Pharmaceutical Compositions Comprising Acylated Insulins

Abstract

The present invention relates to formulations of acylated insulin, pharmaceutical compositions of acylated insulin and long-acting GLP-1 compounds, and medical uses of said formulations and pharmaceutical compositions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Chinese invention patent application No. CN202110709826.5, filed on June 25, 2021, which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to the field of pharmaceutical compositions for treating medical conditions associated with diabetes, in particular to pharmaceutical compositions of acylated insulins, pharmaceutical compositions of acylated insulins and long-acting GLP-1 compounds, as well as medical uses of said pharmaceutical compositions. [Background technology]

[0003] Insulin can be used to treat diabetes and diabetes-related or -caused diseases, and is necessary to maintain normal metabolic regulation. However, natural insulin, such as human insulin, has a short duration of action, so patients need to inject it frequently, which causes many discomforts associated with injections.

[0004] Currently available insulin drugs include, for example, insulin lispro, insulin aspart, insulin aspart 30, insulin detemir, insulin glargine, insulin degludec, etc. However, to date, no insulin products have been approved for sale that are administered less frequently than once a day by subcutaneous injection. Therefore, those skilled in the art have been working to obtain insulin derivatives or analogs with better efficacy, longer duration of action, and less frequent injections, in order to improve the inconvenience and discomfort caused by frequent insulin injections.

[0005] CN101573133B and WO2009 / 010428 disclose formulations containing PEGylated extended insulin, which have a longer duration of action compared to unmodified insulin.

[0006] WO2013086927A1 and WO2018 / 024186 disclose acylated derivatives of long-acting human insulin analogues.

[0007] Moreover, glucagon-like peptide 1 (GLP-1) and its analogs and derivatives are another very important peptides for treating diabetes and diabetes-related diseases. GLP-1 is an intestinal peptide hormone secreted mainly by L cells in the terminal ileum, colon and rectal mucosa, which promotes glucose-dependent insulin secretion, β-cell differentiation and proliferation, and inhibits glucagon secretion. As the number of type 2 diabetes patients is rapidly increasing worldwide, there is an increasing need for more effective and easy-to-administer drugs. For example, a combination formulation containing two active ingredients, insulin and GLP-1 peptide, could be a highly effective therapeutic agent.

[0008] WO2009 / 063072 discloses a pharmaceutical composition comprising a GLP-1 peptide (e.g., liraglutide) and a basal insulin derivative (e.g., insulin degludec), but the composition still fails to achieve an injection frequency lower than once a day.

[0009] Therefore, there is still a need for combination formulations with better physical and chemical stability, longer duration of action, and better efficacy. Summary of the Invention

[0010] To overcome or ameliorate at least one of the disadvantages of the prior art or to provide a useful substitute, a first aspect of the present invention provides a novel pharmaceutical composition comprising an acylated insulin, which has unexpectedly and significantly increased potency, efficacy or potency, longer duration of action, longer in vivo half-life, better bioavailability, better safety, and more satisfactory physical stability, chemical stability and solubility compared to marketed formulations of insulin degludec (trade name "Tresiba" or "Tresiba") or other insulin derivatives.

[0011] The second aspect of the present invention further provides a novel pharmaceutical composition comprising an acylated insulin and a novel long-acting GLP-1 compound, which is not only not weakened but also unexpectedly exhibits better efficacy, physical stability, chemical stability, sustained action time, and in vivo half-life than a single preparation of the acylated insulin and the GLP-1 compound, and in particular, the acylated insulin and the GLP-1 compound in the pharmaceutical composition of the present invention unexpectedly have synergistic efficacy such as synergistic hypoglycemic effect and Hb1Ac lowering effect. In addition, the combination preparation of the present invention unexpectedly has better efficacy, sustained action time, in vivo half-life, physical stability, chemical stability, etc. than other combination preparations of acylated insulin and long-acting GLP-1 compound (e.g., combination preparation of liraglutide and insulin degludec (trade name: Xultophy)). The combination preparation comprising the acylated insulin and a GLP-1 compound provided by the present invention can provide excellent long pharmacokinetic (hereinafter also referred to as PK) properties that enable subcutaneous treatment of diabetic patients twice a week, once a week, once every two weeks, or less frequently.

[0012] The pharmaceutical composition according to the first aspect of the present invention comprises acylated insulin, 2.3 moles of zinc ions / 6 moles of acylated insulin, 45-60 mM phenol, 0-10 mM m-cresol, 10-20 mM NaCl, 1.5% (w / w) glycerin, and 5-10 mM Na2HPO4,

[0013] The insulin base of the acylated insulin is A14E, B16H, B25H, desB30 human insulin (SEQ ID NO:1 and SEQ ID NO:2 represent the A-chain and B-chain, respectively) or A14E, B16E, B25H, desB30 human insulin (SEQ ID NO:3 and SEQ ID NO:4 represent the A-chain and B-chain, respectively), and the acyl group of the acylated insulin is linked to the lysine residue or the amino group of the N-terminal amino acid residue of the insulin base, and the acyl group is represented by the following formula (D): W1-(W2) m -(W3) n - (D), During the ceremony, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; W3 is a neutral, alkylene glycol-containing amino acid residue; W2 is an acidic amino acid residue, W1 is an aliphatic diacid containing 20 to 24 carbon atoms, provided that, formally, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid; W1, W2 and W3 are connected by an amide bond; and The order of appearance of W2 and W3 in formula (D) is independently interchangeable.

[0014] In one embodiment, n is 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18 or 19, preferably n is 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17 or 18, preferably n is 5, 6, 7, 8, 11, 12, 13, 14, 15 or 16, preferably n is 5, 6, 7, 8, 11, 12, 13, 14 or 15.

[0015] In one embodiment, m is an integer from 1 to 6, preferably m is 1, 2, 3 or 4, preferably m is 1 or 2, preferably m is 1.

[0016] In another embodiment, W1 is an aliphatic diacid containing 20-23 carbon atoms, preferably W1 is an aliphatic diacid containing 20, 21 or 22 carbon atoms, provided that, formally, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid.

[0017] In one embodiment, W3 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN- (CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2 )2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2)3-O -(CH2)4-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O-(CH2 )2-O-CH2-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)3-O-(CH2) 2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O-( W3 is -HN-(CH2)2-O-(CH2)2-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)3-O-CH2-CO-, or -HN-(CH2)4-O-(CH2)4-O-CH2-CO-, preferably W3 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-.

[0018] In another embodiment, W2 is an amino acid residue selected from γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp, preferably W2 is selected from γGlu or βAsp.

[0019] In another embodiment, W1 is HOOC-(CH2). 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-, preferably W1 is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0020] In one embodiment, formula (D) is linked by the C-terminus of W3 to a lysine residue or the amino group of the N-terminal amino acid residue of the insulin parent.

[0021] In one embodiment, the acyl moiety is linked to the epsilon amino group of a lysine residue of the insulin parent.

[0022] In one embodiment the acylated insulin is A14E, B16H, B25H, B29K(N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-6xOEG) , desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosane diacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosane diacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K(N(ε)-docosane diacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin Phosphorus, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosane diacyl-γGlu-12xOEG), desB30 human insulin,A14E, B16H, B25H, B29K (N(ε)-tricosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14 E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B 16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H,B25H, B29K (N(ε)-eicosane diacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-24xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25 H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16E, B25H, B 29K(N(ε)-docosane diacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H,B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heneicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tricosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25 H, B29K (N(ε)-tetracosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B2 5H, B29K (N(ε)-docosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25 H, B29K (N(ε)-eicosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H,selected from the insulins B29K(N(ε)-eicosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosane diacyl-γGlu-20xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-20xOEG), desB30 human insulin,

[0023] In one embodiment the acylated insulin is A14E, B16H, B25H, B29K(N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N (ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin,

[0024] In one embodiment the acylated insulin is selected from A14E, B16H, B25H, B29K(N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin or A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin.

[0025] In one embodiment, the content of acylated insulin is higher than about 0.3 mM, preferably about 0.3 to 9 mM, preferably about 0.6 to 8.4 mM, preferably about 0.6 to 7.2 mM, preferably about 0.6 to 6.0 mM, preferably about 0.6 to 4.2 mM, preferably about 0.6 to 3.6 mM, preferably about 0.6 to 3.0 mM, preferably about 0.6 to 2.4 mM, preferably about 0.6 to 2.1 mM, preferably about 0.9 to 1.8 mM, preferably about 0.9 to 1.5 mM, preferably about 1.2 to 1.5 mM.

[0026] In another embodiment, the phenol content is about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, or about 60 mM.

[0027] In another embodiment, the content of m-cresol is about 0 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.

[0028] In another embodiment, the NaCl content is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.

[0029] In another embodiment, the pharmaceutical composition has a pH of 6.5 to 8.5, preferably a pH of 6.5 to 8.0, preferably a pH of 7.0 to 7.8, preferably a pH of 7.2 to 7.6, and more preferably a pH of 7.4.

[0030] In one embodiment, the pharmaceutical composition according to the first aspect of the invention comprises: about 0.9-1.5 mM acylated insulin, about 2.3 moles zinc ion / 6 moles acylated insulin, about 45 mM phenol, about 10 mM m-cresol, about 20 mM NaCl, about 15 mg / mL glycerin, about 5-10 mM Na2HPO4, and a pH value of about 6.5-8.0; Among them, the above acylated insulins are A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl -γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)- docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H , B29K(N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin.

[0031] In one embodiment, the pharmaceutical composition according to the first aspect of the invention comprises: about 1.2 mM acylated insulin, about 2.3 moles zinc ion / 6 moles acylated insulin, about 45 mM phenol, about 10 mM m-cresol, about 20 mM NaCl, about 15 mg / mL glycerin, and about 5 mM Na2HPO4, and has a pH value of about 7.4; Among them, the above acylated insulins are A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl -γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)- docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H , B29K(N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin.

[0032] A second aspect of the invention provides a pharmaceutical composition comprising an insulinotropic GLP-1 compound and an acylated insulin, Among them, the molar ratio of the insulinotropic GLP-1 compound to the acylated insulin is at least about 1:100, preferably at least about 3:100, preferably at least about 5:100, preferably at least about 8:100, preferably about (3:100) to (100:100), preferably about (5:100) to (80:100), preferably about (8:100) to (50:100), preferably about (10:100) to (50:100), preferably about (13:100) to (50:100), preferably about (13:100) to (40:100), preferably about (13:100) to (35:100), preferably about (13:100) to (27:100), preferably about (13:100) to (20:100); The acylated insulin is the acylated insulin in the pharmaceutical composition according to the first aspect of the invention.

[0033] In one embodiment, the insulinotropic GLP-1 compound is an N-ε 26 -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl][Aib8, Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 30-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl](Val 8 Glu 22 Lys 30 Arg 26、34 -GLP-1(7-37)) peptide, or N-ε 23 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl](Val 8 Glu 22 Lys 23 Arg 26、34 -GLP-1(7-37) peptide, In another embodiment, the insulinotropic GLP-1 compound is represented by formula (B): [Acy-(L1) r -(L2) q ]-G1 (B), where G1 is a GLP-1 analog having Arg at position 34 and Ala or Gly at position 8, corresponding to GLP-1(7-37) (SEQ ID NO: 5), and [Acy-(L1) r -(L2) q is a substituent linked to the ε-amino group of the Lys residue at position 26 of the GLP-1 analog, wherein r is an integer of 1 to 10 and q is 0 or an integer of 1 to 10; Acy is an aliphatic diacid containing 20 to 24 carbon atoms, provided that, formally, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; L1 is an amino acid residue selected from γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; L2 is a neutral alkylene glycol-containing amino acid residue; Acy, L1 and L2 are linked by an amide bond; and The order of appearance of L1 and L2 in formula (B) is independently interchangeable.

[0034] The inventors have surprisingly found that when the molar ratio of the insulinotropic GLP-1 compound described in the pharmaceutical composition of the second aspect of the present invention to the acylated insulin is about 8:100, preferably about 10:100, preferably about 13:100, it is possible to achieve a better efficacy than a single formulation containing twice the amount of acylated insulin, and when the molar ratio of the insulinotropic GLP-1 compound described in the pharmaceutical composition of the second aspect of the present invention to the acylated insulin is about 20:100, it is possible to simultaneously achieve an even better efficacy than either a single formulation containing twice the amount of acylated insulin or a single formulation containing twice the amount of GLP-1 compound.

[0035] In one embodiment, G1 is [Gly8, Arg34]GLP-1-(7-37) peptide (SEQ ID NO:6) or [Arg34]GLP-1-(7-37) peptide (SEQ ID NO:7), preferably [Gly8, Arg34]GLP-1-(7-37) peptide.

[0036] In another embodiment, r is 1, 2, 3, 4, 5 or 6, preferably, r is 1, 2, 3 or 4, preferably, r is 1 or 2, preferably, r is 1.

[0037] In another embodiment, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8; preferably, q is 0, 1, 2, 3 or 4; more preferably, q is 0, 1 or 2.

[0038] In another embodiment, Acy is an aliphatic diacid containing 20 to 23 carbon atoms, preferably Acy is an aliphatic diacid containing 20, 21, or 22 carbon atoms, provided that, formally, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid.

[0039] In one embodiment, L2 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN- (CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2 )2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2)3-O -(CH2)4-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O-(CH2 )2-O-CH2-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)3-O-(CH2) 2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O-( L2 is -HN-(CH2)2-O-(CH2)2-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)3-O-CH2-CO-, or -HN-(CH2)4-O-(CH2)4-O-CH2-CO-, preferably L2 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-.

[0040] In another embodiment, L1 is selected from γGlu or βAsp, preferably L1 is γGlu.

[0041] In another embodiment, Acy is HOOC-(CH2). 18 -CO-, HOOC-(CH2) 19-CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-, and preferably, Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0042] In one embodiment, Acy, L1 and L2 in formula (B) are linked in order by an amide bond, and the C-terminus of L2 is linked to the ε-amino group of the Lys residue at position 26 of the above GLP-1 analog.

[0043] In one embodiment, the insulinotropic GLP-1 compound is N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26-[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26-[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26-[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26-(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, or N-ε 26 -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide.

[0044] In another embodiment the acylated insulin is A14E, B16H, B25H, B29K(N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), desB30 human Insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin insulin, A14E, B16H, B25H, B29K(N(ε)-docosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-heneicosane diacyl-γGlu-12xOEG), desB30 human insulin,A14E, B16H, B25H, B29K (N(ε)-tricosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14 E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B 16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H,B25H, B29K (N(ε)-eicosane diacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-24xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25 H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16E, B25H, B 29K(N(ε)-docosane diacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H,B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heneicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tricosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25 H, B29K (N(ε)-tetracosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B2 5H, B29K (N(ε)-docosane diacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25 H, B29K (N(ε)-eicosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosane diacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H,The insulin is selected from the insulins B29K(N(ε)-eicosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosane diacyl-γGlu-20xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K(N(ε)-docosane diacyl-γGlu-20xOEG), desB30 human insulin.

[0045] In one embodiment, the content of acylated insulin is higher than about 0.3 mM, preferably about 0.3 to 9 mM, preferably about 0.6 to 8.4 mM, preferably about 0.6 to 7.2 mM, preferably about 0.6 to 6.0 mM, preferably about 0.6 to 4.2 mM, preferably about 0.6 to 3.6 mM, preferably about 0.6 to 3.0 mM, preferably about 0.6 to 2.4 mM, preferably about 0.6 to 2.1 mM, preferably about 0.9 to 1.8 mM, preferably about 0.9 to 1.5 mM, preferably about 1.2 to 1.5 mM.

[0046] In one embodiment, the composition of the second aspect of the invention further comprises zinc ions, glycerin, phenol, m-cresol, NaCl, and / or Na2HPO4.

[0047] In one embodiment, the zinc ion content is at least about 1.5 moles zinc ions / 6 moles acylated insulin, preferably about 1.5-12 moles zinc ions / 6 moles acylated insulin, preferably about 1.5-10 moles zinc ions / 6 moles acylated insulin, preferably about 1.5-8 moles zinc ions / 6 moles acylated insulin, preferably about 1.5-6 moles zinc ions / 6 moles acylated insulin, preferably about 1.5-4.5 moles zinc ions / 6 moles acylated insulin, preferably about 1.5-3.5 moles zinc ions / 6 moles acylated insulin, preferably about 1.5-2.3 moles zinc ions / 6 moles acylated insulin.

[0048] In one embodiment, the glycerin content is about 2.5% (wt / wt) or less, preferably about 2% (wt / wt) or less, preferably about 0.3% to about 2% (wt / wt), preferably about 0.5% to about 1.8% (wt / wt), preferably about 0.7% to about 1.8% (wt / wt), preferably about 1% to about 1.8% (wt / wt), preferably about 1.5% to about 1.7% (wt / wt).

[0049] In another embodiment, the phenol content is about 30 to 70 mM, preferably about 40 to 65 mM, preferably about 45 to 60 mM, preferably about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, or about 65 mM.

[0050] In another embodiment, the content of m-cresol is about 0 to 35 mM, preferably about 0 to 20 mM, preferably about 0 to 10 mM, preferably about 0 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM.

[0051] In another embodiment, the NaCl content is about 0 to 150 mM, preferably about 5 to 120 mM, preferably about 10 to 120 mM, preferably about 10 to 100 mM, preferably about 10 to 75 mM, preferably about 10 to 50 mM, preferably about 10 to 30 mM, preferably about 10 to 20 mM, preferably about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.

[0052] In another embodiment, the content of Na2HPO4 is about 0 to 75 mM, preferably about 5 to 60 mM, preferably about 5 to 50 mM, preferably about 5 to 25 mM, preferably about 5 to 15 mM, preferably about 5 to 10 mM.

[0053] In another embodiment, the pH of the pharmaceutical composition according to the second aspect of the present invention is about 6.5 to 8.5, preferably about 6.8 to 8.2, preferably about 7.0 to 8.2, preferably 7.2 to 7.6, more preferably 7.4 or 7.6.

[0054] In one embodiment, the pharmaceutical composition of the second aspect of the invention comprises: About 0.9 to 1.5 mM acylated insulin; an insulinotropic GLP-1 compound having a molar ratio to the acylated insulin of at least about 8:100, preferably about (8:100) to (50:100), preferably about (10:100) to (50:100), preferably about (13:100) to (50:100), preferably about (13:100) to (40:100), preferably about (13:100) to (35:100), preferably about (13:100) to (27:100), preferably about (13:100) to (20:100); Approximately 1.5-2.3 moles of zinc ions per 6 moles of acylated insulin; Approximately 45-60 mM phenol, About 0-10 mM m-cresol; Approximately 10-20 mM NaCl, Approximately 15-17 mg / mL glycerin, About 5-10 mM Na2HPO4, The pH value is about 6.5 to 8.0. Among them, the above acylated insulins are A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl -γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B 29K(N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, and The insulinotropic GLP-1 compounds are N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26-[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide.

[0055] In one embodiment, the pharmaceutical composition of the second aspect of the invention comprises: About 1.2 mM acylated insulin; at least about 0.096 mM, preferably about 0.096 to 0.6 mM, preferably about 0.12 to 0.6 mM, preferably about 0.16 to 0.6 mM, preferably about 0.16 to 0.48 mM, preferably about 0.16 to 0.42 mM, preferably about 0.16 to 0.32 mM, preferably about 0.16 to 0.24 mM of an insulinotropic GLP-1 compound; Approximately 1.5-2.3 moles of zinc ions per 6 moles of acylated insulin; Approximately 45-60 mM phenol, About 0-10 mM m-cresol; Approximately 10-20 mM NaCl, Approximately 15-17 mg / mL glycerin, About 5-10 mM Na2HPO4, The pH value is about 6.5 to 8.0. Among them, the above acylated insulins are A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl -γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosane diacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B 29K(N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, and The insulinotropic GLP-1 compounds are N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26-[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide.

[0056] A third aspect of the present invention provides the use of a pharmaceutical composition according to the first and second aspects of the present invention for use as a medicament.

[0057] A fourth aspect of the invention provides a pharmaceutical composition according to the first and second aspects of the invention for treating or preventing diabetes, hyperglycemia and / or impaired glucose tolerance.

[0058] A fifth aspect of the invention provides the use of a pharmaceutical composition according to the first and second aspects of the invention in the manufacture of a medicament for the treatment or prevention of diabetes, hyperglycemia and / or impaired glucose tolerance.

[0059] A sixth aspect of the invention provides a method of treating or preventing diabetes, hyperglycemia and / or impaired glucose tolerance comprising administering an effective amount of a pharmaceutical composition according to the first and second aspects of the invention. [Brief description of the drawings]

[0060] [Figure 1a] 1 shows the hypoglycemic effect on db / db mice of a single formulation of Compound 2 and a single formulation of Compound 11 at a medium dose, a pharmaceutical composition containing Compound 2 and Compound 11 at a low dose, and a solvent. [Figure 1b] Corresponding to Figure 1a, the ΔAUC of the hypoglycemic effect on db / db mice of a single formulation of compound 2 and a single formulation of compound 11 at a medium dose, a pharmaceutical composition containing compound 2 and compound 11 at a low dose, and a solvent are shown. [Figure 1c] 1 shows the hypoglycemic effect of a single formulation of Compound 2, a single formulation of Compound 11, a pharmaceutical composition containing Compound 2 and Compound 11, and a solvent at a moderate dose in db / db mice. [Figure 1d] Corresponding to FIG. 1c, the ΔAUC of the hypoglycemic effect on db / db mice of a single formulation of compound 2, a single formulation of compound 11, a pharmaceutical composition containing compound 2 and compound 11, and a solvent at a moderate dose is shown. [Figure 1e] 1 shows the HbA1c lowering effect in db / db mice of a single formulation of compound 2 and a single formulation of compound 11 at a medium dose, a pharmaceutical composition containing compound 2 and compound 11 at a low dose, and a solvent. [Figure 1f] 1 shows the HbA1c lowering effect in db / db mice of a single formulation of Compound 2, a single formulation of Compound 11, a pharmaceutical composition containing Compound 2 and Compound 11, and a solvent at a moderate dose. [Figure 2a] The blood glucose lowering effects on db / db mice of a single formulation of compound 2 at a high dose, a single formulation of compound 11 and a degludecryraglutide composition (control composition), a pharmaceutical composition containing compound 2 and compound 11 at a low dose, and a solvent are shown. [Figure 2b]Corresponding to Figure 2a, the AUC of the hypoglycemic effect on db / db mice of a single formulation of compound 2, a single formulation of compound 11 and a degludecryraglutide composition at high doses, a pharmaceutical composition containing compound 2 and compound 11 at low doses, and a solvent are shown. [Figure 2c] The HbA1c lowering effects in db / db mice of a single formulation of compound 2 at a high dose, a single formulation of compound 11, a degludecryraglutide composition (control composition), a pharmaceutical composition containing compound 2 and compound 11, a pharmaceutical composition containing compound 2 and compound 11 at a medium dose, a pharmaceutical composition containing compound 2 and compound 11 at a low dose, and a solvent are shown. [Figure 2d] The TG lowering effect in db / db mice of a single formulation of compound 2, a single formulation of compound 11 and a degludecryraglutide composition at high doses, a pharmaceutical composition containing compound 2 and compound 11 at low doses, and a solvent is shown. [Figure 3a] 1 shows the hypoglycemic effect of a single preparation of Compound 2, a single preparation of Control Example 1, a degludec preparation, and the solvent in db / db mice. [Figure 3b] 1 shows ΔAUC of the hypoglycemic effect on db / db mice of a single formulation of Compound 2, a single formulation of Control Example 1, a degludec formulation, and the solvent. [Figure 3c] 1 shows the hypoglycemic effect of a single preparation of Compound 2, a single preparation of Control Example 1, a degludec preparation, and a solvent in db / db mice, in which an OGTT was performed 48 hours after the first administration. [Figure 3d] 1 shows the HbA1c lowering effects of a single formulation of Compound 2, a single formulation of Control Example 1, a degludec formulation, and a solvent in db / db mice. [Figure 3e] 1 shows the TG lowering effect of a single preparation of Compound 2, a single preparation of Control Example 1, a degludec preparation, and a solvent in db / db mice. [Figure 4a] 1 shows the hypoglycemic effect of a single formulation of Compound 2, a degludec formulation, and a solvent in GK rats. [Figure 4b] FIG. 4a corresponds to FIG. 4a, and shows the AUC of the hypoglycemic effect of a single formulation of Compound 2, a degludec formulation, and a vehicle on GK rats. [Figure 4c]1 shows the HbA1c lowering effect of a single formulation of Compound 2, a degludec formulation, and a solvent in GK rats. [Figure 4d] 1 shows the TG lowering effect of a single formulation of Compound 2, a degludec formulation, and a vehicle in GK rats. [Figure 4e] 1 shows the GSP lowering effect of a single formulation of Compound 2, a degludec formulation, and a vehicle in GK rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] definition Here, the term insulin includes naturally occurring insulin, such as human insulin, as well as insulin analogues and insulin derivatives thereof.

[0062] The term insulin analogue formally includes polypeptides having a molecular structure derivable from the structure of naturally occurring insulin (e.g. human insulin) by omission and / or substitution (replacement) of one or more amino acid residues present in natural insulin and / or by addition of one or more amino acid residues. The added and / or substituted amino acid residues may be codable amino acid residues, or other naturally occurring amino acid residues, or purely synthetic amino acid residues. Preferably, the added and / or substituted amino acid residues are codable amino acid residues.

[0063] The term "insulin derivative" herein refers to naturally occurring insulin or insulin analogues that have been chemically modified, which modification may be, for example, the introduction of a side chain at one or more positions of the insulin backbone, or the oxidation or reduction of groups of amino acid residues on insulin, or the conversion of free carboxy groups into ester groups, or the acylation of free amino or hydroxy groups. The acylated insulins of the present invention belong to the insulin derivatives.

[0064] The term "parent insulin" refers to the insulin portion of an insulin derivative or acylated insulin (also referred to herein as parent insulin), e.g., in the present invention, to the portion of an insulin derivative or acylated insulin that is not linked to a side chain or has an additional acyl group. The parent insulin may be a naturally occurring insulin, such as human insulin or porcine insulin. Alternatively, the parent insulin may be an insulin analogue.

[0065] Herein, the term "amino acid residue" includes amino acids in which a hydrogen atom has been removed from an amino group, and / or a hydroxyl group has been removed from a carboxy group, and / or a hydrogen atom has been removed from a sulfhydryl group. Less precisely, an amino acid residue may be referred to as an amino acid.

[0066] Unless otherwise specified, all amino acids referred to herein are L-amino acids.

[0067] Here, the term alkylene glycol includes oligo / polyalkylene glycol moieties and monoalkylene glycol moieties. Monoalkylene glycol and polyalkylene glycol include, for example, chains based on monoethylene glycol and polyethylene glycol, chains based on monopropylene glycol and polypropylene glycol, and chains based on monobutylene glycol and polybutylene glycol, i.e., chains based on the repeating units -CH2CHO-, -CH2CH2CHO- or -CH2CH2CH2CHO-. The alkylene glycol moieties may be monodisperse (having a clearly defined length / molecular weight) or polydisperse (having an ill-defined length / average molecular weight). The monoalkylene glycol moieties include -OCH2CHO-, -OCH2CH2CHO- or -OCH2CH2CH2CHO- with different groups at each end.

[0068] The term "aliphatic diacid" includes straight or branched chain aliphatic dicarboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non-limiting examples of aliphatic diacids are adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, eicosane diacid, docosane diacid, and tetracosane diacid.

[0069] The term "basal insulin" refers to insulin that has a longer duration of action than regular or normal human insulin.

[0070] As used herein, "efficacy" or "potency" refers to the ability of a drug or active compound to produce a certain action or effect (e.g., hypoglycemic effect). For example, when administered at the same dose, the insulin derivatives of the present invention produce a higher hypoglycemic effect or effect than insulin degludec or other existing insulin derivatives.

[0071] The term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other conditions that cause hyperglycemia. The term is used for metabolic disorders in which the pancreas produces insufficient insulin or the body's cells fail to respond properly to insulin, preventing cells from absorbing glucose. As a result, glucose accumulates in the blood.

[0072] Type 1 diabetes, also called insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, is caused by the destruction of β-cells and generally results in absolute insulin deficiency. Type 2 diabetes, also called non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with primary insulin resistance and thus relative insulin deficiency and / or a primary insulin secretory disorder with insulin resistance.

[0073] The term "GLP-1 analog" or "analog of GLP-1" as used herein refers to a peptide or compound that is a variant of human glucagon-like peptide-1 (GLP-1(7-37)), in which one or more amino acid residues of GLP-1(7-37) have been substituted and / or one or more amino acid residues have been deleted and / or one or more amino acid residues have been added thereto. Specifically, the sequence of GLP-1(7-37) is set forth in SEQ ID NO:5 in the Sequence Listing. The peptide having the sequence set forth in SEQ ID NO:5 may be referred to as "native" GLP-1 or "native" GLP-1(7-37).

[0074] In the sequence listing, the first amino acid residue (histidine) of SEQ ID NO:5 is numbered as 1. However, in the following, in accordance with established convention in the field, the histidine residue is numbered as 7 and subsequent amino acid residues are numbered accordingly, ending with glycine numbered as 37. Thus, in general, the amino acid residue numbering or position numbering of the GLP-1(7-37) sequence referred to herein is the sequence starting with His at position 7 and ending with Gly at position 37.

[0075] [Gly8, Arg34]GLP-1-(7-37) peptide is a GLP-1 analog having Gly and Arg at positions corresponding to positions 8 and 34 of GLP-1(7-37) (SEQ ID NO:5), respectively. [Arg34]GLP-1-(7-37) peptide is a GLP-1 analog having Arg at positions corresponding to position 34 of GLP-1(7-37) (SEQ ID NO:5). Specifically, the amino acid sequences of [Gly8, Arg34]GLP-1-(7-37) peptide and [Arg34]GLP-1-(7-37) peptide are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively, in the sequence listing.

[0076] In the context of a GLP-1 peptide or analog thereof, the term "derivative" as used herein refers to a chemically modified GLP-1 peptide or analog in which one or more substituents are already covalently attached to said peptide. The substituents may also be referred to as side chains.

[0077] In this specification, the naming of insulin or GLP-1 compounds is performed according to the following principles: according to mutations and modifications (e.g., acylation) of human insulin or natural GLP-1(7-37), the name is given. The naming of the acyl moiety is according to the IUPAC nomenclature and in other cases, according to the peptide nomenclature. For example, the following acyl moieties are named:

[0078] [ka]

[0079] For example, it can be named as "eicosane diacyl-γGlu-OEG-OEG", "eicosane diacyl-γGlu-2xOEG" or "eicosane diacyl-gGlu-2xOEG" or "19-carboxynonadecanoyl-γGlu-OEG-OEG", where OEG stands for the abbreviation for the group -NH(CH2)2O(CH2)2OCH2CO- (i.e., 2-[2-(2-aminoethoxy)ethoxy]acetyl) and γGlu (and gGlu) is the abbreviation for the amino acid γ-glutamic acid in the L-configuration. Alternatively, the acyl group moiety can be named according to IUPAC nomenclature (OpenEye, IUPAC format). In accordance with this nomenclature, the acyl group moiety of the present invention is referred to as [2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl] or [2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butyryl]-amino]-ethoxy]-ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl].

[0080] For example, the sequence / structure shown below is "A14E, B16H, B25H, B29K(N εGlu-2-Glu-2xOEG), desB30 human insulin” or “A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-2xOEG), desB30 human insulin”, in which the amino acid Y at position A14 in human insulin has been mutated to E, the amino acid Y at position B16 in human insulin has been mutated to H, the amino acid F at position B25 in human insulin has been mutated to H, and the amino acid K at position B29 in human insulin has been mutated to the ε nitrogen (N ε Glu-2xOEG) and is missing the amino acid T at position B30 in human insulin.

[0081] [ka]

[0082] Insulin is a polypeptide hormone secreted from β-cells in the pancreas and consists of two polypeptide chains, A and B, which are linked by two interchain disulfide bonds, and the A chain is characterized by having one intrachain disulfide bond.

[0083] The nucleic acid sequence encoding each insulin analog polypeptide can be prepared synthetically by established standard methods, such as those described in Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869, or Matthes et al. (1984) EMBO Journal 3:801-805.

[0084] In a specific embodiment, the pharmaceutical composition includes at least one pharma- ceutically acceptable excipient. The term "excipient" as used herein broadly refers to any ingredient other than the active therapeutic ingredient. An excipient may be an inert, inactive, and / or non-pharmaceutical active ingredient. Excipients may be used for various purposes, such as, for example, carriers, solvents, binders, lubricants, glidants, diluents, and / or to improve the administration and / or absorption of the active ingredient. The formulation of a drug active ingredient with different excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th Edition (1995) and any updated editions).

[0085] In some embodiments, a particular value referred to herein and given in relation to a number or interval may be understood to be at or near that particular value. In some embodiments, the term "about" refers to ±10% of the stated value, e.g., about 100 mM includes 100 mM ± 10 mM, 10% includes 10% ± 1%, etc.

[0086] As used herein, the term "effective amount" refers to a dose sufficient to effect treatment of a patient compared to non-treatment.

[0087] For the convenience of the patient, it is assumed that the patient prefers the time interval (time delay) between the administration of the acylated insulin pharmaceutical composition of the present invention and the next administration of the acylated insulin pharmaceutical composition of the present invention to be the same or approximately the same length in days. Furthermore, it may be expected that the patient prefers to administer the acylated insulin pharmaceutical composition once a week, i.e., on the same day of the week, such as every Sunday. Averaged over a period of one month, six months or one year, this amounts to administering the acylated insulin pharmaceutical composition every sixth day, and no more frequently. For some patients, it may be necessary to administer the acylated insulin pharmaceutical composition every fifth day or approximately every fifth day, and no more frequently, averaging over a period of one month, six months or one year. For other patients, it may be necessary to administer the acylated insulin pharmaceutical composition every fourth day or approximately every fourth day, and no more frequently, averaging over a period of one month, six months or one year. For other patients, it may be necessary to administer the acylated insulin pharmaceutical composition every third day or about every third day on average over a period of one month, six months or one year, and not more frequently. Still other patients may find it advantageous to administer the acylated insulin pharmaceutical composition twice a week, for example about 3-4 days between each administration, on average over a period of one month, six months or one year. For some patients, it may be necessary to administer the acylated insulin pharmaceutical composition every second day or about every second day on average over a period of one month, six months or one year, and not more frequently. For other patients, it may be necessary to administer the acylated insulin pharmaceutical composition every other day or about every other day on average over a period of one month, six months or one year, and not more frequently. For some patients, it may be necessary to administer the acylated insulin pharmaceutical composition every seventh day or about every seventh day on average over a period of one month, six months or one year, and not more frequently. Furthermore, other patients may not administer the acylated insulin pharmaceutical composition at the exact same length of time intervals (number of days) every week, month, or year.Averaging over a one month, six month or year period, some patients may administer the acylated insulin pharmaceutical composition at intervals of every 5 to 7 days, and no more frequently, as the case may be. Averaging over a one month, six month or year period, other patients may administer the acylated insulin pharmaceutical composition at intervals of every 4 to 6 days, and no more frequently, as the case may be. Averaging over a one month, six month or year period, still other patients may administer the acylated insulin pharmaceutical composition at intervals of every 3 to 7 days, and no more frequently, as the case may be.

[0088] The primary target diseases and conditions of the present invention are diabetes (type 1 or type 2) or other conditions characterized by hyperglycemia, but generally are metabolic diseases and conditions in which the metabolic actions of insulin have clinical relevance or benefit, such as prediabetes, impaired glucose tolerance, metabolic syndrome, obesity, cachexia, in vivo β-cell damage / death, hyperphagia, inflammation, etc. All of these types of conditions are known or believed to benefit from a stable metabolic state in subjects suffering from said diseases or conditions.

[0089] Abbreviation Na2HPO4 is disodium hydrogen phosphate. NaOH is sodium hydroxide. OEG is the amino acid residue -NH(CH2)2O(CH2)2OCH2CO-. OSu is succinimid-1-yloxy-2,5-dioxo-pyrrolidin-1-yloxy. OtBu is oxy-tert-butyl. HCl is hydrogen chloride. γGlu or gGlu is a γL-glutamyl radical. NHS is N-hydroxysuccinimide. DCC is dicyclohexylcarbodiimide. AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid. OH is the hydroxyl radical. Gly is glycine. Arg is arginine. TFA is trifluoroacetic acid. HbA1c is glycosylated hemoglobin. GSPs are glycosylated serum proteins. TG is triglyceride. EXAMPLES

[0090] The following examples are offered by way of illustration and not by way of limitation.

[0091] Control Example 1 A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-2xOEG), desB30 human insulin (control compound 1) [ka]

[0092] 1. Preparation of A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-2xOEG), and desB30 human insulin A14E, B16H, B25H, and desB30 human insulin were prepared by a conventional method for preparing insulin analogues (see Glendorf T, Sorensen AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding; Biochemistry 2008 47 4743-4751 for the detailed method). A14E, B16H, B25H, and desB30 human insulin (5 g, 0.888 mmol) were dissolved in 100 mM Na2HPO4 aqueous solution (150 mL), acetonitrile (100 mL) was added, and the pH was adjusted to pH 10-12.5 with 1 N NaOH. tert-Butyl eicosane diacyl-γGlu-(2xOEG-OSu)-OtBu (0.948 g, 0.976 mmol) was dissolved in acetonitrile (50 mL) and slowly added to the insulin solution. The pH was maintained at 10-12.5. After 120 min, the reaction mixture was added to water (150 mL) and the pH was adjusted to 5.0 with 1N aqueous HCl. The precipitate was separated by centrifugation and lyophilized. The lyophilized crude product was added to a mixed solution of trifluoroacetic acid (60 mL) and dichloromethane (60 mL) and stirred at room temperature for 30 min. The mixture was concentrated to about 30 mL, poured into ice-cold n-heptane (300 mL), and the precipitated product was separated by filtration and washed twice with n-heptane. After drying in vacuum, it was purified by ion exchange chromatography (Resource Q, 0.25% to 1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), the purified fractions were combined, the pH was adjusted to 5.2 with 1 N HCl, the precipitate was isolated and lyophilized to give the title compound 5. LC-MS (electrospray): m / z=1063.6852[M+6H]6 +

[0093] 2. Preparation of intermediate tert-butyl eicosane diacyl-γGlu-(2xOEG-OSu)-OtBu 2.1 tert-Butyl eicosane diacyl-OSu Under nitrogen gas protection, mono-tert-butyl eicosane diacid (20g, 50.17mmol) and NHS (5.77g, 50.17mmol) were mixed in dichloromethane, triethylamine (13.95mL) was added, the resulting cloudy mixture was stirred at room temperature, DCC (11.39g, 55.19mmol) was added, and further stirred overnight. After filtration, the obtained filtrate was concentrated to near dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated saline, and after separation, the upper organic phase was dried with anhydrous sodium sulfate, and after filtration, the filtrate was concentrated under reduced pressure to near dryness and dried in vacuum overnight to obtain 24.12g (yield 97%) of tert-butyl eicosane diacyl-OSu. LC-MS(Scie×100API): m / z=496.36(M+1) +

[0094] 2.2 tert-Butyl eicosane diacyl-γGlu-OtBu tert-Butyl eicosane diacyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred, followed by the addition of H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water, which were heated to obtain a clear solution, and the solution was stirred at room temperature for 4 hours. Next, 10% aqueous citric acid (200 mL) was added, the mixture was separated, and the lower organic phase was washed with saturated saline, and after separation, the lower organic phase was dried over anhydrous sodium sulfate, and after filtration, the filtrate was concentrated under reduced pressure until almost dry, and then dried overnight in vacuum. 27.27 g (yield 96%) of tert-butyl eicosane diacyl-γGlu-OtBu was obtained. LC-MS(Scie×100API): m / z=584.44(M+1) +

[0095] 2.3 tert-Butyl eicosane diacyl-γGlu-(OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosane diacyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added and stirred for 10 minutes, NHS (5.38 g, 50.17 mmol) was added, and DCC (10.60 g, 51.38 mmol) was added. The mixture was stirred at room temperature overnight. After filtration, the filtrate was concentrated to near dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was added with saturated saline to wash, and after separation, the upper organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness, methyl tert-butyl ether was added, stirred for 30 minutes, suction filtered, and the filter cake was dried overnight in vacuum to obtain 25.76 g (yield 81%) of tert-butyl eicosane diacyl-γGlu-(OSu)-OtBu. LC-MS(Scie×100API): m / z=681.46(M+1) +

[0096] 2.4 tert-Butyl eicosane diacyl-γGlu-(2xOEG-OH)-OtBu tert-Butyl eicosane diacyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred, and 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in order, and the mixture was heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Next, 10% aqueous citric acid (200 mL) was added, the mixture was separated, and the lower organic phase was washed with saturated saline. After separation, the lower organic phase was dried over anhydrous sodium sulfate, and after filtration, the filtrate was concentrated under reduced pressure until almost dry, and then dried overnight in vacuum. 30.75 g (yield 93%) of tert-butyl eicosane diacyl-γGlu-(2xOEG-OH)-OtBu was obtained. LC-MS(Scie×100API): m / z=874.59(M+1) +

[0097] 2.5 tert-Butyl eicosane diacyl-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosane diacyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added and stirred for 10 minutes, NHS (4.05 g, 35.18 mmol) was added, and DCC (7.98 g, 38.70 mmol) was added. The mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated to near dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated saline, and after separation, the upper organic phase was dried over anhydrous sodium sulfate, and after filtration, the filtrate was concentrated under reduced pressure to near dryness and dried in vacuum overnight to obtain 31.09 g (91% yield) of tert-butyl eicosane diacyl-γGlu-(2xOEG-OSu)-OtBu. LC-MS(Scie×100API): m / z=971.61(M+1) +

[0098] Example 1 A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin (compound 1) [ka]

[0099] Following steps similar to those in Control Example 1, part 1, compounds A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin were prepared. LC-MS (electrospray): m / z=1305.4716[M+6H]6 +

[0100] The preparation of the intermediate tert-butyl eicosane diacyl-γGlu-(12xOEG-OSu)-OtBu followed similar steps as in Control Example 1, Part 2. LC-MS(Scie×100API): m / z=2423.35(M+1) +

[0101] Example 2 A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin (compound 2) [ka]

[0102] Following steps similar to those in Control Example 1, part 1, compounds A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin were prepared. LC-MS (electrospray): m / z=1310.1425[M+6H]6 +

[0103] The preparation of the intermediate tert-butyldocosane diacyl-γGlu-(12xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 1, Part 2. LC-MS(Scie×100API): m / z=2451.38(M+1) + .

[0104] Example 3 A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin (compound 3) [ka]

[0105] Following steps similar to those in Control Example 1, part 1, compounds A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin were prepared. LC-MS (electrospray): m / z=1247.47[M+7H]7 +

[0106] The preparation of the intermediate tert-butyldocosane diacyl-γGlu-(18xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 1, Part 2. LC-MS(Scie×100API): m / z=3320.83(M+1) +

[0107] Example 4 A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-24xOEG), desB30 human insulin (compound 4) [ka]

[0108] Following steps similar to those in Control Example 1, part 1, compounds A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-24xOEG), desB30 human insulin were prepared. LC-MS (electrospray): m / z=873.35[M+11H]11 +

[0109] The preparation of the intermediate tert-butyldocosane diacyl-γGlu-(24xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 1, Part 2. LC-MS(Scie×100API): m / z=4192.27(M+1) +

[0110] Example 5 N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (compound 5) [ka]

[0111] 1. Preparation of N-ε26-[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide [Gly8, Arg34]GLP-1-(7-37) peptide was produced by a general protein recombinant expression method (for specific methods, see Molecular Cloning: A Laboratory Manual (Fourth Edition), Michael R. Green, Cold Spring Harbor Press, 2012). [Gly8, Arg34]GLP-1-(7-37) peptide (5 g, 1.48 mmol) was dissolved in 100 mM Na2HPO4 aqueous solution (150 mL), acetonitrile (100 mL) was added, and the pH was adjusted to pH 10-12.5 with 1 N NaOH. tert-Butyl eicosane diacyl-γGlu(2xOEG-OSu)-OtBu (1.59 g, 1.63 mmol) was dissolved in acetonitrile (50 mL) and gradually added to the [Gly8, Arg34]GLP-1-(7-37) peptide solution. The pH was maintained at 10-12.5. After 120 min, the reaction mixture was added to water (150 mL) and the pH was adjusted to 5.0 with 1N aqueous HCl. The precipitate was separated by centrifugation and lyophilized. The crude product was added to a mixture of trifluoroacetic acid (60 mL) and dichloromethane (60 mL) and stirred at room temperature for 30 min. The mixture was concentrated to about 30 mL, poured into ice-cold n-heptane (300 mL), and the precipitated product was separated by filtration and washed twice with n-heptane. After drying in vacuum, the product was purified by ion exchange chromatography (Resource Q, 0.25% to 1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), and the purified fractions were combined, the pH was adjusted to 5.2 with 1N HCl, the precipitate was separated, and lyophilized to give the title compound. LC-MS (electrospray): m / z=1028.79[M+4H]4 +

[0112] 2. Preparation of intermediate tert-butyl eicosane diacyl-γGlu-(2xOEG-OSu)-OtBu 2.1 tert-Butyl eicosane diacyl-OSu Under nitrogen gas protection, mono-tert-butyl eicosanedioate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane (400 mL), triethylamine (13.95 mL) was added, the resulting cloudy mixture was stirred at room temperature, DCC (11.39 g, 55.19 mmol) was added, and it was further stirred overnight. After filtration, the obtained filtrate was concentrated to near dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was added with saturated saline and washed with water, after separation, the upper organic phase was dried with anhydrous sodium sulfate, after filtration, the filtrate was concentrated under reduced pressure to near dryness, and dried in vacuum overnight to obtain 24.12 g (yield 97%) of tert-butyl eicosanediacil-OSu. LC-MS(Scie×100API): m / z=496.36(M+1) +

[0113] 2.2 tert-Butyl eicosane diacyl-γGlu-OtBu tert-Butyl eicosane diacyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred, followed by the addition of H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water (25 mL), which was heated to obtain a clear solution, and the solution was stirred at room temperature for 4 hours. Next, 10% aqueous citric acid (200 mL) was added, the solution was separated, and the lower organic phase was washed with saturated saline, and after separation, the lower organic phase was dried over anhydrous sodium sulfate, and after filtration, the filtrate was concentrated under reduced pressure until almost dry, and then dried overnight in vacuum. 27.27 g (yield 96%) of tert-butyl eicosane diacyl-γGlu-OtBu was obtained. LC-MS(Scie×100API): m / z=584.44(M+1) +

[0114] 2.3 tert-Butyl eicosane diacyl-γGlu(OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosane diacyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added and stirred for 10 minutes, NHS (5.38 g, 50.17 mmol) was added, and DCC (10.60 g, 51.38 mmol) was added. The mixture was stirred at room temperature overnight. After filtration, the filtrate was concentrated to near dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was added with saturated saline and washed with water, the upper organic phase was dried over anhydrous sodium sulfate after separation, the filtrate was concentrated under reduced pressure to near dryness, methyl tert-butyl ether was added, stirred for 30 minutes, suction filtered, and the filter cake was dried overnight in vacuum to obtain 25.76 g (yield 81%) of tert-butyl eicosane diacyl-γGlu-(OSu)-OtBu. LC-MS(Scie×100API): m / z=681.46(M+1) +

[0115] 2.4 tert-Butyl eicosane diacyl-γGlu-(2xOEG-OH)-OtBu tert-Butyl eicosane diacyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred, and 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in order, and the mixture was heated to obtain a clear solution, which was then stirred at room temperature for 4 hours. Next, 10% aqueous citric acid (200 mL) was added, the mixture was separated, and the lower organic phase was washed with saturated saline solution, and after separation, the lower organic phase was dried over anhydrous sodium sulfate, and after filtration, the filtrate was concentrated under reduced pressure until almost dry, and then dried overnight in vacuum. 30.75 g (yield 93%) of tert-butyl eicosane diacyl-γGlu-(2xOEG-OH)-OtBu was obtained. LC-MS(Scie×100API): m / z=874.59(M+1) +

[0116] 2.5 tert-Butyl eicosane diacyl-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosane diacyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added and stirred for 10 minutes, NHS (4.05 g, 35.18 mmol) was added, and DCC (7.98 g, 38.70 mmol) was added. The mixture was stirred at room temperature overnight. After filtration, the filtrate was concentrated to near dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was added with saturated saline and washed with water, and after separation, the upper organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure to near dryness, and dried in vacuum overnight to obtain 31.09 g (91% yield) of tert-butyl eicosane diacyl-γGlu-(2xOEG-OSu)-OtBu. LC-MS(Scie×100API): m / z=971.61(M+1) +

[0117] Example 6 N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (compound 6) [ka]

[0118] Following steps similar to those in part 1 of Example 5, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z=992.52[M+4H]4+

[0119] The preparation of the intermediate tert-butyl eicosane diacyl-γGlu-(OEG-OSu)-OtBu was carried out following steps similar to those in Example 5, part 2. LC-MS(Scie×100API): m / z=826.54(M+1) +

[0120] Example 7 N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide (compound 7) [ka]

[0121] Following steps similar to those in part 1 of Example 5, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8, Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z=956.25[M+4H]4 +

[0122] The preparation of the intermediate tert-butyl eicosane diacyl-γGlu-(OSu)-OtBu was carried out following steps similar to those in Example 5, part 2. LC-MS(Scie×100API): m / z=681.46(M+1) +

[0123] Example 8 N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide (compound 8) [ka]

[0124] Following steps similar to those in part 1 of Example 5, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z=959.75[M+4H]4 +

[0125] The preparation of the intermediate tert-butyl eicosane diacyl-γGlu-(OSu)-OtBu was carried out following steps similar to those in Example 5, part 2. LC-MS(Scie×100API): m / z=681.46(M+1) +

[0126] Example 9 N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (compound 9) [ka]

[0127] Following steps similar to those in part 1 of Example 5, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z=1021.78[M+4H]4 +

[0128] Example 10 N-ε 26-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide (compound 10) [ka]

[0129] Following steps similar to those in part 1 of Example 5, N-ε 26 -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8, Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z=949.24[M+4H]4 +

[0130] The preparation of the intermediate tert-butyloctadecanediacyl-γGlu-(OSu)-OtBu was carried out following steps similar to those in Example 5, Part 2. LC-MS(Scie×100API): m / z=653.43 (M+1) +

[0131] Example 11 N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (compound 11) [ka]

[0132] Following steps similar to those in part 1 of Example 5, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z=1035.80[M+4H]4 +

[0133] The preparation of the intermediate tert-butyldocosane diacyl-γGlu-(2xOEG-OSu)-OtBu was carried out following steps similar to those in Example 5, part 2. LC-MS(Scie×100API): m / z=999.64(M+1) +

[0134] Example 12 This experiment was aimed at determining the chemical stability of the acylated insulin formulations of this invention.

[0135] Acylated insulin preparations Compound 2 was dissolved in 10 mM anhydrous disodium hydrogen phosphate solution to a concentration twice the final insulin concentration in each formulation in the table below. Phenol, m-cresol, glycerin and sodium chloride were mixed according to the amounts of each component in the table below, and then added to the solution of compound 2 and the pH was adjusted to 7.4. Then zinc acetate was slowly added all at once to the solution of compound 2 according to the amount in the table below and the pH was adjusted to 7.4, finally producing the acylated insulin formulations shown in Table 1, where the Zn content is expressed as Zn / 6 moles of acylated insulin (abbreviated as "Zn / 6ins").

[0136] [Table 1]

[0137] In this example, the chemical stability of the formulation may be demonstrated by the change in high molecular weight protein (HMWP) relative to day 0 after storage at 25° C. and 37° C. for 14 and 21 days, and also by the change in the amount of related substances after storage at 25° C. and 37° C. for 14 and 21 days.

[0138] Measurement of high molecular weight proteins (HMWP) The content of high molecular weight protein (HMWP) was measured by high performance liquid chromatography (HPLC), and tested on a column of type and specification Shodex PROTEIN kw-802.5, with a column temperature of 30°C, a sample cell temperature of 5°C, and a mobile phase containing 3L of 0.1% arginine solution, 750mL of glacial acetic acid, and 1250mL of acetonitrile at a flow rate of 0.5mL / min. The detection wavelength was 276nm, and the sample injection volume was 10μL. Table 2 shows the increase in HMWP on the 14th and 21st days relative to the 0th day at 25°C and 37°C.

[0139] [Table 2]

[0140] From the above table, it can be seen that the amount of HMWP increased very slowly over time in the acylated insulin preparations of this invention at different concentrations, indicating that all of the acylated insulin preparations have excellent chemical stability.

[0141] Measurement of the amount of related substances The content of insulin-related substances was measured by high performance liquid chromatography (HPLC) and tested on a Kromasil 300A-5μm-C4 (4.6×150 mm) column at a column temperature of 35° C., a sample cell temperature of 5° C., and a flow rate of 1.0 mL / min with an elution phase. Elution was performed with a mobile phase consisting of: Phase A contained 0.18 M anhydrous sodium sulfate, 10% acetonitrile (v / v), and was adjusted to a pH value of 2.3 with 85% phosphoric acid. Phase B was 75% acetonitrile (v / v). Gradient: 64% / 36% A / B linear change from 0 to 40 min, 10% / 90% A / B linear change from 40 to 65 min, 64% / 36% A / B linear change from 65 to 66 min, isogradient of 64% / 36% A / B from 66 to 70 min. The detection wavelength was 214 nm.

[0142] Table 3 shows the increase in related substances on days 14 and 21 relative to day 0 at 25°C and 37°C.

[0143] [Table 3]

[0144] From the above table, it can be seen that the amount of insulin-related substances in the acylated insulin preparations of the present invention under the different concentration conditions also increased very slowly over time, indicating that the acylated insulin preparations are very stable.

[0145] Example 13 This experiment was aimed at determining the chemical stability of the acylated insulin formulations of this invention.

[0146] According to the amounts of each component in Table 4 below, the acylated insulin formulations in Table 4 were prepared according to steps similar to those in Example 12. Also, according to steps similar to those in Example 12, the changes in HMWP and related substances were measured on days 14 and 35 relative to day 0. Tables 5 and 6 below show the changes in HMWP and related substances for acylated insulin formulations with different formulation methods.

[0147] [Table 4]

[0148] [Table 5]

[0149] [Table 6]

[0150] From the above table it can be seen that the amount of insulin HMWP and related substances in the acylated insulin preparation of the present invention also increased very slowly over time, indicating that the acylated insulin preparation is very stable.

[0151] Example 14 This experiment is aimed at determining the chemical stability of compositions of the present invention comprising acylated insulin and a GLP-1 compound.

[0152] Compositions Comprising Acylated Insulin and a GLP-1 Compound According to the amounts of each component in Table 7 below, compositions containing acylated insulin (compound 2) and GLP-1 (compound 11) were prepared according to steps similar to those in Example 12. Also, according to steps similar to those in Example 12, changes in HMWP of acylated insulin in the compositions were measured on days 14 and 35 relative to day 0. Table 8 below shows the changes in HMWP of acylated insulin in compositions with different formulation methods.

[0153] [Table 7]

[0154] [Table 8]

[0155] From the above table, it can be seen that the amount of HMWP of the acylated insulin in the above acylated insulin and GLP-1 composition of the present invention increases very slowly over time, indicating that the presence of the GLP-1 compound does not affect the stability of the acylated insulin.

[0156] Example 15 Pharmacodynamic study in type 2 diabetic db / db mice The objective of this study is to demonstrate the long-term hypoglycemic and HbA1c effects of a composition of the present invention comprising acylated insulin and a GLP-1 compound in type 2 diabetic db / db mice in the context of diabetes.

[0157] In type 2 diabetic db / db mice, compositions 1 to 3 and 5 to 7 containing compound 2 and compound 11, as well as single formulation 10 containing compound 2, single formulation 11 containing compound 11, and a solvent group were tested as references. The compositions of the formulations or compositions used are shown in Table 9.

[0158] [Table 9-1]

[0159] [Table 9-2]

[0160] Male db / db (Cavens) mice aged 8–9 weeks were housed in suitable cages in a barrier environment with free access to standard food and purified water, and the environmental conditions were controlled at a relative humidity of 40%–60% and a temperature of 22°C–24°C. After an acclimation period of 1–2 weeks, the mice were used in the experiments.

[0161] Before the start of the experiment on the day, random blood glucose was evaluated and the mice were weighed. According to random blood glucose and body weight, the mice were randomly allocated to a vehicle group or a treatment group, with 13 groups in total, 5 mice per group, and the mice were treated as follows: Subcutaneous injection of vehicle, or subcutaneous injection of low dose (5 U / kg of compound 2) of pharmaceutical composition 1, composition 2, composition 5, composition 6, composition 7, or subcutaneous injection of medium dose (10 U / kg of compound 2) of pharmaceutical composition 1, composition 3, composition 5, composition 6, composition 7, or subcutaneous injection of formulation 10 (10 U / kg of compound 2), or subcutaneous injection of formulation 11 (calculated at 96.8 μg / kg of compound 11).

[0162] The drug was administered by subcutaneous injection (5 mL / kg) in the dorsal region of the neck on days 0, 3, 6, 9, 12, 15, 18, and 21, respectively, and blood glucose was detected randomly at 3, 6, 9, 24, 48, and 72 hours after the first administration, and then monitored once daily. After the study was completed, the percentage of glycated hemoglobin (Hb1Ac) in whole blood was measured using EDTA anticoagulant.

[0163] The rat's tail was cleaned with an alcohol cotton ball, and a drop of blood was collected from the tail with a disposable blood collection needle, and measured with a blood glucose meter and attached test strips (Roche). A dose-response curve of blood glucose versus time was plotted for each pharmaceutical composition. To explain the effect of the pharmaceutical composition of the present invention on blood glucose, the difference in the area under the blood glucose-time curve (ΔAUC) from 0 to the monitoring end point was calculated for each dose-response curve. The smaller the ΔAUC value, the better the blood glucose lowering effect and the better the drug efficacy.

[0164] Figures 1a to 1f show that the pharmaceutical composition of the present invention comprising an acylated insulin and a GLP-1 compound unexpectedly has synergistic glucose-reducing and Hb1Ac-lowering effects in type 2 diabetic db / db mice after administration, compared with acylated insulin alone and GLP-1 alone.

[0165] Specifically, Figures 1a and 1b show that the hypoglycemic effect of pharmaceutical composition 7 containing acylated insulin and GLP-1 compound at a low dose is obviously superior to both single preparation 10 and preparation 11, in which the amount of acylated insulin contained in single preparation 10 is twice that of acylated insulin in composition 7 at a low dose, and the amount of GLP-1 compound contained in single preparation 11 is twice that of GLP-1 compound in composition 7 at a low dose, showing that the acylated insulin and GLP-1 compound in the pharmaceutical composition of the present invention have an unexpected synergistic hypoglycemic effect. Compositions 1 and 2 at low doses have the same or better hypoglycemic effect compared to preparation 10, and compositions 5 and 6 at low doses have the same or better hypoglycemic effect compared to preparation 11, showing that in the presence of the synergistic effect of acylated insulin and GLP-1 compound, a lower total amount of acylated insulin and GLP-1 compound can be used to achieve the same or better hypoglycemic effect.

[0166] Figures 1c and 1d show that in the presence of the synergistic hypoglycemic effect of acylated insulin and GLP-1 compound, when the dose of acylated insulin contained in each composition is the same, the composition containing a small amount of GLP-1 compound has an obviously improved hypoglycemic effect compared to a pharmaceutical composition containing only acylated insulin alone, and Compositions 1, 3, 5, 6, and 7 at moderate doses all have the same or better hypoglycemic effect compared to Preparation 11.

[0167] FIG. 1e showed that composition 7 at low doses had better Hb1Ac lowering effect than formulations 10 and 11, and the acylated insulin and GLP-1 compounds unexpectedly had a synergistic Hb1Ac lowering effect.

[0168] Figure 1f shows that in the presence of synergistic Hb1Ac lowering effects of acylated insulin and GLP-1 compound, when the dose of acylated insulin contained in each composition was the same, the composition containing a small amount of GLP-1 compound had a clearly improved Hb1Ac lowering effect compared to a pharmaceutical composition containing acylated insulin alone or only a GLP-1 compound.

[0169] Example 16 Long-term pharmacodynamic study in type 2 diabetic db / db mice With reference to the experimental steps similar to those in Example 15, a long-term efficacy study was carried out in type 2 diabetic db / db mice.

[0170] In type 2 diabetic db / db mice, compositions 5, 9 and 10 containing compound 2 and compound 11, as well as single formulation 10 containing compound 2, single formulation 11 containing compound 11, degludecryraglutide composition and solvent groups were tested.

[0171] In the above formulations or compositions, the degludecryraglutide composition served as a control composition and was insulin degludecryraglutide injection (trade name: Xultophy) purchased from Novo Nordisk, and the specific compositions of the remaining formulations or compositions are shown in Table 10.

[0172] [Table 10]

[0173] Male db / db (Cavens) mice aged 8–9 weeks were housed in suitable cages in a barrier environment with free access to standard food and purified water, and the environmental conditions were controlled at a relative humidity of 40%–60% and a temperature of 22°C–24°C. After an acclimation period of 1–2 weeks, the mice were used in the experiments.

[0174] Before the start of the experiment on the day, random blood glucose was evaluated and the mice were weighed. According to random blood glucose and body weight, the mice were randomly allocated to a vehicle group or a treatment group, with 14 groups in total, 7 mice per group, and the mice were given the following treatments: vehicle subcutaneous injection, or low dose of pharmaceutical composition 5, composition 9, composition 10 subcutaneous injection, or medium dose of pharmaceutical composition 5, composition 9, composition 10 subcutaneous injection, or high dose of pharmaceutical composition 5, composition 9, composition 10 subcutaneous injection, or medium dose of formulation 10 subcutaneous injection, or high dose of formulation 10 subcutaneous injection, or high dose of formulation 11 subcutaneous injection, or high dose of degludecryraglutide composition subcutaneous injection. Among these, in the case of low doses, the dose was calculated based on a dose of acylated insulin in the composition of 5 U / kg, in the case of medium doses, the dose was calculated based on a dose of acylated insulin in the composition or formulation of 10 U / kg, and in the case of high doses, the dose was calculated for the first time based on a dose of acylated insulin in composition 5, composition 9, composition 10, and formulation 10 of 20 U / kg, a dose of insulin degludec in the degludecrilaglutide composition of 7 U / kg, and a dose of the GLP-1 compound in formulation 11 of 250 μg / kg, and for the second to eleventh times, the dose was calculated based on a dose of acylated insulin in composition 5, composition 9, composition 10, and formulation 10 of 15 U / kg, a dose of insulin degludec in the degludecrilaglutide composition of 5.25 U / kg, and a dose of the GLP-1 compound in formulation 11 of 189 μg / kg.

[0175] The mice were administered subcutaneously (5 mL / kg body weight) in the neck and back, and the composition of the solvent and degludecrylaglutide was administered once a day for a total of 33 times. Composition 5, Composition 9, Composition 10, Formulation 10 and Formulation 11 were administered on days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30, respectively. The mice were evaluated for blood glucose at 3, 6, 9, 12, 24 and 48 hours after the first administration. Blood glucose was monitored every day before administration from day 3 to day 33, and the area under the blood glucose-time curve (AUC) was calculated. The proportion of glycated hemoglobin (Hb1Ac) of the mice was measured before grouping and on day 16 after administration, and the change in glycated hemoglobin (△Hb1Ac) was calculated. At the end of the experiment, non-anticoagulated whole blood was collected from the mice, and serum was collected after centrifugation. Triglyceride (TG) levels in the serum were detected using an ELISA reagent kit.

[0176] Figures 2a to 2d show that the pharmaceutical composition of the present invention comprising acylated insulin and a GLP-1 compound unexpectedly has synergistic glucose-reducing, Hb1Ac-lowering and triglyceride-lowering effects in type 2 diabetic db / db mice after administration compared with acylated insulin alone or GLP-1 alone, and that the pharmaceutical composition of the present invention administered once every three days has better efficacy compared with a high-dose degludecrylaglutide composition administered once daily.

[0177] Specifically, Figures 2a and 2b show that the hypoglycemic effect of pharmaceutical composition 5, composition 9, and composition 10 containing acylated insulin and GLP-1 compound at low doses is equivalent to that of formulation 10 at high doses, and the dose of acylated insulin in formulation 10 is more than three times that of acylated insulin in composition 5 at low doses, and that the administration of low doses of acylated insulin in the presence of a small amount of GLP-1 compound can achieve hypoglycemic effect equivalent to or greater than that of high doses of acylated insulin. The hypoglycemic effect of composition 9 at low doses administered once every three days is equivalent to that of the control composition (degludecrylaglutide composition) at high doses administered once a day, indicating that the pharmaceutical composition of the present invention containing acylated insulin and GLP-1 compound has unexpectedly better hypoglycemic effect than the commercially available composition product degludecrylaglutide. The hypoglycemic effect of composition 10 at a low dose is obviously superior to that of the single formulation 10 at a high dose, and is equivalent to that of formulation 11 at a high dose, in which the content of acylated insulin in formulation 10 is more than three times that of the acylated insulin in composition 10, and the content of GLP-1 compound in formulation 11 is more than three times that of the GLP-1 compound in composition 10. From the above, it is found that the acylated insulin and GLP-1 compound in the pharmaceutical composition of the present invention have an unexpectedly synergistic hypoglycemic effect.

[0178] Figure 2c shows that the HbA1c lowering effect of composition 5 at a medium dose administered once every three days is equivalent to that of the degludecrylaglutide composition at a high dose administered once a day, and by using a small amount of the pharmaceutical composition of the present invention, the HbA1c lowering effect equivalent to that of the degludecrylaglutide composition can be achieved, and the pharmaceutical composition of the present invention has better efficacy. The HbA1c lowering effect of composition 10 at a low dose is obviously superior to that of preparations 10 and 11 at a high dose, indicating that the acylated insulin and GLP-1 compound in the pharmaceutical composition of the present invention have a synergistic HbA1c lowering effect.

[0179] Figure 2d shows that the TG lowering effect of composition 10 at a low dose is obviously superior to that of the degludecrylaglutide composition at a high dose, and the TG lowering effect of the pharmaceutical composition of the present invention is superior to that of the degludecrylaglutide composition. The TG lowering effect of composition 10 at a low dose is equivalent to that of formulation 10 at a high dose and formulation 11 at a high dose, indicating that the acylated insulin and GLP-1 compound in the pharmaceutical composition of the present invention have a synergistic TG lowering effect.

[0180] Example 17 Long-term pharmacodynamic study in type 2 diabetic db / db mice The purpose of this study is to demonstrate the long-term blood glucose lowering, HbA1c lowering and TG lowering effects of the acylated insulin preparation of the present invention in type 2 diabetic db / db mice in the case of diabetes.

[0181] Formulations containing Compound 2 were tested in type 2 diabetic db / db mice, as well as formulations containing the control Compound 1 as a reference, a degludec formulation, a normal control group and a vehicle control group.

[0182] In the above formulations, the degludec formulation was control formulation 2, which was insulin degludec injection (Tresiba) purchased from Novo Nordisk, and the specific compositions of the remaining formulations are shown in Table 11.

[0183] [Table 11]

[0184] Eight to nine-week-old db / db (Cavens) mice and normal mice were housed in suitable cages in a barrier environment with free access to standard food and purified water, and the environmental conditions were controlled at a relative humidity of 40% to 60% and a temperature of 22°C to 24°C. After an acclimation period of 1 to 2 weeks, they were used in the experiments.

[0185] Before the start of the experiment on the day, random blood glucose was evaluated and the mice were weighed. According to the random blood glucose and body weight, the mice were randomly allocated to a control group or a treatment group with 10 mice in each group, for a total of 4 groups, and the mice were subjected to the following treatments. The solvent was subcutaneously injected, or the formulation 2 containing compound 2 was subcutaneously injected, or the control formulation 1 containing control compound 1 was subcutaneously injected, or the control formulation 2 containing insulin degludec was subcutaneously injected. In the cases of 1 to 4 administrations, the dosage was 30 U / kg in all cases, and in the cases of 5 to 10 administrations, the dosage was 37.5 U / kg in all cases.

[0186] The mice were administered the drug by subcutaneous injection into the back of the neck by subcutaneous administration (5mL / kg body weight) on days 0, 3, 7, 10, 13, 16, 19, 22, 25, and 28, respectively. The blood glucose of the mice was evaluated before each administration and 72 hours after the final administration, and the change in the area under the blood glucose-time curve (ΔAUC) was calculated. To simulate a meal, an oral glucose tolerance test (OGTT) was performed 48 hours after the first administration during the test, and blood glucose was measured 30, 60, and 120 minutes after forced oral administration of a glucose solution (100mg / mL, 10mL / kg). After the study was completed, the proportion of glycated hemoglobin (Hb1Ac) in whole blood was detected using an EDTA anticoagulant, and non-anticoagulated whole blood was collected at the same time. After centrifugation, serum was collected and the triglyceride (TG) level in serum was measured using an ELISA reagent kit.

[0187] Figures 3a to 3f show that the formulation of the present invention containing acylated insulin unexpectedly exhibits improved blood glucose lowering, Hb1Ac lowering, and TG lowering effects in type 2 diabetic db / db mice at the same dose, compared to the degludec formulation and the formulation containing control compound 1.

[0188] Example 18 Long-term pharmacodynamic study in a rat model of early stage type 2 diabetes mellitus (T2DM) (GK rat) The purpose of this study is to demonstrate the long-term blood glucose lowering, HbA1c lowering, TG lowering and GSP lowering effects of the acylated insulin preparation of the present invention in non-obese model rats (GK rats) with early stage type 2 diabetes mellitus (T2DM).

[0189] In GK rats, formulations containing Compound 2 were tested, as well as a degludec formulation as a reference, a normal control group and a solvent control group. The composition of the formulations is shown in Table 12.

[0190] GK rats (with random blood glucose and glycated hemoglobin higher than 20% and 30% of those of normal Wistar rats, respectively) and normal rats that passed the screening were housed in suitable breeding boxes in a barrier environment with free access to standard food and purified water, and the environmental conditions were controlled at a relative humidity of 40% to 60% and a temperature of 22°C to 24°C. After an acclimation period of 1 to 2 weeks, they began to be used in the experiments.

[0191] Before the start of the experiment on the day, random blood glucose was evaluated and the weight of the GK rats was measured. According to the random blood glucose, weight and glycated hemoglobin, the rats were randomly allocated to a control group or a treatment group, with a total of 3 groups, each group consisting of 6 rats, and the rats were given the following treatments: subcutaneous injection of the solvent, subcutaneous injection of formulation 2 containing compound 2, or subcutaneous injection of control formulation 2 containing degludec, each dose was 10 U / kg.

[0192] The mice were administered subcutaneously (SC) in the neck and back (1 mL / kg body weight), and the solvent and degludec formulations were administered once a day for a total of 32 times. Compound 2-containing formulation 2 was administered on days 0, 4, 8, 12, 16, 20, 24, and 28, respectively. The blood glucose of the mice was evaluated before each administration and 72 hours after the final administration, and the change in the area under the blood glucose-time curve (ΔAUC) was calculated. The rats were fasted for 4 hours on the day before the test, and the rats were subjected to glycated hemoglobin (HbA1c) detection. After the study, the proportion of glycated hemoglobin (Hb1Ac) in whole blood was detected using an EDTA anticoagulant. At the same time, non-anticoagulated whole blood was collected, and the serum was collected after centrifugation to measure the serum glycated serum albumin (GSP) and triglyceride (TG) values ​​using an ELISA reagent kit.

[0193] Figures 4a to 4e show that the formulation of the present invention containing acylated insulin unexpectedly exhibits improved blood glucose lowering, Hb1Ac lowering, GSP lowering and TG lowering effects in non-obese early stage type 2 diabetes mellitus (T2DM) model rats at the same dose compared to the degludec formulation.

[0194] Example 19 This experiment is aimed at determining the chemical stability of compositions of the present invention comprising acylated insulin and a GLP-1 compound. According to the amount of each component in Table 12 below, the composition in Table 12 was prepared according to the steps similar to those in Example 12. Also, according to the steps similar to those in Example 12, the change in HMWP on the 19th day and the 33rd day relative to the day 0, and the change in related substances on the 19th day relative to the day 0 were measured. Tables 13 and 14 below show the change in HMWP and related substances of Compound 2 in different compositions.

[0195] [Table 12]

[0196] [Table 13]

[0197] [Table 14]

[0198] From the above table it can be seen that the amount of HMWP and related substances in the acylated insulin in the above acylated insulin and GLP-1 composition of this invention increased very slowly over time, indicating that the presence of the GLP-1 compound does not affect the stability of the acylated insulin.

[0199] Example 20 A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin (compound 12) [ka]

[0200] Following steps similar to those in Control Example 1, part 1, compounds A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin were prepared. LC-MS (electrospray): m / z=1165.0674[M+11H]6 +

[0201] The preparation of the intermediate tert-butyldocosane diacyl-γGlu-(6xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 1, Part 2. LC-MS(Scie×100API): m / z=1579.94 (M+1) +

[0202] Example 21 A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin (compound 13) [ka]

[0203] Following steps similar to those in Control Example 1, part 1, compounds A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin were prepared. LC-MS (electrospray): m / z=1160.3997[M+6H]6 +

[0204] The preparation of the intermediate tert-butyl eicosane diacyl-γGlu-(6xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 1, Part 2.

[0205] Example 22 Pharmacokinetics This example is intended to illustrate the in vivo pharmacokinetic properties of the formulations of the present invention.

[0206] Pharmacokinetics in SD rats Forty-eight SD rats, 12 in each group (half female and half male), were divided into a low-dose group of formulation 2, a medium-dose group of formulation 2, and a high-dose group of formulation 2, and were administered 15, 30, and 60 nmol / kg by subcutaneous injection, respectively, and further, 30 nmol / kg was administered by intravenous injection to the intravenous injection group. Blood samples were taken from the low-, medium-, and high-dose groups of formulation 2 before administration (0 min), and 3, 6, 9, 12, 24, 36, 48, 72, 96, and 120 hours after administration to measure the drug blood concentrations, and blood samples were taken from the intravenous injection group of formulation 2 before administration (0 min), and 5, 15, 0.5, 3, 9, 24, 36, 48, 72, 96, and 120 hours after administration to measure the drug blood concentrations. The pharmacokinetic parameters were calculated using the noncompartmental model of WinNonLin (8.0.0.3176) software. 1 / 2 , T max , C max , AUC last , AUC inf , Vd, Cl, MRT last , and F were calculated.

[0207] [Table 15]

[0208] From the above experimental results, it was found that formulation 2 of the present invention exhibits a longer half-life, a longer MRT, and a higher bioavailability in SD rats.

[0209] Sequence Listing SEQ ID NO:1: A14E, B16H, B25H, desB30 human insulin A chain: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO:2: A14E, B16H, B25H, desB30 human insulin B chain: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys SEQ ID NO:3: A14E, B16E, B25H, desB30 human insulin A chain: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO:4: A14E, B16E, B25H, desB30 human insulin B chain: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys SEQ ID NO:5: GLP-1-(7-37) peptide His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly sequence number 6: [Gly8、Arg34]GLP-1-(7-37) peptide His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly sequence number 7: [Arg34]GLP-1-(7-37) peptide His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

Claims

1. Acylated insulin, 2.3 moles of zinc ions / 6 moles of acylated insulin, 45 to 60 mM of phenol, 0 to 10 mM of m-cresol, 10 mM to 20 mM of NaCl, 1.5% (weight / weight) of glycerin, 5 to 10 mM of Na ２ HPO ４ and A pharmaceutical composition comprising: Among them, The insulin moiety of the acylated insulin is A14E, B16H, B25H, desB30 human insulin or A14E, B16E, B25H, desB30 human insulin, and the acyl group portion of the acylated insulin is linked to the amino group of the lysine residue or N-terminal amino acid residue of the insulin moiety, and the acyl group portion is represented by the following formula (D): W1-(W2) ｍ -(W3) ｎ -(D), In the formula, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, W3 is neutral and is an amino acid residue containing an alkylene glycol, W2 is an acidic amino acid residue, W1 is an aliphatic diacid containing 20 to 24 carbon atoms, provided that formally, a hydroxy group has been removed from one of the carboxy groups of the aliphatic diacid, W1, W2 and W3 are linked by an amide bond, and The order of appearance of W2 and W3 in formula (D) is independently interchangeable, Pharmaceutical composition.

2. Said n is 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18 or 19, preferably, n is 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17 or 18, preferably, n is 5, 6, 7, 8, 11, 12, 13, 14, 15 or 16, preferably, n is 5, 6, 7, 8, 11, 12, 13, 14 or 15, and / or m is an integer from 1 to 6, preferably, m is 1, 2, 3 or 4, preferably, m is 1 or 2, preferably, m is 1, and / or W1 is an aliphatic diacid containing 20 to 23 carbon atoms, preferably, W1 is an aliphatic diacid containing 20, 21 or 22 carbon atoms, provided that formally, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid. The pharmaceutical composition according to claim 1.

3. Said W3 is -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-, -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-, -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-, -HN-(CH ２ ) ２ -O-(CH ２ ) ２-O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ２-O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ３ -O-CH ２ -CO-、or -HN-(CH ２ ) ４ -O-(CH ２ ) ４ -O-CH ２ -CO- and preferably, W3 is -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO- and / or W2 is an amino acid residue selected from γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp, preferably, W2 is selected from γGlu or βAsp, and / or W1 is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) １９ -CO-, HOOC-(CH ２ ) ２０ -CO-, HOOC-(CH ２ ) ２１ -CO- or HOOC-(CH ２ ) ２２ -CO-, and preferably, W1 is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) ２０ -CO- or HOOC-(CH ２ ) ２２ -CO-. The pharmaceutical composition according to claim 1.

4. In the formula (D), it is linked to the amino group of the lysine residue or N-terminal amino acid residue of the insulin parent body by the C-terminus of W3. The pharmaceutical composition according to claim 1.

5. The acyl group moiety is linked to the ε-amino group of the lysine residue of the insulin parent body. The pharmaceutical composition according to claim 1.

6. The acylated insulin is A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E,B16H, B25H, B29K (N(ε)-tricosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosandiacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H, B25H,B29K(N(ε)-eicosanediacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-24xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H,B29K(N(ε)-eicosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-heneicosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-tricosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-tetracosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H,B29K (N(ε)-eicosanediacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosanediacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanediacyl-γGlu-20xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K (N(ε)-docosanediacyl-γGlu-20xOEG), desB30 human insulin, selected from the group consisting of insulin, Preferably, the acylated insulin is selected from insulins such as A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin. Preferably, the acylated insulin is selected from insulins such as A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin. The pharmaceutical composition according to claim 1.

7. The content of the acylated insulin is higher than 0.3 mM, preferably 0.3 to 9 mM, preferably 0.6 to 8.4 mM, preferably 0.6 to 7.2 mM, preferably 0.6 to 6.0 mM, preferably 0.6 to 4.2 mM, preferably 0.6 to 3.6 mM, preferably 0.6 to 3.0 mM, preferably 0.6 to 2.4 mM, preferably 0.6 to 2.1 mM, preferably 0.9 to 1.8 mM, preferably 0.9 to 1.5 mM, preferably 1.2 to 1.5 mM, and / or The content of phenol is 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, or 60 mM, and / or The content of m-cresol is 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM, and / or The content of NaCl is 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, and / or The pH of the pharmaceutical composition is 6.5 to 8.5, preferably the pH is 6.5 to 8.0, preferably the pH is 7.0 to 7.8, preferably the pH is 7.2 to 7.6, and more preferably the pH is 7.

4. The pharmaceutical composition according to claim 1.

8. 0.9 to 1.5 mM of acylated insulin, and 2.3 moles of zinc ions / 6 moles of acylated insulin, and 45 mM of phenol, and 10 mM of m-cresol, and 20 mM of NaCl, and 15 mg / mL of glycerin, and 5 to 10 mM of Na ２ HPO ４ and a pharmaceutical composition comprising, having a pH value of 6.5 to 8.0, Among them, the acylated insulin is A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, a pharmaceutical composition. **Claim 9**:

1. A pharmaceutical composition comprising 1.2 mM of acylated insulin, 2. 2.3 moles of zinc ions / 6 moles of acylated insulin, 45 mM of phenol, 10 mM of m-cresol, 20 mM of NaCl, 15 mg / mL of glycerin, 5 mM of Na ２ HPO ４ and having a pH value of 7.4, and Among them, the acylated insulin is A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, a pharmaceutical composition.

10. A pharmaceutical composition comprising an insulin-secretory GLP-1 compound and acylated insulin, Among them, the molar ratio of the insulin-secreting GLP-1 compound to the acylated insulin is at least 1:100, preferably at least 3:100, preferably at least 5:100, preferably at least 8:100, preferably (3:100) to (100:100), preferably (5:100) to (80:100), preferably (8:100) to (50:100), preferably (10:100) to (50:100), preferably (13:100) to (50:100), preferably (13:100) to (40:100), preferably (13:100) to (35:100), preferably (13:100) to (27:100), preferably (13:100) to (20:100), and the acylated insulin is as defined in claim 1, the insulin-secreting GLP-1 compound is N-ε ２６ -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37) peptide, or N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ３０ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl](Val ８ Glu ２２Ｌ ys ３０Ａ rg ２６、３４ -GLP-1(7-37)) peptide, or N-ε ２３ -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoyl amino)-4(s)-carboxybutyryl amino]ethoxy)ethoxy]acetamide)ethoxy]ethoxy)acetyl](Val ８ Glu ２２ Lys ２３ Arg ２６，３４ -GLP-1(7-37)) peptide, or the insulin-secreting GLP-1 compound is represented by the following formula (B), [Acy-(L1) ｒ -(L2) ｑ -G1 (B), wherein G1 is a GLP-1 analog having Arg at position 34 corresponding to GLP-1(7-37) (SEQ ID NO: 5) and having Ala or Gly at position 8, [Acy-(L1) ｒ -(L2) ｑ is a substituent linked to the ε-amino group of the Lys residue at position 26 of the GLP-1 analog, wherein r is an integer of 1 to 10, q is 0 or an integer of 1 to 10, Acy is an aliphatic diacid containing 20 to 24 carbon atoms, provided that formally, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid, L1 is an amino acid residue selected from γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp, L2 is a neutral amino acid residue containing an alkylene glycol, Acy, L1 and L2 are linked by an amide bond, and the order of appearance of L1 and L2 in formula (B) is independently interchangeable, Pharmaceutical composition.

11. G1 is the [Gly8, Arg34]GLP-1-(7-37) peptide (SEQ ID NO: 6) or the [Arg34]GLP-1-(7-37) peptide (SEQ ID NO: 7), preferably the [Gly8, Arg34]GLP-1-(7-37) peptide, and / or r is 1, 2, 3, 4, 5 or 6, preferably r is 1, 2, 3 or 4, preferably r is 1 or 2, preferably r is 1, and / or q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably q is 0, 1, 2, 3 or 4, more preferably q is 0, 1 or 2, and / or Acy is an aliphatic diacid containing 20 to 23 carbon atoms, preferably Acy is an aliphatic diacid containing 20, 21 or 22 carbon atoms, provided that formally a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid. The pharmaceutical composition according to claim 10.

12. L2 is -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-, -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-, -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-, -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-(CH ２ ) ２ -CO-,-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-,-HN-(CH ２ ) ３ -O-(CH ２ ) ３ -O-CH ２ -CO-、or -HN-(CH ２ ) ４ -O-(CH ２ ) ４ -O-CH ２ -CO- and preferably, L2 is -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO- and / or L1 is selected from γGlu or βAsp, preferably L1 is γGlu, and / or Acy is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) １９ -CO-, HOOC-(CH ２ ) ２０ -CO-, HOOC-(CH ２ ) ２１ -CO- or HOOC-(CH ２ ) ２２ -CO- and preferably, Acy is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) ２０ -CO- or HOOC-(CH ２ ) ２２ -CO-. The pharmaceutical composition according to claim 10.

13. In formula (B), between Acy, L1 and L2 are sequentially linked by an amide bond, and the C-terminus of L2 is linked to the ε-amino group of the Lys residue at position 26 of the GLP-1 analog. The pharmaceutical composition according to claim 10.

14. The insulinotropic GLP-1 compound is N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(21-carboxyhenicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(4-[21-carboxyheneicosanoyl amino]-4(S)-carboxybutyryl amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoyl amino)-4(S)-carboxybutyryl amino]ethoxy)ethoxy]acetyl amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(4-[23-carboxytricosanoyl amino]-4(S)-carboxybutyryl amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - (23-carboxytricosanoyl amino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - (19-carboxynonadecanoyl amino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - (21-carboxyheneicosanoyl amino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoyl amino)-4(S)-carboxybutyryl amino]ethoxy)ethoxy]acetyl amino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(4-[19-carboxynonadecanoyl amino]-4(S)-carboxybutyryl amino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６-[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６- [2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - (20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - (22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ - [2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, or N-ε ２６ -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, selected from the group consisting of said compounds, and / or The acylated insulin is A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E,B16H, B25H, B29K (N(ε)-tricosanediasyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosanediasyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-15xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediasyl-γGlu-13xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediasyl-γGlu-14xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-15xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-17xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediasyl-γGlu-16xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediasyl-γGlu-17xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-icosanediasyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediasyl-γGlu-18xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediasyl-γGlu-19xOEG), desB30 human insulin, A14E, B16H, B25H,B29K(N(ε)-eicosanediacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-20xOEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-docosanediacyl-γGlu-24xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-9xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-9xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-10xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H,B29K(N(ε)-icosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-heneicosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-tricosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-tetracosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-icosanediacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-icosanediacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-icosanediacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-13xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-14xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-icosanediacyl-γGlu-15xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-icosanediacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-icosanediacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-16xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-17xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-icosanediacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H,Selected from insulin such as B29K(N(ε)-eicosandiacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-19xOEG), desB30 human insulin, A14E, B16E, B25H, B29K(N(ε)-eicosandiacyl-γGlu-20xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K(N(ε)-docosanediacyl-γGlu-20xOEG), desB30 human insulin, The pharmaceutical composition according to claim 10.

15. The insulin-secretory GLP-1 compound is N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε ２６-[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε ２６ selected from the compound of -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, and / or the acylated insulin is selected from insulin such as A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandiacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosandiacyl-γGlu-18xOEG), desB30 human insulin The pharmaceutical composition according to claim 10.

16. The content of the acylated insulin is higher than 0.3 mM, preferably 0.3 - 9 mM, preferably 0.6 - 8.4 mM, preferably 0.6 - 7.2 mM, preferably 0.6 - 6.0 mM, preferably 0.6 - 4.2 mM, preferably 0.6 - 3.6 mM, preferably 0.6 - 3.0 mM, preferably 0.6 - 2.4 mM, preferably 0.6 - 2.1 mM, preferably 0.9 - 1.8 mM, preferably 0.9 - 1.5 mM, preferably 1.2 - 1.5 mM. The pharmaceutical composition according to claim 10.

17. The composition further contains zinc ions, glycerin, phenol, m - cresol, NaCl, and / or Na ２ HPO ４ and further includes. The pharmaceutical composition according to claim 10.

18. The content of the zinc ions is at least 1.5 moles of zinc ions / 6 moles of acylated insulin, preferably 1.5 - 12 moles of zinc ions / 6 moles of acylated insulin, preferably 1.5 - 10 moles of zinc ions / 6 moles of acylated insulin, preferably 1.5 - 8 moles of zinc ions / 6 moles of acylated insulin, preferably 1.5 - 6 moles of zinc ions / 6 moles of acylated insulin, preferably 1.5 - 4.5 moles of zinc ions / 6 moles of acylated insulin, preferably 1.5 - 3.5 moles of zinc ions / 6 moles of acylated insulin, preferably 1.5 - 2.3 moles of zinc ions / 6 moles of acylated insulin, and / or The content of the glycerin is 2.5% (weight / weight) or less, preferably 2% (weight / weight) or less, preferably 0.3% - 2% (weight / weight), preferably 0.5% - 1.8% (weight / weight), preferably 0.7% - 1.8% (weight / weight), preferably 1% - 1.8% (weight / weight), preferably 1.5% - 1.7% (weight / weight), and / or The content of the phenol is 30 to 70 mM, preferably 40 to 65 mM, preferably 45 to 60 mM, preferably 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, or 65 mM, and / or The content of the m-cresol is 0 to 35 mM, preferably 0 to 20 mM, preferably 0 to 10 mM, preferably 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM, and / or The content of the NaCl is 0 to 150 mM, preferably 5 to 120 mM, preferably 10 to 120 mM, preferably 10 to 100 mM, preferably 10 to 75 mM, preferably 10 to 50 mM, preferably 10 to 30 mM, preferably 10 to 20 mM, preferably 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, and / or The Na ２ HPO ４ The content of is 0 to 75 mM, preferably 5 to 60 mM, preferably 5 to 50 mM, preferably 5 to 25 mM, preferably 5 to 15 mM, preferably 5 to 10 mM, and / or The pH of the pharmaceutical composition is 6.5 to 8.5, preferably the pH is 6.8 to 8.2, preferably the pH is 7.0 to 8.2, preferably the pH is 7.2 to 7.6, more preferably the pH is 7.4 or 7.6, The pharmaceutical composition according to claim 17.

19. 0.9 to 1.5 mM of acylated insulin, and an insulin-secreting GLP-1 compound in which the molar ratio to the acylated insulin is at least 8:100, preferably (8:100) to (50:100), preferably (10:100) to (50:100), preferably (13:100) to (50:100), preferably (13:100) to (40:100), preferably (13:100) to (35:100), preferably (13:100) to (27:100), preferably (13:100) to (20:100), and 1.5 to 2.3 moles of zinc ions / 6 moles of acylated insulin, and 45 to 60 mM of phenol, and 0 to 10 mM of m-cresol, and 10 to 20 mM of NaCl, and 15 to 17 mg / mL of glycerin, and 5 to 10 mM of Na ２ HPO ４ and a pharmaceutical composition comprising the same, wherein the pH value is 6.5 to 8.0, and Among them, the acylated insulin is A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, and the insulin-secretory GLP-1 compound is N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(19-Carboxynonadecanoyl amino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(19-Carboxynonadecanoyl amino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(21-Carboxyheneicosanoyl amino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]-[Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε ２６ -[2-(2-[2-(4-[21-Carboxyheneicosanoyl amino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl]-[Gly8,Arg34]GLP-1-(7-37) peptide, A pharmaceutical composition.

20. 1. 2 mM of acylated insulin, At least 0.096 mM, preferably 0.096 - 0.6 mM, preferably 0.12 - 0.6 mM, preferably 0.16 - 0.6 mM, preferably 0.16 - 0.48 mM, preferably 0.16 - 0.42 mM, preferably 0.16 - 0.32 mM, preferably 0.16 - 0.24 mM of insulin-secretory GLP-1 compound, 1.5 - 2.3 moles of zinc ions / 6 moles of acylated insulin, 45 - 60 mM of phenol, 0 - 10 mM of m-cresol, 10 - 20 mM of NaCl, 15 - 17 mg / mL of glycerin, 5 - 10 mM of Na ２ HPO ４ And a pharmaceutical composition containing the same, The pH value is 6.5 - 8.0, Among them, the acylated insulin is A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanediacyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-12xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosanediacyl-γGlu-18xOEG), desB30 human insulin, and the insulin-secreting GLP-1 compound is N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(19-carboxynonadecanoyl amino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε ２６ -(19-carboxynonadecanoyl amino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε ２６ -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoyl amino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε ２６ -[2-(2-[2-(4-[21-carboxyheneicosanoyl amino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, A pharmaceutical composition.

21. Used as a drug, The pharmaceutical composition according to any one of claims 1 to 20.

22. Used for treating or preventing diabetes, hyperglycemia, and / or glucose intolerance, The pharmaceutical composition according to any one of claims 1 to 20.

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