阅读说明：本技术 含有蛋白的水性液体制剂 (Aqueous liquid preparation containing protein ) 是由 安川秀仁 花田崇 谷纯也 冈部真二 浅野友香 于 2019-06-25 设计创作，主要内容包括：课题是提供在溶液状态下储存稳定且注射时的疼痛减轻的、含有蛋白作为有效成分的水性液体制剂。解决手段为含有浓度为1～20mM的磷酸缓冲剂和作为有效成分的蛋白的水性液体制剂。例如,一种水性液体制剂,含有作为缓冲剂的浓度为1～20mM的磷酸缓冲剂和作为有效成分的人生长激素,还含有作为非离子性表面活性剂的泊洛沙姆和作为等渗剂的苯酚。(The object is to provide an aqueous liquid preparation containing a protein as an active ingredient, which is stable in storage in a solution state and reduces pain upon injection. The solution is an aqueous liquid preparation containing a phosphate buffer at a concentration of 1 to 20mM and a protein as an active ingredient. For example, an aqueous liquid preparation contains a phosphate buffer at a concentration of 1 to 20mM as a buffer, human growth hormone as an active ingredient, poloxamer as a nonionic surfactant, and phenol as an isotonic agent.)

1. An aqueous liquid preparation comprising a phosphate buffer at a concentration of 1 to 20mM and a protein as an active ingredient.

2. The aqueous liquid preparation according to claim 1, wherein the concentration of the phosphate buffer is 5 to 16 mM.

3. The aqueous liquid preparation according to claim 2, wherein the concentration of the phosphate buffer is 8 to 12 mM.

4. The aqueous liquid preparation according to any one of claims 1 to 3, wherein the concentration of the protein is 1 to 50 mg/mL.

5. The aqueous liquid preparation according to any one of claims 1 to 4, further comprising a nonionic surfactant.

6. The aqueous liquid preparation according to claim 5, wherein the nonionic surfactant is a polysorbate or poloxamer.

7. The aqueous liquid preparation according to claim 5, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80 and poloxamer 188.

8. The aqueous liquid preparation according to any one of claims 5 to 7, wherein the concentration of the nonionic surfactant is 1 to 10 mg/mL.

9. The aqueous liquid preparation according to any one of claims 1 to 8, further comprising a preservative.

10. The aqueous liquid preparation according to claim 9, wherein the preservative is benzyl alcohol or phenol.

11. The aqueous liquid preparation according to claim 9, wherein the preservative is benzyl alcohol, and the concentration of the benzyl alcohol is 2 to 20 mg/mL.

12. The aqueous liquid preparation according to claim 9, wherein the preservative is phenol, and the concentration of phenol is 1 to 10 mg/mL.

13. The aqueous liquid preparation according to any one of claims 1 to 12, which further comprises an isotonic agent.

14. The aqueous liquid preparation as claimed in claim 13, wherein the isotonic agent is a neutral salt or a sugar alcohol.

15. The aqueous liquid preparation as claimed in claim 13, wherein the isotonic agent is sodium chloride or D-mannitol.

16. The aqueous liquid preparation according to any one of claims 1 to 15, which has a pH of 5.5 to 7.2.

17. The aqueous liquid preparation according to claim 1, which comprises 1 to 50mg/mL of the protein, 5 to 16mM of phosphate buffer, 1.8 to 2.2mg/mL of poloxamer 188, 2.8 to 3.8mg/mL of phenol, and 35 to 45mg/mL of D-mannitol, and has a pH of 6.0 to 6.4.

18. The aqueous liquid preparation according to claim 1 to 17, wherein the protein is selected from the group consisting of growth hormone, somatomedin, insulin, glucagon, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of antibody and other protein, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, bepotin, tissue plasminogen activator (t-PA), thrombomodulin, Follicle Stimulating Hormone (FSH), gonadotropin releasing hormone (GnRH), gonadotropin, DNasel, Thyroid Stimulating Hormone (TSH), Nerve Growth Factor (NGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), Neurotrophin 3, neurotrophin 4/5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2(FGF2), epithelial cell growth factor (EGF), Vascular Endothelial Growth Factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), an enzyme having an activity of decomposing beta amyloid, etanercept, pegvisomant, metriptine, adynapt, and Asfotase, GLP-1 receptor agonists.

19. The aqueous liquid preparation according to claim 1 to 17, wherein the protein is selected from the group consisting of a mouse antibody, a humanized antibody, a human/mouse chimeric antibody and a human antibody.

20. The aqueous liquid preparation according to claim 1 to 17, wherein the protein is a substance selected from the group consisting of an anti-IL-6 antibody, an anti-amyloid β antibody, an anti-BACE antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-HER 2 antibody, an anti-PCSK 9 antibody and an anti-TNF- α antibody.

21. The aqueous liquid preparation according to claim 1 to 17, wherein the protein is a lysosomal enzyme selected from the group consisting of α -L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β -galactosidase, GM2 activating protein, β -hexosaminidase A, β -hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α -mannosidase, β -mannosidase, galactosylceramidase, saponified protein C, arylsulfatase A, α -L-fucosidase, aspartylglucamine, α -N-acetylgalactosaminidase, acid sphingomyelinase, α -galactosidase A, β -glucuronidase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl CoA alpha-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, starch-1, 6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidylpeptidase-1, and hyaluronidase-1.

22. The aqueous liquid preparation according to claim 1 to 17, wherein the protein is a fusion protein of an antibody and another protein selected from the group consisting of growth hormone, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of antibody and another protein, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, dabigatran, tissue plasminogen activator (t-PA), thrombomodulin, follicle stimulating hormone, DNasel, Thyroid Stimulating Hormone (TSH), Nerve Growth Factor (NGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4/5, and combinations thereof, Neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2(FGF2), epithelial cell growth factor (EGF), Vascular Endothelial Growth Factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor) and substances having an enzyme that decomposes beta amyloid.

23. The aqueous liquid preparation according to claim 1 to 17, wherein the protein is a fusion protein of an antibody and a lysosomal enzyme selected from the group consisting of α -L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β -galactosidase, GM2 activating protein, β -hexosaminidase A, β -hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α -mannosidase, β -mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α -L-fucosidase, aspartylase, α -N-acetylglucosaminidase, acid sphingomyelinase, α -galactosidase A, β -glucuronidase, Heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl CoA alpha-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, starch-1, 6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidylpeptidase-1, and hyaluronidase-1.

24. The aqueous liquid preparation according to claim 1 to 23, which is a preparation to be administered by subcutaneous injection or intramuscular injection.

Technical Field

The present invention relates to an aqueous liquid preparation containing a protein as an active ingredient, which is stable in storage in a solution state and reduces pain upon injection, and more particularly, to an aqueous liquid preparation containing a phosphate buffer as a buffer at a concentration of 1 to 20mM and containing a protein as an active ingredient.

Background

Various preparations containing proteins as active ingredients have been developed. Many of these proteins are recombinant proteins produced by gene recombination techniques. Since a protein preparation as a polymer is not absorbed by oral administration, it is specifically administered by subcutaneous injection, intramuscular injection, intravenous injection, or the like. Therefore, the administration of the drug is accompanied by pain due to penetration of the injection needle. The components contained in the preparation have an influence on the pain level.

In human growth hormone (hGH), the wild type is a single chain polypeptide hormone consisting of 191 amino acid residues. hGH is used as a therapeutic agent for growth hormone deficiency dwarfism (growth hormone deficiency dwarfism), dwarfism in Turner syndrome, adult growth hormone deficiency (adult growth hormone deficiency), and the like (non-patent document 1).

hGH-based treatments typically require long term use in units of years, and during this period hGH needs to be injected intramuscularly in 2-4 times per week, or subcutaneously in 6-7 times. Therefore, in order to reduce the burden of patient's entrance and exit to the hospital, hGH-based therapies are usually self-administered at home. In addition, since a conventional formulation of hGH is a lyophilized formulation, and the lyophilized formulation needs to be dissolved in a dissolving solution at the time of use, an aqueous liquid formulation in which hGH is dissolved in advance has been developed in order to improve convenience for patients (non-patent document 1).

hGH is administered by injection into the muscle or subcutaneous tissue of a patient. Moreover, hGH is often used in children. Therefore, it is more strongly desired that the injection of hGH formulations is accompanied by less pain.

Documents of the prior art

Non-patent document

Non-patent document 1: growth subcutaneous injection 6 mg/growth subcutaneous injection 12mg package insert (2017).

Disclosure of Invention

Problems to be solved by the invention

Disclosed is an aqueous liquid preparation which is stable in storage in a solution state and reduces pain upon injection and contains a protein as an active ingredient.

Means for solving the problems

In the studies for the above object, the present inventors found that: the present inventors have completed the present invention by using a phosphate buffer with a concentration of 1 to 20mM as a buffer contained in an aqueous liquid preparation, which can reduce pain during injection while maintaining stability of growth hormone in the aqueous liquid preparation. That is, the present invention includes the following.

1. An aqueous liquid preparation contains a phosphate buffer at a concentration of 1 to 20mM and a protein as an active ingredient.

2. The aqueous liquid preparation according to the above 1, wherein the concentration of the phosphate buffer is 5 to 16 mM.

3. The aqueous liquid preparation according to the above 2, wherein the concentration of the phosphate buffer is 8 to 12 mM.

4. The aqueous liquid preparation according to any one of 1 to 3, wherein the concentration of the protein is 1 to 50 mg/mL.

5. The aqueous liquid preparation according to any one of the above 1 to 4, further comprising a nonionic surfactant.

6. The aqueous liquid preparation of the above 5, wherein the nonionic surfactant is polysorbate or poloxamer.

7. The aqueous liquid preparation of the above 5, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80 and poloxamer 188.

8. The aqueous liquid preparation according to any one of the above 5 to 7, wherein the concentration of the nonionic surfactant is 1 to 10 mg/mL.

9. The aqueous liquid preparation according to any one of the above items 1 to 8, further comprising a preservative.

10. The aqueous liquid preparation according to the above 9, wherein the preservative is benzyl alcohol or phenol.

11. The aqueous liquid preparation of the above 9, wherein the preservative is benzyl alcohol, and the concentration of the benzyl alcohol is 2 to 20 mg/mL.

12. The aqueous liquid preparation of the above 9, wherein the preservative is phenol, and the concentration of the phenol is 1 to 10 mg/mL.

13. The aqueous liquid preparation according to any one of 1 to 12, further comprising an isotonic agent.

14. The aqueous liquid preparation of the above item 13, wherein the isotonic agent is a neutral salt or a sugar alcohol.

15. The aqueous liquid preparation of the above item 13, wherein the isotonic agent is sodium chloride or D-mannitol.

16. The aqueous liquid preparation according to any one of 1 to 15, wherein the aqueous liquid preparation has a pH of 5.5 to 7.2.

17. The aqueous liquid preparation of the above 1, which contains 1 to 50mg/mL of protein, 5 to 16mM of phosphate buffer, 1.8 to 2.2mg/mL of poloxamer 188, 2.8 to 3.8mg/mL of phenol, and 35 to 45mg/mL of D-mannitol, and has a pH of 6.0 to 6.4.

18. The aqueous liquid preparation according to 1 to 17, wherein the protein is selected from the group consisting of growth hormone, somatomedin, insulin, glucagon, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of antibody and other protein, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, bepotin, tissue plasminogen activator (t-PA), thrombomodulin, Follicle Stimulating Hormone (FSH), gonadotropin releasing hormone (GnRH), gonadotropin, DNasel, Thyroid Stimulating Hormone (TSH), Nerve Growth Factor (NGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophin 3, and combinations thereof, Neurotrophin 4/5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2(FGF2), epithelial cell growth factor (EGF), Vascular Endothelial Growth Factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), enzyme having an activity of decomposing beta amyloid, etanercept, pegvisomant, metreleptin, arbitracin, Asfotase, and GLP-1 receptor agonists.

19. The aqueous liquid preparation according to 1 to 17, wherein the protein is selected from the group consisting of a mouse antibody, a humanized antibody, a human/mouse chimeric antibody and a human antibody.

20. The aqueous liquid preparation according to 1 to 17, wherein the protein is a substance selected from the group consisting of an anti-IL-6 antibody, an anti-amyloid β antibody, an anti-BACE antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-HER 2 antibody, an anti-PCSK 9 antibody and an anti-TNF- α antibody.

21. The aqueous liquid preparation according to 1 to 17, wherein the protein is a lysosomal enzyme selected from the group consisting of α -L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β -galactosidase, GM 2-activating protein, β -hexosaminidase A, β -hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α -mannosidase, β -mannosidase, galactosylceramide enzyme, saponified protein C, arylsulfatase A, α -L-fucosidase, aspartylglucamine enzyme, α -N-acetylgalactosaminidase, acid sphingomyelinase, α -galactosidase A, β -glucuronidase, heparan N-sulfatase, acetyl-D-glucosidase, and mixtures thereof, alpha-N-acetylglucosaminidase, acetyl CoA alpha-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, starch-1, 6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidylpeptidase-1, and hyaluronidase-1.

22. The aqueous liquid preparation according to 1 to 17, wherein the protein is a fusion protein of an antibody and another protein selected from the group consisting of growth hormone, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of an antibody and another protein, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, bepotin, tissue plasminogen activator (t-PA), thrombomodulin, follicle stimulating hormone, DNasel, Thyroid Stimulating Hormone (TSH), Nerve Growth Factor (NGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4/5, and, Neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2(FGF2), epithelial cell growth factor (EGF), Vascular Endothelial Growth Factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor) and substances having an enzyme that decomposes beta amyloid.

23. The aqueous liquid preparation according to any one of the above 1 to 17, wherein the protein is a fusion protein of an antibody and a lysosomal enzyme selected from the group consisting of α -L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β -galactosidase, GM 2-activating protein, β -hexosaminidase A, β -hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α -mannosidase, β -mannosidase, galactosylceramidase, saponified protein C, arylsulfatase A, α -L-fucosidase, aspartylglucamine, α -N-acetylgalactosaminidase, acid sphingomyelinase, α -galactosidase A, β -glucuronidase, acid sphingomyelinase, acid-galactosidase A, β -glucuronidase, and the like, Heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl CoA alpha-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, starch-1, 6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidylpeptidase-1, and hyaluronidase-1.

24. The aqueous liquid preparation 1 to 23, which is a preparation to be administered by subcutaneous injection or intramuscular injection.

Effects of the invention

According to the present invention, pain of a preparation containing a protein as an active ingredient upon injection can be alleviated.

Drawings

FIG. 1 is a gauge for use in examples 1 and 2 to express pain in a subject.

Detailed Description

The present invention relates to an aqueous liquid preparation containing a protein as an active ingredient. Here, the kind of the protein as the active ingredient is not particularly limited, and examples thereof include: growth hormones, growth regulators (including growth regulators A, B and C), insulin, glucagon, lysosomal enzymes, cytokines, lymphokines, coagulation factors (including coagulation factor VII, coagulation factor VIII and coagulation factor IX), antibodies, fusion proteins of antibodies with other proteins, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, darbepotin (darbepoetin), tissue plasminogen activator (t-PA), thrombomodulin, Follicle Stimulating Hormone (FSH), gonadotropin releasing hormone (GnRH), gonadotropin, DNasel, Thyroid Stimulating Hormone (TSH), Nerve Growth Factor (NGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and, Neurotrophin 3, neurotrophin 4/5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2(FGF2), epithelial cell growth factor (EGF), Vascular Endothelial Growth Factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), or an enzyme having an activity of decomposing beta amyloid, etanercept (etanercept), pegvisomant (pegvisomant), metreleptin (metreleptin), abamectin (abatacept), Asfotase, glucagon-like peptide-1 receptor agonists.

When the active ingredient is an antibody, the biological species of the antibody is not particularly limited, and examples thereof include a mouse antibody, a humanized antibody, a human/mouse chimeric antibody, and a human antibody. In addition, the antigen to which the antibody specifically binds is not particularly limited, and is, for example, an anti-IL-6 antibody, an anti- β amyloid antibody, an anti-BACE antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-HER 2 antibody, an anti-PCSK 9 antibody, or an anti-TNF- α antibody. The antibody is not limited to an antibody having a basic structure consisting of 4 polypeptide chains in total of 2 immunoglobulin light chains and 2 immunoglobulin heavy chains, and may be a single-chain antibody, Fab, F (ab')₂Or an antigen-binding fragment.

When the active ingredient is a lysosomal enzyme, the type of lysosomal enzyme is not particularly limited, and examples thereof include: alpha-L-iduronidase, iduronic acid-2-sulfatase, glucocerebrosidase, beta-galactosidase, GM2 activating protein, beta-hexosaminidase A, beta-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, alpha-mannosidase, beta-mannosidase, galactosylceramidase, saposin C, allyl sulfatase A, (allyl sulfatase A), alpha-L-fucosidase, aspartylglucamine, alpha-N-acetylgalactosaminidase, acid sphingomyelinase, alpha-galactosidase A, beta-glucuronidase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, Acetyl CoA alpha-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, starch-1, 6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, or hyaluronidase-1.

When the active ingredient is a fusion protein of an antibody and another protein, the type of the other protein is not particularly limited, and examples thereof include: growth hormone, lysosomal enzyme, cytokine, lymphokine, coagulation factor, antibody, fusion protein of antibody with other protein, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, dabigatran, tissue plasminogen activator (t-PA), thrombomodulin, follicle stimulating hormone, DNasel, Thyroid Stimulating Hormone (TSH), Nerve Growth Factor (NGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4/5, neurotrophin 6, neuregulin 1, activator, basic fibroblast growth factor (bFGF), fibroblast growth factor 2(FGF2), Epithelial cell growth factor (EGF), Vascular Endothelial Growth Factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), or an enzyme having an activity of decomposing beta amyloid.

When the other protein is a lysosomal enzyme, the type of lysosomal enzyme is not particularly limited, and examples thereof include: alpha-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, beta-galactosidase, GM2 activating protein, beta-hexosaminidase A, beta-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, alpha-mannosidase, beta-mannosidase, galactosylceramidase, saponin C, arylsulfatase A, alpha-L-fucosidase, aspartylglucamidase, alpha-N-acetylgalactosaminidase, acid sphingomyelinase, alpha-galactosidase A, beta-glucuronidase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl CoA alpha-glucosaminidase, acetyl CoA, alpha-glucosaminidase, alpha-D-glucosaminidase, beta-galactosidase-glucuronidase, acetyl-D-N-acetyltransferase, alpha-D-glucosidase, beta-galactosaminyl-N-acetyltransferase, beta-galactosidase-D-L-sulfatase, n-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, starch-1, 6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidylpeptidase-1, or hyaluronidase-1.

The biological species of the protein contained as an active ingredient in the aqueous liquid preparation is not particularly limited, and is preferably a human protein. The protein can be produced in the form of recombinant protein by using gene recombination technology. The recombinant protein can be produced, for example, by: a gene encoding the protein is incorporated into an expression vector, and mammalian cells (e.g., CHO cells derived from chinese hamster ovary), escherichia coli, and the like are transformed using the same, and the transformed cells are cultured.

In the present invention, the term "growth hormone" is used to refer to human growth hormone in particular, but the term "growth hormone" is not limited thereto, and includes growth hormones of mammals including domestic animals such as cattle and horses, and pets such as dogs and cats. "growth hormones" also include: an analog of growth hormone obtained by substituting, deleting or inserting 1 or more amino acid residues constituting growth hormone.

In the present invention, growth hormone can be produced by gene recombination technology. A method of manufacturing a growth hormone having biological activity by incorporating a growth hormone gene into an expression vector, transforming mammalian cells (e.g., CHO cells derived from chinese hamster ovary) or escherichia coli using the same, and culturing the transformed cells is well known to those skilled in the art (US2010/0227819, US4342832, etc.).

In the present invention, the concentration of the protein contained in the aqueous liquid preparation is not particularly limited. However, the concentration is preferably 1 to 50mg/mL, more preferably 1 to 10mg/mL, and still more preferably 2 to 8 mg/mL. A suitable protein concentration is, for example, 4mg/mL or 8 mg/mL. In the case where the protein is a growth hormone, these concentrations are particularly preferred.

The aqueous liquid preparation of the protein of the present invention is formed by containing a phosphate buffer as a buffer. The concentration of the phosphate buffer contained in the aqueous liquid preparation is preferably 1 to 20mM, more preferably 5 to 16mM, still more preferably 8 to 15mM, and still more preferably 8 to 12 mM. For example, the final concentration in the aqueous liquid preparation is adjusted to 10mM, 11mM, or 12 mM. The pH of the aqueous liquid preparation containing a buffering agent is preferably 5.5 to 7.2, more preferably 6.0 to 6.4, still more preferably 6.0 to 6.3, and particularly preferably 6.2.

The aqueous liquid formulation of the protein of the present invention further contains a nonionic surfactant. The nonionic surfactant contained in the aqueous liquid preparation may be used alone or in combination with polysorbate, poloxamer, or the like. As polysorbates, polysorbate 20, polysorbate 80 are particularly suitable, and as poloxamers, poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) diol) is particularly suitable. The concentration of the nonionic surfactant contained in the aqueous liquid preparation is preferably 1 to 10mg/mL, more preferably 1 to 5mg/mL, still more preferably 1 to 3mg/mL, yet more preferably 1.5 to 2.5mg/mL, yet more preferably 1.8 to 2.2mg/mL, and is adjusted to 2mg/mL, for example. When poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is used as the nonionic surfactant, the concentration thereof in the aqueous liquid preparation is preferably 1 to 10mg/mL, more preferably 1 to 5mg/mL, even more preferably 1 to 3mg/mL, even more preferably 1.5 to 2.5mg/mL, even more preferably 1.8 to 2.2mg/mL, and is adjusted to 2mg/mL, for example.

It is also envisaged that the aqueous liquid formulation of the protein is not disposable and is not single use, but is used repeatedly after unsealing. In this case, it is desirable to prevent deterioration of quality due to contamination of bacteria during repeated use. Thus, aqueous liquid formulations of the proteins of the invention may contain a preservative. The preservative contained in the aqueous liquid preparation is not particularly limited as long as it is a pharmaceutically acceptable substance, and benzyl alcohol, phenol, benzoic acid, or a mixture thereof can be suitably used.

When phenol is used as a preservative, the concentration of phenol in the aqueous liquid preparation is preferably 1 to 10mg/mL, more preferably 2.5 to 4.0mg/mL, still more preferably 2.8 to 3.8mg/mL, for example 3.3 mg/mL. When benzyl alcohol is used as a preservative, the concentration of benzyl alcohol in the aqueous liquid preparation is preferably 2 to 20mg/mL, more preferably 7 to 12mg/mL, still more preferably 8 to 10mg/mL, for example 9 mg/mL. When benzoic acid is used as a preservative, the concentration of benzoic acid in the aqueous liquid preparation is preferably 1-20 mg/mL.

The aqueous liquid preparation of the protein of the present invention may further contain an isotonic agent. The isotonic agent contained in the aqueous liquid preparation is not particularly limited as long as it is a pharmaceutically acceptable substance, and a sugar alcohol, a neutral salt, or a mixture thereof can be suitably used. The sugar alcohol may be D-mannitol as a suitable sugar alcohol, and the neutral salt may be sodium chloride as a suitable neutral salt. In the case of using D-mannitol as an isotonic agent, the concentration thereof in the aqueous liquid preparation is preferably 35 to 70mg/mL, more preferably 35 to 45mg/mL, for example 40 mg/mL. When sodium chloride is used as an isotonic agent, the concentration thereof in the aqueous liquid preparation is preferably 5.5 to 11.5mg/mL, more preferably 5.5 to 7.5mg/mL, for example 6.5 mg/mL. However, the concentration of the isotonic agent is not limited to these, and it is appropriately adjusted depending on the relationship with the concentration of other components in the aqueous liquid preparation. The concentration of the isotonic agent is usually adjusted so that the osmotic pressure ratio of the aqueous liquid preparation with respect to physiological saline is 0.9 to 1.6, more preferably 0.9 to 1.1. In the present invention, the term "osmotic pressure ratio" refers to an osmotic pressure ratio relative to physiological saline.

Suitable examples of the aqueous liquid preparation of the protein of the present invention are shown below.

(suitable example 1) an aqueous liquid preparation comprising 1 to 50mg/mL of a protein, 5 to 16mM of a phosphate buffer, 1.8 to 2.2mg/mL of poloxamer 188, 2.8 to 3.8mg/mL of phenol, and 35 to 45mg/mL of D-mannitol and having a pH of 6.0 to 6.4. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 8-10 mg/mL benzyl alcohol can be added instead of phenol.

(suitable example 2) an aqueous liquid preparation comprising 1 to 10mg/mL of a protein, 8 to 15mM of a phosphate buffer, 1.8 to 2.2mg/mL of poloxamer 188, 2.8 to 3.8mg/mL of phenol, and 35 to 45mg/mL of D-mannitol and having a pH of 6.0 to 6.4. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 8-10 mg/mL benzyl alcohol can be added instead of phenol.

(suitable example 3) an aqueous liquid preparation comprising 2 to 8mg/mL of a protein, 10mM of a phosphate buffer, 2mg/mL of poloxamer 188, 3.3mg/mL of phenol, and 40mg/mL of D-mannitol, and having a pH of 6.2. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 8-10 mg/mL benzyl alcohol can be added instead of phenol.

The aqueous liquid preparation of the protein of the present invention does not deteriorate the protein even when stored at 2 to 8 ℃ for a long period of time. Here, the deterioration of protein means that the quantitative value of protein becomes 98% or less, for example, 95% or less, as compared with the aqueous liquid preparation immediately after filling into a vial or a syringe as a preparation. In addition, protein degradation is also considered to occur when the activity originally possessed by protein is 98% or less, for example 95% or less, as compared with the activity immediately after filling of a vial or a syringe with an aqueous liquid preparation as a preparation.

The period over which the storage can be performed without deterioration of the protein is preferably at least 18 months, more preferably 24 months, further preferably 30 months, further preferably 36 months.

The aqueous liquid preparation of the protein of the present invention is administered to a patient by intramuscular injection, subcutaneous injection, or the like. If pain is accompanied by injection, the patient's medication compliance may be reduced, and a sufficient therapeutic effect may not be obtained. According to the aqueous liquid preparation of the present invention, since pain felt by a patient at the time of injection is reduced, the patient's medication compliance is increased and the patient does not evade medication.

Although pain felt by injection is subjective, a Visual Analog Scale (VAS) is generally used as a method for objectively evaluating pain. The VAS is a method that generally includes the following steps. (1) The subject was presented with a 100mm straight line showing no pain at the left end and the highest pain imaginable at the right end, (2) pain felt by the subject was shown on the straight line, and (3) the pain shown by the subject was quantified and summed up.

In addition, as a method for objectively evaluating pain, a Numerical Rating Scale (NRS) is known. A numerical rating scale is a method that generally includes the following steps. (1) The subject was presented with a line of 11 grades that divided the pain from no pain to the most severe pain, (2) the pain felt by the subject himself was expressed by substituting the numerical values shown on the line, and (3) the numerical values were summed up.

In addition, as a method for objectively evaluating Pain, Faces Pain Scale (facial expression Scale) is also known. VAS and NRS are methods by which a subject expresses pain perceived by himself/herself with a numerical value shown on a gauge, and a facial expression scale is a method by which pain perceived by a subject is expressed with an expression of a person corresponding to the pain shown on the gauge.

As a method for objectively evaluating pain, a method of appropriately combining VAS, NRS, and a facial expression scale or a method of changing the same may be used, and the method is not limited to these.

When the aqueous liquid preparation of the present invention is used for evaluating pain felt by a subject upon subcutaneous injection by the above-described method for objectively evaluating pain, the pain is, for example, equal to or lower than that upon subcutaneous injection of physiological saline.

Hereinafter, a case where the protein contained in the aqueous liquid preparation is a growth hormone will be described in detail.

In the present invention, the concentration of the growth hormone contained in the aqueous liquid preparation is not particularly limited. However, the concentration is preferably 1 to 12mg/mL, more preferably 2 to 8 mg/mL. Suitable concentrations of growth hormone are, for example, 4mg/mL or 8 mg/mL.

The aqueous liquid preparation of growth hormone of the present invention is formed by containing a phosphate buffer as a buffer. The concentration of the phosphate buffer contained in the aqueous liquid preparation is preferably 1 to 20mM, more preferably 5 to 16mM, still more preferably 8 to 15mM, and still more preferably 8 to 12 mM. For example, the final concentration of phosphate buffer in an aqueous liquid formulation is adjusted to 10mM, 11mM, or 12 mM. The pH of the aqueous liquid preparation containing a buffering agent is preferably 5.5 to 7.2, more preferably 6.0 to 6.4, still more preferably 6.0 to 6.3, yet more preferably 6.0 to 6.2, particularly 6.2.

The aqueous liquid preparation of growth hormone of the present invention further contains a nonionic surfactant. The nonionic surfactant contained in the aqueous liquid preparation may be used alone or in combination with polysorbate, poloxamer, or the like. As polysorbates, polysorbate 20, polysorbate 80 are particularly suitable, and as poloxamers, poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) diol) is particularly suitable. The concentration of the nonionic surfactant contained in the aqueous liquid preparation is preferably 1 to 10mg/mL, more preferably 1 to 5mg/mL, still more preferably 1 to 3mg/mL, yet more preferably 1.5 to 2.5mg/mL, yet more preferably 1.8 to 2.2mg/mL, and is adjusted to 2mg/mL, for example. When poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is used as the nonionic surfactant, the concentration of poloxamer 188 in the aqueous liquid preparation is preferably 1-5 mg/mL, more preferably 1-3 mg/mL, even more preferably 1.5-2.5 mg/mL, even more preferably 1.8-2.2 mg/mL, and is adjusted to 2mg/mL, for example.

It is also envisaged that the aqueous liquid formulation of growth hormone is not disposable and is not single use, but is used repeatedly after opening. In this case, it is desirable to prevent deterioration of quality due to contamination of bacteria during repeated use. Accordingly, the aqueous liquid formulation of growth hormone of the present invention may contain a preservative. The preservative contained in the aqueous liquid preparation is not particularly limited as long as it is a pharmaceutically acceptable substance, and benzyl alcohol, phenol, benzoic acid, or a mixture thereof can be suitably used.

When phenol is used as a preservative, the concentration of phenol in the aqueous liquid preparation is preferably 1 to 10mg/mL, more preferably 2.5 to 4.0mg/mL, even more preferably 2.8 to 3.8mg/mL, for example 3.3 mg/mL. When benzyl alcohol is used as a preservative, the concentration of benzyl alcohol in the aqueous liquid preparation is preferably 2 to 20mg/mL, more preferably 7 to 12mg/mL, even more preferably 8 to 10mg/mL, for example 9 mg/mL. When benzoic acid is used as a preservative, the concentration of benzoic acid in the aqueous liquid preparation is preferably 1-20 mg/mL.

The aqueous liquid preparation of growth hormone of the present invention may further contain an isotonic agent. The isotonic agent contained in the aqueous liquid preparation is not particularly limited as long as it is a pharmaceutically acceptable one, and a sugar alcohol, a neutral salt, or a mixture thereof can be suitably used. The sugar alcohol may be D-mannitol as a suitable sugar alcohol, and the neutral salt may be sodium chloride as a suitable neutral salt. In the case of using D-mannitol as an isotonic agent, the concentration thereof in the aqueous liquid preparation is preferably 35 to 70mg/mL, more preferably 35 to 45mg/mL, for example 40 mg/mL. When sodium chloride is used as an isotonic agent, the concentration thereof in the aqueous liquid preparation is preferably 25 to 35mg/mL, more preferably 5.5 to 7.5mg/mL, for example 6.5 mg/mL. However, the concentration of the isotonic agent is not limited to these, and it is appropriately adjusted depending on the relationship with the concentration of other components in the aqueous liquid preparation. The concentration of the isotonic agent is usually adjusted so that the osmotic pressure ratio of the aqueous liquid preparation to physiological saline is 0.9 to 1.6, more preferably 0.9 to 1.1.

Suitable examples of the aqueous liquid preparation of growth hormone of the present invention are shown below.

(suitable example 1') an aqueous liquid preparation comprising 1 to 12mg/mL of growth hormone, 5 to 16mM of phosphate buffer, 1.8 to 2.2mg/mL of poloxamer 188, 2.8 to 3.8mg/mL of phenol, and 35 to 45mg/mL of D-mannitol and having a pH of 6.0 to 6.3. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 8-10 mg/mL benzyl alcohol can be added instead of phenol.

(suitable example 2') an aqueous liquid preparation comprising 4 to 8mg/mL of growth hormone, 5 to 16mM of phosphate buffer, 1.8 to 2.2mg/mL of poloxamer 188, 2.8 to 3.8mg/mL of phenol, and 35 to 45mg/mL of D-mannitol and having a pH of 6.0 to 6.3. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 8-10 mg/mL benzyl alcohol can be added instead of phenol.

(suitable example 3') an aqueous liquid preparation comprising 4 to 8mg/mL of growth hormone, 8 to 12mM of phosphate buffer, 2mg/mL of poloxamer 188, 3.3mg/mL of phenol and 40mg/mL of D-mannitol and having a pH of 6.2. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 9mg/mL of benzyl alcohol may be added instead of phenol.

(suitable example 4') an aqueous liquid preparation comprising 4mg/mL or 8mg/mL of growth hormone, 10mM of phosphate buffer, 2mg/mL of Poloxamer 188, 3.3mg/mL of phenol and 40mg/mL of D-mannitol and having a pH of 6.2. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 9mg/mL of benzyl alcohol may be added instead of phenol.

(suitable example 5') an aqueous liquid preparation comprising 4mg/mL or 8mg/mL of growth hormone, 10mM of phosphate buffer, 2mg/mL of Poloxamer 188, 3.3mg/mL of phenol and 40mg/mL of D-mannitol and having a pH of 6.2. However, the aqueous liquid preparation may further contain phosphoric acid and/or sodium hydroxide for adjusting pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. In addition, 9mg/mL of benzyl alcohol may be added instead of phenol.

The aqueous liquid formulation of growth hormone of the present invention may be used as a formulation filled in a vial, or as a formulation pre-filled in a syringe, i.e., a pre-filled syringe-type or cartridge-type formulation. When a preparation is prepared in a prefilled syringe type or cartridge type, the amount of liquid to be filled in 1 syringe is usually adjusted to 1 to 2mL, and for example, 1 syringe is filled so that the amount expressed becomes 1.5 mL.

Most of the growth hormone exists in the form of a monomer in an aqueous solution immediately after the growth hormone is prepared into an aqueous liquid preparation. However, when stored in the form of an aqueous liquid preparation, dimers are produced with the lapse of time, and the ratio of growth hormone present in the form of a monomer decreases. In addition, the quantitative value of growth hormone also decreases with the passage of time.

The aqueous liquid preparation of growth hormone of the present invention does not deteriorate even when stored at 2 to 8 ℃ for a long period of time. Here, the deterioration of growth hormone means that the ratio of growth hormone present in a monomer form becomes 98% or less, for example 95% or less, as compared with immediately after filling the aqueous liquid preparation as a preparation into a vial or a syringe. Further, the quantitative value of growth hormone is 98% or less, for example 95% or less, as compared with the case where the aqueous liquid preparation is filled into a vial or a syringe as a preparation, and this case is also considered to be deterioration of growth hormone.

The period during which storage can be performed without deterioration of growth hormone is preferably at least 18 months, more preferably 24 months, further preferably 30 months, and further preferably 36 months.

The aqueous liquid formulation of growth hormone of the present invention is administered to a patient by intramuscular injection or subcutaneous injection. Administration to a patient is performed by a physician, but is also contemplated by the patient himself or a patient's guardian. Such a type of drug requires the patient himself or a patient's guardian to follow the administration schedule, and if pain is involved in the injection, the patient's medication compliance may be reduced, resulting in failure to obtain a sufficient therapeutic effect. According to the aqueous liquid preparation of growth hormone of the present invention, since pain felt by a patient at the time of injection is reduced, the patient's medication compliance does not decrease, and all patients can enjoy the original therapeutic effect.

Although pain felt at the time of injection is subjective, a Visual Analogue Scale (VAS) is generally used as a method for objectively evaluating pain. The VAS is a method that generally includes the following steps. (1) A line drawn in the horizontal direction showing no pain at the left end and the highest conceivable pain at the right end is shown to the subject, (2) pain felt by the subject himself is shown on the line, and (3) the pain shown by the subject is quantified and summed up. The length of the straight line is typically 100 mm.

In addition, as a method for objectively evaluating pain, a numerical evaluation scale (NRS) is known. A numerical rating scale is a method that generally includes the following steps. (1) The subject was presented with a line of 11 grades that divided the pain from no pain to the most severe pain, (2) the pain felt by the subject himself was expressed by substituting the numerical values shown on the line, and (3) the numerical values were summed up.

In addition, as a method for objectively evaluating Pain, Faces Pain Scale (facial expression Scale) is also known. VAS and NRS are methods by which a subject expresses self-perceived pain by replacing the numerical values shown on the gauge, and the facial expression scale is a method by which a subject expresses self-perceived pain by replacing the human expression corresponding to the pain shown on the gauge.

As a method for objectively evaluating pain, a method of appropriately combining VAS, NRS, and a facial expression scale or a method of changing the same may be used, and the method is not limited to these.

In the case where the aforementioned method for objectively evaluating pain is used to evaluate pain felt by a subject when the subject is injected subcutaneously, the pain is equal to or less than that when physiological saline is injected subcutaneously.

Examples

The present invention will be described in further detail with reference to examples, but the present invention is not limited to the examples.

EXAMPLE 1 evaluation of pain in aqueous liquid preparation (1)

Aqueous liquid preparations of formulations 1 and 2 containing phosphate buffer and D-mannitol shown in Table 1 were prepared (Table 1). The phosphate buffer concentrations for formulations 1 and 2 were 20mM and 50mM, respectively. The osmotic pressure ratio of any formula is 1.0-1.1.

[ Table 1]

(Table 1) composition of aqueous liquid preparations (formulation 1 and formulation 2)

The evaluation of pain was carried out using the gauge shown in fig. 1, on which schematic expressions expressing pain were displayed. Using this gauge, pain was quantified on 10 scales with "no pain felt" as 1 and "pain considered most severe" as 10. The experiment was performed with 3 subjects. For subjects (males aged 20, 40 and 50), formulation 1 with a phosphate buffer concentration of 20mM, formulation 2 with a phosphate buffer concentration of 50mM and physiological saline were each administered 1 time avoiding the same administration site. Administration is carried out in such a way that the subject cannot discern the solution administered. The dosage is 125μL, the injection needle (BD micro-fine plus 31 G.times.5 mm, Becton, Dickinson and Company) was used to administer the drug subcutaneously to the thigh or upper arm of the subject. The gauge shown in fig. 1 is shown to the subject immediately after administration, and the expression most suitable for expressing pain at the time of injection is selected from the schematic expressions shown on the gauge. The selected pain values were summed up, and the average was obtained as a pain score. As a result, the pain score of formula 1 was 3.0, the pain score of formula 2 was 3.7, and the pain score of physiological saline was 2.0. These results show that: by reducing the concentration of phosphate buffer, pain upon injection of the aqueous liquid preparation can be reduced. In consideration of the safety of the subjects, the test was carried out using an aqueous liquid preparation containing no protein component.

EXAMPLE 2 evaluation of pain in aqueous liquid preparation (2)

Aqueous liquid preparations of formulations 3 to 5 (table 2) containing phosphate buffer, D-mannitol, poloxamer 188 and phenol shown in table 2 were prepared. However, only formulation 5 added an appropriate amount of sodium hydroxide to adjust the pH. The phosphate buffer concentrations for formulations 3, 4 and 5 were 10mM, 20mM and 50mM, respectively. The osmotic pressure ratio of any formula is 1.0-1.1.

[ Table 2]

(Table 2) composition of aqueous liquid preparation (formulation 3 to formulation 5)

The experiment was performed with 3 subjects. For subjects (males aged 20, 40 and 50), formulation 3 with a phosphate buffer concentration of 10mM, formulation 4 with a phosphate buffer concentration of 15mM, formulation 5 with a phosphate buffer concentration of 50mM, and physiological saline were each administered 1 time avoiding the same administration site. Administration is carried out in such a way that the subject cannot discern the solution administered. The dosage is set to 125μL, the administration is performed subcutaneously to the thigh or upper arm of the subject using a syringe needle (BD micro-fine plus 31Gx5mm, Becton, Dickinson and Company). Then, the pain score of each formulation was determined in the same manner as in example 1. As a result, the pain score of formula 3 was 2.0, the pain score of formula 4 was 2.0, the pain score of formula 5 was 4.3, and the pain score of physiological saline was 2.7. These results show that: by setting the concentration of the phosphate buffer to 15mM or less, pain upon injection of the aqueous liquid preparation can be clearly reduced. Surprisingly, particularly by setting the concentration of phosphate buffer to a concentration of 15mM or less, pain at the time of injection can be reduced even compared with physiological saline. In consideration of the safety of the subjects, the test was carried out using an aqueous liquid preparation containing no protein component.

EXAMPLE 3 evaluation of pain in aqueous liquid preparation (3)

Aqueous liquid preparations of formulations 6 to 7 containing phosphate buffer, D-mannitol, poloxamer 188 and phenol shown in table 3 were prepared (table 3). However, only formulation 6 added the appropriate amount of sodium hydroxide to adjust the pH. The phosphate buffer concentrations for formulations 6 and 7 were 10mM and 50mM, respectively. The osmotic pressure ratio of any formula is 1.0-1.1.

[ Table 3]

(Table 3) composition of aqueous liquid preparations (formulation 6 to formulation 7)

The pain of formulation 6 having a phosphate buffer concentration of 10mM, formulation 7 having a phosphate buffer concentration of 50mM and physiological saline was compared with 31 subjects. For subjects (including men and women aged 20-70), avoiding the same site of administration, saline was administered first, followed by formulations 6 and 7. Administration is carried out in such a way that the subject cannot discern the solution administered. The dosage is 125μL, the injection needle (BD micro-fine plus 31 G.times.5 mm, Becton, Dickinson and Company) was used to administer the drug subcutaneously to the thigh or upper arm of the subject. Then, the pain score of each formulation was determined in the same manner as in example 1. As a result, the pain score of formula 6 was 2.6, the pain score of formula 7 was 4.9, and the pain score of physiological saline was 2.7. Still show that: by reducing the concentration of phosphate buffer, pain upon injection can be reduced; and in the case where the concentration of phosphate buffer is 10mM, pain at the time of injection can be reduced even as compared with physiological saline. In consideration of the safety of the subjects, the test was carried out using an aqueous liquid preparation containing no protein component.

EXAMPLE 4 evaluation of stability of aqueous liquid preparation of human growth hormone (1)

Aqueous liquid preparations of 4 growth hormones were prepared according to GH formulations 1 to 4 shown in table 4. The concentrations of phosphate buffer for GH formula 1 and GH formula 2 were 50mM and 16mM, respectively. GH formula 3 and GH formula 4 differ only in the concentration of growth hormone, and the concentration of phosphate buffer is 10 mM.

[ Table 4]

(Table 4) composition of aqueous liquid preparation of growth hormone (GH formulation 1 to GH formulation 4)

The aqueous liquid preparations of GH formulations 1 to 4 were filled in 1.5mL each in a glass cartridge, and stored in the dark at a temperature of 2 to 8 ℃ (long-term storage test) or at 25 ℃ (accelerated test). During the storage, the pH, monomer (%) and quantitative (%) of the solution were successively determined. The monomer (%) and the quantitative (%) were calculated by the method described in example 6.

Table 5 shows the results of stability evaluation of GH formulation 1 at a phosphate buffer concentration of 50 mM. In the long-term storage test, the pH, monomer (%) and quantitative (%) were measured at the beginning of storage, 3 months after the beginning of storage, 6 months after, 9 months after, 12 months after, 18 months after and 24 months after. During the storage period in the long-term storage test, the pH and the quantitative (%) were hardly changed, and the monomer (%) was maintained at 99% or more. In the accelerated test, the pH, monomer (%) and quantitative (%) were measured 1 month after the start of storage, 2 months after storage, and 3 months after storage. During storage in the accelerated test, the pH and the quantitative (%) were hardly changed, and the monomer (%) was also maintained at 98% or more. These results show that: GH formula 1 is stable at temperatures of 2-8 ℃ for at least 24 months in the dark. In addition, when the standard values of the monomer amount (%) and the quantitative amount (%) were 98% or more and 100% or more, respectively, the following values were predicted from the values obtained by the long-term storage test: GH formula 1 satisfied the standard value even when stored in the dark at a temperature of 2 to 8 ℃ after 36 months from the start of storage.

[ Table 5]

(Table 5) stability of aqueous liquid preparation (GH formulation 1) (reference example)

Table 6 shows the results of stability evaluation of GH formulation 2 at a phosphate buffer concentration of 16 mM. In the long-term storage test, the pH, monomer (%) and quantitative (%) were measured at the start of storage, 1 month after the start of storage and 3 months after the start of storage. During the storage period in the long-term storage test, pH, monomer (%) and quantitative (%) were hardly changed, and values equivalent to those of GH formulation 1 were shown. In the accelerated test, the pH, monomer (%) and quantitative (%) were measured 1 month after the start of storage, 2 months after storage, and 3 months after storage. During storage in the accelerated test, pH, monomer (%) and quantitative (%) were hardly changed, and values equivalent to those of GH formulation 1 were shown. These results show that: GH formulation 2 has the same stability as GH formulation 1, and GH formulation 2 is predicted to be stable at temperatures of 2-8 ℃ in the dark for at least 24 months. Also shown is: when the standard values of the monomer amount (%) and the quantitative amount (%) are 98% or more and 100% or more, respectively, it is predicted that GH formulation 2 satisfies the standard values even after 36 months when stored in the dark at a temperature of 2 to 8 ℃.

[ Table 6]

(Table 6) stability of aqueous liquid formulation (GH formulation 2)

Tables 7 and 8 show the stability evaluation results of GH formulation 3 and GH formulation 4, respectively, at a phosphate buffer concentration of 10 mM. In the long-term storage test, the pH, monomer (%) and quantitative (%) were measured at the start of storage and about 1 month after the start of storage (after 4 weeks). In all formulations, pH, monomer (%) and quantitative (%) were hardly changed during the storage period of the long-term storage test, and showed the same values as those of GH formulation 1. In the accelerated test, pH, monomer (%) and quantitative (%) were measured about 1 month after the start of storage (after 4 weeks). In all formulations, pH, monomer (%) and quantitative (%) were hardly changed during storage in the accelerated test, and showed the same values as those of GH formulation 1. These results show that: GH formulations 3 and 4 have equivalent stability to GH formulation 1, with GH formulations 3 and 4 predicted to be stable at temperatures of 2-8 ℃ in the dark for at least 24 months. Also shown is: in the case where the standard values of the monomer amount (%) and the quantitative amount (%) are 98% or more and 100% or more, respectively, it is predicted that GH formulations 3 and 4 satisfy the standard values even after 36 months when stored in the dark at a temperature of 2 to 8 ℃.

[ Table 7]

(Table 7) stability of aqueous liquid formulation (GH formulation 3)

[ Table 8]

(Table 8) stability of aqueous liquid formulation (GH formulation 4)

EXAMPLE 5 evaluation of stability of aqueous liquid preparation of human growth hormone (2)

The long-term storage test from the start of storage to 9 months and the accelerated test from the start of storage to 3 months were performed for GH formula 3 and GH formula 4 at a phosphate buffer concentration of 10 mM. Table 9 and table 10 show the stability evaluation results of GH formulation 3 and GH formulation 4, respectively. In the long-term storage test, the pH, monomer (%) and quantitative (%) were measured at the beginning of storage, 1 month after the beginning of storage, 2 months after, 3 months after, 6 months after and 9 months after. In all formulations, pH, monomer (%) and quantitative (%) were hardly changed during the storage period of the long-term storage test, and showed the same values as those of GH formulation 1. In the accelerated test, pH, monomer (%) and quantitative (%) were measured 1 month after the start of storage, 2 months after and 3 months after. In all formulations, pH, monomer (%) and quantitative (%) were hardly changed during the storage period of the accelerated test, and showed the same values as those of GH formulation 1. These results show that: GH formulations 3 and 4 have equivalent stability to GH formulation 1, with GH formulations 3 and 4 predicted to be stable at temperatures of 2-8 ℃ in the dark for at least 24 months. Also shown is: in the case where the standard values of the monomer amount (%) and the quantitative amount (%) are 98% or more and 100% or more, respectively, it is predicted that GH formulations 3 and 4 satisfy the standard values even after 36 months when stored in the dark at a temperature of 2 to 8 ℃.

[ Table 9]

(Table 9) stability of aqueous liquid formulation (GH formulation 3)

[ Table 10]

(Table 10) stability of aqueous liquid formulation (GH formulation 4)

EXAMPLE 6 measurement of monomer (%) of growth hormone and quantification of growth hormone

The determination of monomer (%) was carried out by analyzing the sample by size exclusion HPLC (SE-HPLC). A high performance liquid chromatography device LC-20A (including a system controller CBM-20A, an online degassing unit DGU-20A5R, a liquid feeding unit LC-20AB, an automatic sampler SIL-20AC, a column temperature box CTO-20AC, an ultraviolet visible detector SPD-20AV or SPD-20A, Shimadzu corporation) is provided with a filling material with an exclusion limit molecular weight of 5 multiplied by 10⁵A particle diameter of 5μm.TSKgel G3000SWXL (internal diameter 7.8 mm. times.30 cm, Tosoh Corp.) as a column of hydrophilic silica gel. The solution was dissolved in water to 1000mL using a mobile phase (15.6 g of sodium dihydrogen phosphate dihydrate, 35.8g of disodium hydrogen phosphate dodecahydrate, and 11.7g of sodium chloride, and the solution was used with a pore size of 0.22μm membrane filter) to make the column in equilibrium, applying a sample diluted with pure water to a growth hormone concentration of 1-2 mg/mL to the column, and monitoring the absorbance at 215nm to prepare an elution curve. The flow rate was set to 0.5 mL/min. The monomer (%) was determined from the area of the peak corresponding to the monomer (monomer peak area) and the area of the peak corresponding to the dimer (dimer peak area) on the elution curve using the formula of monomer (%) = monomer peak area/(monomer peak area + dimer peak area) × 100 (%).

In addition, a standard curve was prepared by analyzing growth hormone with a known concentration by SE-HPLC, and the monomer area was interpolated thereon, thereby quantifying the growth hormone present in the form of a monomer in the sample. The quantitative value (%) of each sample was determined as the quantitative value of the growth hormone contained in the solution at the start of storage, which was determined from the calibration curve, assuming that the theoretical value was 100%.

Industrial applicability

According to the present invention, an aqueous liquid preparation containing a protein as an active ingredient, which is stable in storage in a solution state and reduces pain upon injection, can be provided.