meter (manufactured by Horiba, Ltd.).

[0115] (Experimental results) The experimental results are shown in Table 3. As a result, the lyophilized formulation showed a reduction in the amount of dimers and total related substances as the pH of the preparation solution decreased, improving the quality. In particular, it was revealed that the amount of dimers and total related substances could be reduced at a pH of around 3 to 4.

[0116] [Table 3]

[0117] (2) Study of stabilizers Stabilizers for the formulation of the present invention were investigated. Refined sucrose, a non-reducing disaccharide, was selected as the stabilizer, and the relationship between the amount added and the solid state stability was evaluated.

[0118] (Experimental Method) a. Preparation manufacturing method Tables 4 and 5 show the formulations. Predetermined amounts of citric acid hydrate and sodium citrate hydrate were measured and dissolved in distilled water for injection. The pH of this solution was adjusted to 4.0 to prepare a citrate buffer solution of the specified concentration. Weighed excipients and active pharmaceutical ingredients were added to this citrate buffer solution and dissolved, and the final pH was adjusted to 4.0 with aqueous sodium hydroxide solution and stirred. The liquid volume was then adjusted to the final weight with the citrate buffer solution. The prepared solution was filtered through a hydrophilic PVDF filter (manufactured by Merck Millipore), and the filtrate was filled into a 3 mL vial (manufactured by Taisei Kako Co., Ltd.), which was then partially sealed with a rubber stopper. The solution was then freeze-dried using a freeze-dryer under the following conditions. After freeze-drying was completed, the pressure inside the freeze-dryer was restored with nitrogen, and the rubber stopper was fully sealed.

[0119] (Freeze-drying conditions) The freeze-dried product was produced by the following steps: 1) cooling at 5°C, 2) cooling at -5°C for 1 hour, 3) freezing at -40°C for 4 hours, 4) primary drying at -35°C for 24 hours or more under a vacuum pressure of 10 Pa, and 6) secondary drying at 25°C for 5 hours or more under a vacuum pressure of 2 Pa.

[0120] [Table 4] *1: As a free form *2: Prepared by mixing 20mmol / L citric acid hydrate solution and 20mmol / L sodium citrate hydrate solution to a pH of 4.0 *3: Taking into account the conversion factor of 1.41 for the drug substance, the solute concentration was calculated from the added refined sucrose and 20 mmol / L citric acid buffer (molecular weight of anhydrous citric acid: 192.1235).

[0121] [Table 5] *1: As a free form *2: Prepared by mixing 20mmol / L citric acid hydrate solution and 20mmol / L sodium citrate hydrate solution to a pH of 4.0 *3: Taking into account the conversion factor of 1.41 for the drug substance, the solute concentration was calculated from the added refined sucrose and 20 mmol / L citric acid buffer (molecular weight of anhydrous citric acid: 192.1235).

[0122] b. Stability test over time The freeze-dried preparation was stored in a thermo-hygrostat at 40°C / 75% RH (relative humidity) for one month, and the initial and post-storage amounts of dimer and total related substances were measured.

[0123] c. Measurement method for related substances As in Examples 1 and 2, measurements were made by UPLC.

[0124] d.pH measurement method While stirring the prepared solution, the pH was measured using a pH meter (manufactured by Horiba, Ltd.).

[0125] (Experimental results) The experimental results are shown in Tables 6 to 8. As a result, compared to Comparative Example 3, which did not contain a stabilizer, the preparations of Examples 3 to 6, which contained refined sucrose, had improved stability.

[0126] [Table 6]

[0127] [Table 7]

[0128] [Table 8]

[0129] (3) Solution stability at various pH levels It was revealed that differences in the pH of the preparation solution affect the stability of the active ingredient, and that the amount of related substances can be reduced particularly at a pH of 3 to 4. Therefore, the stability was actually examined using preparation solutions around this pH range.

[0130] (Experimental Method) a. Preparation manufacturing method Tables 9 and 10 show the formulations. Water for injection was weighed into a beaker, and then the additives were added and dissolved. This solution was cooled to 5°C using a cool stirrer, after which the specified amount of active ingredient was added, and the pH of the solution was adjusted to 2.7, 3.2, 3.5, 3.7, or 4.0 with 0.5N sodium hydroxide. After adjusting the pH, the final weight was adjusted with water for injection. The refined sucrose was blended in a molar ratio of 25 times that of the active ingredient. This solution was stored at 25°C for 24 hours.

[0131] [Table 9] *1 : As a free form

[0132] [Table 10] *1 : As a free form

[0133] b. Measurement method for related substances As in Examples 1 and 2, measurements were made by UPLC.

[0134] c.pH measurement method While stirring the prepared solution, the pH was measured using a pH meter (manufactured by Horiba, Ltd.).

[0135] (Experimental results) The experimental results are shown in Tables 11 to 13. As a result, the higher the pH of the solution, the more significant the increase in both the dimer and total related substances. In particular, the solution prepared with a pH of 4.0 had the worst stability.

[0136] [Table 11]

[0137] [Table 12]

[0138] [Table 13]

[0139] (4) Examination of the effects of additives The effects of the types and amounts of additives in the formulation of the present invention on the stability of the freeze-dried formulation were evaluated.

[0140] (Experimental Method) a. Preparation manufacturing method Tables 14 and 15 show the formulations. Water for injection was weighed into a beaker, and then the additives were added and dissolved. This solution was cooled to 5°C using a cool stirrer, after which a predetermined amount of the active ingredient was added and the pH was measured. The pH was then adjusted to 2.7 with 0.5N sodium hydroxide solution. After adjusting the pH, the final weight was adjusted with water for injection. The prepared solution was filtered through a hydrophilic PVDF filter (manufactured by Merck Millipore), and the filtrate was filled into a 3 mL vial (manufactured by Taisei Kako Co., Ltd.), which was then partially sealed with a rubber stopper. Then, freeze-drying was carried out under the following conditions. After freeze-drying was completed, the pressure inside the freeze-dryer was restored with nitrogen, and the rubber stopper was fully sealed.

[0141] (Freeze-drying conditions) The freeze-dried product was produced by the following steps: 1) cooling at 5°C, 2) cooling at -5°C for 1 hour, 3) freezing at -40°C for 4 hours, 4) primary drying at -35°C for 24 hours or more under a vacuum pressure of 10 Pa, and 6) secondary drying at 40°C for 5 hours or more under a vacuum pressure of 2 Pa.

[0142] [Table 14] *1 : As a free form

[0143] [Table 15] *1 : As a free form

[0144] b. Stability test over time The freeze-dried preparation was stored in a thermo-hygrostat at 40°C / 75% RH (relative humidity) for 2 weeks, and the amounts of dimer and total related substances were measured.

[0145] c. Measurement method for related substances As in Examples 1 and 2, measurements were made by UPLC.

[0146] d.pH measurement method While stirring the prepared solution, the pH was measured using a pH meter (manufactured by Horiba, Ltd.).

[0147] (Experimental results) The experimental results are shown in Tables 16 to 18. As a result, the amounts of dimers and total related substances increased after storage in the trehalose-containing preparations of Comparative Examples 4 and 5 compared to the preparations of Examples 11 to 14 containing refined sucrose. On the other hand, the amounts of dimers and total related substances were almost the same in Examples 11 and 12, which contained polysorbate 80 and refined sucrose as stabilizers, and Examples 13 and 14, which contained refined sucrose but no polysorbate 80. Therefore, it was revealed that the amount of related substances could be sufficiently reduced using refined sucrose alone as a stabilizer.

[0148] [Table 16]

[0149] [Table 17]

[0150] [Table 18]

[0151] (5) Examination of the concentration of the preparation solution The effect of the concentration of the preparation solution in the preparation of the present invention on the stability of the freeze-dried preparation was evaluated.

[0152] (Experimental Method) a. Preparation manufacturing method The formulation is shown in Table 19. Water for injection was weighed into a beaker, and then citric acid hydrate, sodium citrate hydrate, and refined sucrose were added and dissolved. The pH of this solution was measured, and then the active ingredient was added and dissolved, and the pH was measured again. Sodium hydroxide solution was then added to adjust the pH to 3.2, and the final weight was adjusted with water for injection. The prepared solution was filtered through a hydrophilic PVDF filter (Merck Millipore), and the filtrate was filled into a vial (Fuji Glass Co., Ltd.), which was then partially sealed with a rubber stopper. The solution was then freeze-dried using a freeze-dryer under the following conditions. After freeze-drying was completed, the pressure inside the freeze-dryer was restored with nitrogen, and the rubber stopper was fully sealed.

[0153] [Table 19] *1 As a free body

[0154] (Freeze-drying conditions) As shown in Tables 20 and 21, the freeze-dried product was produced by the following steps: 1) cooling at 5°C, 2) cooling at -5°C for 2 hours, 3) freezing at -40°C for 4 hours, 4) primary drying at -33°C for 45 hours or more under a vacuum pressure of 10 Pa, and 6) secondary drying at 40°C for 8 hours or more under a vacuum pressure of 5 Pa.

[0155] [Table 20]

[0156] [Table 21]

[0157] b. Stability test over time The freeze-dried preparation was stored in a thermo-hygrostat at 25°C / 60% RH (relative humidity) for 26 weeks, and the amount of dimer and total related substances was measured.

[0158] c. Measurement method for related substances Related substances were measured by HPLC (model: Alliance (manufactured by Waters)). (Preparation of sample dilution solvent) For a preparation volume of 500 mL, use Cetyltrimethylammonium Chloride was dissolved in a proportion of 0.16 g to prepare a 0.001 M sample dilution solvent. (Sample solution preparation method) Add 3 mL of sample dilution solvent to one vial and redissolve. Take 1.5 mL and make 3.5 mL of sample dilution. The solvent was added to prepare a sample solution, and the amount of related substances was calculated by the area percentage method. Test conditions Detector: UV spectrophotometer (measurement wavelength 215 nm) Column: XBridge Peptide BEH C18, 300Å, 4.6×250mm, 5μm (Waters) Column temperature: constant temperature around 55°C Mobile phase A: Trifluoroacetic acid TS Mobile phase B: Acetonitrile for liquid chromatography / 2-propanol for liquid chromatography / trifluoroacetic acid mixture (500:500:1) The gradient program for mobile phases A and B is as shown in Table 22.

[0159] [Table 22] Flow rate: 1.0 mL / min Injection volume: 20μL Sample cooler temperature: Constant temperature around 5℃ Area measurement range: 70 minutes after sample injection

[0160] d.pH measurement method While stirring the prepared solution, the pH was measured using a pH meter (manufactured by Horiba, Ltd.).

[0161] (Experimental results) The experimental results are shown in Table 23. As a result, it was revealed that the concentration of the preparation solution did not affect stability even when the final concentration of the preparation solution was changed and a lyophilized formulation was produced using that solution and stored over time.

[0162] [Table 23]

[0163] (6) Study of formulations with higher concentration of the preparation solution and increased amount of sucrose In the preparation of the present invention, a preparation in which the concentration of the preparation solution was increased and the amount of sucrose was increased was investigated.

[0164] (Experimental Method) a. Preparation manufacturing method Tables 24 to 26 show the formulations. Water for injection was weighed into a beaker, and then citric acid hydrate, sodium citrate hydrate, and refined sucrose were added and dissolved. The pH of this solution was measured, and then the active pharmaceutical ingredient was added and dissolved, and the pH was measured again. The final weight was then adjusted with water for injection and stirred. The prepared solution was filtered through a hydrophilic PVDF filter (manufactured by Merck Millipore), and the filtrate was filled into vials (manufactured by Taisei Kako) in 0.25 g portions, which were then partially sealed with rubber stoppers. The solution was then freeze-dried using a freeze-dryer under the following conditions. After freeze-drying was completed, the pressure inside the freeze-dryer was restored with nitrogen, and the rubber stoppers were fully sealed.

[0165] [Table 24] *1 As a free form

[0166] [Table 25] *1 As a free form

[0167] [Table 26] *1 As a free form

[0168] (Freeze-drying conditions) Lyophilization was carried out to produce freeze-dried products in Examples 17, 18, 22, and 23 as shown in Tables 27 and 28, and in Examples 19, 20, 21, and 24 as shown in Tables 29 and 30.

[0169] [Table 27]

[0170] [Table 28]

[0171] [Table 29]

[0172] [Table 30]

[0173] b. Stability test over time The freeze-dried preparation was stored in a thermo-hygrostat at 40°C / 75% RH (relative humidity) for one month, and the amount of dimer and total related substances was measured.

[0174] c. Measurement method for related substances As in Examples 15 and 16, measurements were made by HPLC.

[0175] (Experimental results) Tables 31 and 32 show the amount of dimer (initial, after 1 month of storage at 40°C / 75% RH (relative humidity), increase from initial), and Tables 33 and 34 show the amount of total related substances (initial, after 1 month of storage at 40°C / 75% RH (relative humidity), increase from initial). As a result, there was almost no difference in the increase in the amount of dimer from the initial between the formulations, but the total amount of related substances tended to decrease as the amount of sucrose increased.

[0176] [Table 31]

[0177] [Table 32]

[0178] [Table 33]

[0179] [Table 34]

[0180] (7) Study of high-dose formulations with highly concentrated preparations A quality assessment was conducted on a high-dose formulation (120 mg formulation) that is expected to be used in clinical trials.

[0181] (Experimental Method) a. Preparation manufacturing method Table 35 shows the formulation of the prepared solution, and Table 36 shows the formulation for 120 mg of active ingredient per vial. Water for injection was weighed into a beaker, followed by the addition and dissolution of citric acid hydrate, sodium citrate hydrate, and refined sucrose. The pH of this solution was measured, and the active ingredient was added and dissolved. The pH was then measured again. Sodium hydroxide solution was then added to adjust the pH to 3.2, and the final weight was adjusted with water for injection. The prepared solution was filtered through a hydrophilic PVDF filter (Merck Millipore), and the filtrate was filled into vials (Fuji Glass Co., Ltd.), which were partially sealed with rubber stoppers. The solution was then freeze-dried using a freeze dryer under the conditions shown in Tables 37 and 38. After freeze-drying, the freeze dryer chamber was restored to pressure with nitrogen, and the rubber stoppers were fully sealed. The amount of material filled in one vial was 3.0 g in Example 25, 4.0 g in Example 26, and 0.25 g in Example 22 as a control.

[0182] [Table 35] *1 As a free form

[0183] [Table 36] *1 As a free form

[0184] [Table 37]

[0185] [Table 38]

[0186] b. Stability test over time The freeze-dried preparation was stored in a thermo-hygrostat at 40°C / 75% RH (relative humidity) for one month, and the amount of dimer and total related substances was measured.

[0187] c. Measurement method for related substances As in Examples 15 and 16, measurements were made by HPLC.

[0188] (Experimental results) Table 39 shows the amount of dimer (initial, after 1 month of storage at 40°C / 75% RH (relative humidity), increase from initial), and Table 40 shows the amount of total related substances (initial, after 1 month of storage at 40°C / 75% RH (relative humidity), increase from initial). As a result, there was almost no difference between the formulations in either the increase in the amount of dimer from the initial or the increase in the amount of total related substances.

[0189] [Table 39]

[0190] [Table 40] [Industrial Applicability]

[0191] The stability of the formulation of the present invention containing the active pharmaceutical ingredient, which is a peptide, can be improved by adding a stabilizer, particularly a sugar, and adjusting the pH to a specific range, thereby stabilizing even unstable peptides over time.

Claims

1. A pharmaceutical composition comprising the substance described in (a) below, citric acid, sodium citrate, and sucrose, wherein the pharmaceutical composition has a pH of 3.0 to 4.5 when dissolved in distilled water for injection; (a) A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an acid addition salt thereof.

2. 2. The pharmaceutical composition according to claim 1, wherein the pH of the composition when dissolved in distilled water for injection is 3.0 to 4.

0.

3. 2. The pharmaceutical composition according to claim 1, wherein the pH of the composition when dissolved in distilled water for injection is 3.0 to 3.

6.

4. 2. The pharmaceutical composition according to claim 1, wherein the pH of the composition when dissolved in distilled water for injection is 3.1 to 3.

3.

5. 5. The pharmaceutical composition according to claim 1, wherein the amount of sucrose in the pharmaceutical composition is 10 to 300 times the molar amount of the substance described in (a).

6. 6. The pharmaceutical composition according to claim 5, wherein the amount of sucrose in the pharmaceutical composition is 50 to 300 times the molar amount of the substance described in (a).

7. 7. The pharmaceutical composition according to claim 1, wherein the pH is adjusted using a hydroxide of one or more metals selected from the group consisting of alkali metals, alkaline earth metals, and magnesium.

8. 8. The pharmaceutical composition according to claim 7, wherein the hydroxide is one or more selected from the group consisting of potassium hydroxide, calcium hydroxide, sodium hydroxide and magnesium hydroxide.

9. 9. The pharmaceutical composition of claim 8, wherein the hydroxide is sodium hydroxide.

10. 10. The pharmaceutical composition according to any one of claims 1 to 9, comprising the acid addition salt according to (a), wherein the acid addition salt is a trifluoroacetate salt.

11. The pharmaceutical composition according to any one of claims 1 to 10, which is a lyophilized product.

12. 12. The pharmaceutical composition according to claim 11, wherein the amount of the substance described in (a) is 6.0 to 50.0 mg per 1 g of the lyophilized product.

13. A method for producing a pharmaceutical composition, comprising the steps of: 1) dissolving citric acid hydrate, sodium citrate hydrate, and sucrose in distilled water for injection and adjusting the pH to 3.0 to 4.0; 2) A step of cooling the liquid produced in step 1) to 18°C ​​or less; 3) a step of dissolving the substance according to claim 1 (a) in the liquid obtained in step 2); 4) adjusting the pH of the liquid obtained in step 3) to 2.5 to 4.0 with a hydroxide of one or more metals selected from the group consisting of alkali metals, alkaline earth metals, and magnesium; and 5) A step of freeze-drying the liquid obtained in step 4).

14. A method for producing a pharmaceutical composition, comprising the steps of: 1) dissolving citric acid hydrate, sodium citrate hydrate, and sucrose in distilled water for injection and adjusting the pH to 3.0 to 4.0; 2) A step of cooling the liquid produced in step 1) to 18°C ​​or less; 3) a step of dissolving the substance according to (a) of claim 1 in the liquid obtained in step 2); 4) adjusting the pH of the liquid obtained in step 3) to 2.9 to 3.5 with sodium hydroxide; and 5) A process of freeze-drying the liquid obtained in the process 4).

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