Insulin derivative

Abstract

To provide a novel insulin derivative, a pharmaceutical formulation thereof, a pharmaceutical composition thereof with a long-acting GLP-1 compound, and a medical use of the acylated insulin, the pharmaceutical formulation, and the pharmaceutical composition.SOLUTION: The novel acylated insulin has an unexpected, significantly increased drug effect, a longer duration of action, a longer in vivo half-life, excellent bioavailability, as well as better physical and chemical stabilities compared with insulin degludec or other insulin derivatives.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of therapeutic peptides, and in particular to a novel insulin derivative, its pharmaceutical formulation, pharmaceutical composition thereof with a long-acting GLP-1 compound, pharmaceutical composition thereof with a fast-acting insulin, and the medical uses of the insulin derivative, pharmaceutical formulation and pharmaceutical composition. [Background technology]

[0002] Insulin is a polypeptide hormone secreted by pancreatic beta cells. It consists of two polypeptide chains, designated A and B, which are linked by two interchain disulfide bonds. In human, porcine, and bovine insulin, the A and B chains contain 21 and 30 amino acid residues, respectively. However, the amino acid residues present at different positions in the two chains vary among species. The widespread application of genetic engineering allows for the preparation of analogs of naturally occurring insulin by substitution, deletion, or addition of one or more amino acid residues.

[0003] Insulin can be used to treat diabetes and related or resulting diseases, and is necessary for maintaining normal metabolic regulation. However, natural insulins such as human insulin have a short duration of action, which requires frequent injections, causing patients a lot of discomfort due to the injections. Therefore, efforts have been made to obtain insulin derivatives or analogs that have better efficacy, longer duration of action, and require less frequent injections, so as to alleviate the inconvenience and discomfort caused by frequent insulin injections.

[0004] WO 1995007931 A1 discloses insulin detemir, a commercially available long-acting insulin, whose molecular structure is characterized by the removal of the threonine at position 30 of the human insulin B chain and the linkage of a 14-carbon aliphatic monoacid to the lysine residue at position 29 of the B chain. WO 2005012347 A2 discloses insulin degludec, another commercially available long-acting insulin. Insulin degludec is a novel ultra-long-acting insulin with a longer duration of action than insulin detemir, whose molecular structure is characterized by the removal of the threonine at position 30 of the human insulin B chain and the linkage of a 16-carbon aliphatic diacid side chain to the lysine residue at position B29 by a single glutamic acid molecule. CN 101573133 B and WO 2009 / 010428 disclose PEGylated insulin, which has a longer duration of action than conventional unmodified insulin. WO2013086927A1 and WO2018 / 024186 disclose acylated derivatives of long-acting human insulin analogues.

[0005] However, to date, no basal insulin product has yet been approved for marketing at a frequency lower than once-daily subcutaneous injection. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there remains a need for insulin derivatives or analogs that have better efficacy or potency, longer duration of action, less frequent administration, and better physicochemical properties than commercially available insulins (e.g., insulin degludec) or known insulin derivatives. [Means for solving the problem]

[0007] The present invention provides novel insulin derivatives (e.g., acylated insulins). Through extensive testing, the inventors have unexpectedly discovered that the novel insulin derivatives (e.g., acylated insulins) have significantly improved efficacy or potency, longer duration of action, longer in vivo half-life, better bioavailability, and better safety, as well as more favorable physical stability, chemical stability, and solubility, compared to commercially available insulin degludec (trade name "Tresiba") or several other insulin derivatives.

[0008] In one aspect, the present invention provides an insulin derivative comprising a parent insulin, an albumin binding residue and a linking group Lin, wherein the parent insulin is a naturally occurring insulin or an insulin analogue, and the albumin binding residue is linked to the parent insulin via a linking group Lin, with the proviso that: the linking group Lin is a hydrophilic linking group having at least 10, preferably at least 15, preferably at least 25, preferably at least 30, preferably at least 36, preferably 15 to 100, preferably 25 to 90, preferably 30 to 80, preferably 30 to 59, preferably 30 to 54 carbon atoms, or the linking group Lin comprises at least 5 neutral amino acid residues containing alkylene glycol, preferably the linking group Lin comprises at least 6 neutral amino acid residues containing alkylene glycol, preferably the linking group Lin comprises 5 to 9 neutral amino acid residues containing alkylene glycol, or the linking group Lin comprises an alkylene glycol having at least 15, preferably at least 20, preferably at least 24, preferably 15 to 50, preferably 20 to 39 carbon atoms, and The albumin binding residue comprises 20 to 40 carbon atoms, preferably the albumin binding residue comprises a linear or branched lipophilic group having 20 to 40 carbon atoms, preferably the albumin binding residue is a fatty acid or aliphatic diacid comprising 20 to 26 carbon atoms (more preferably a fatty acid or aliphatic diacid comprising 20 to 24 carbon atoms), provided that in form the hydroxy group has already been removed from one of the carboxy groups of the fatty acid and the aliphatic diacid.

[0009] Through extensive experiments, the inventors have unexpectedly discovered that by combining an albumin-binding residue of a certain length with a hydrophilic linking group of a certain length in the insulin derivatives of the present invention, the insulin derivatives of the present invention have comparable or longer durations of action compared to existing insulin derivatives, as well as significantly improved efficacy beyond expectations, and that in the presence of albumin, the effect of albumin on the insulin receptor-binding ability is significantly reduced, resulting in significantly improved insulin receptor-binding ability.

[0010] In some embodiments, the parent insulin comprises at least one lysine residue and the albumin binding residue is linked to the lysine residue or the amino group of the N-terminal amino acid residue of the parent insulin via a linking group Lin.

[0011] In some embodiments, the insulin derivative further comprises one or more linking groups II, which are acidic amino acid residues and which are linked between the albumin binding residue and the linking group Lin and / or between the linking group Lin and the insulin parent, preferably between the albumin binding residue and the linking group Lin.

[0012] In another aspect of the present invention, there is provided an insulin derivative which is an acylated insulin, wherein the parent insulin of the acylated insulin is a naturally occurring insulin or insulin analogue and comprises at least one lysine residue, and the acyl moiety of the acylated insulin is linked to the amino group of a lysine residue or the N-terminal amino acid residue of the parent insulin, and the acyl moiety has the formula (A), i.e. III-(II) m -(I) n - (A) however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is 5, 6, 7, 8, or 9; I is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is a fatty acid or aliphatic diacid containing 20 to 26 (preferably 20 to 24) carbon atoms, provided that, in form, a hydroxy group has been removed from one of the carboxy groups of the fatty acid and the carboxy group of the aliphatic diacid; III, II and I are connected by an amide bond, and The order of appearance of II and I in formula (A) is independently interchangeable; Or, The acyl group moiety is represented by formula (A'), i.e., III-(II) m -(I') n’ - (A') however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n' is an integer; I' is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is a fatty acid or aliphatic diacid containing 20 to 26 (preferably 20 to 24) carbon atoms, provided that, in form, a hydroxy group has been removed from one of the carboxy groups of the fatty acid and the carboxy group of the aliphatic diacid; III, II and I' are connected by an amide bond, The order of appearance of II and I' in formula (A') is independently interchangeable, and (I') n’ The total number of carbon atoms is 15 to 100, preferably 20 to 100, preferably 25 to 90, preferably 30 to 80, preferably 30 to 59, preferably 30 to 54.

[0013] In another aspect of the present invention, there is provided an insulin derivative which is an acylated insulin, wherein the parent insulin of the acylated insulin is a naturally occurring insulin or insulin analogue and comprises at least one lysine residue, and the acyl moiety of the acylated insulin is linked to the amino group of a lysine residue or the N-terminal amino acid residue of the parent insulin, and the acyl moiety has the formula (A), i.e. III-(II) m -(I) n - (A) however, m is 0 or an integer from 1 to 10, and n is 5, 6, 7, 8, or 9; I is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is an aliphatic diacid containing 20 to 26 (preferably 20 to 24) carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; III, II and I are connected by an amide bond, and The order of appearance of II and I in formula (A) is independently interchangeable; Or, The acyl group moiety is represented by formula (A'), i.e., III-(II) m -(I') n’ - (A') however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n' is an integer; I' is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is an aliphatic diacid containing 20 to 26 (preferably 20 to 24) carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; III, II and I' are connected by an amide bond, The order of appearance of II and I' in formula (A') is independently interchangeable, and (I') n’ The total number of carbon atoms is 25 to 90, preferably 30 to 80, preferably 30 to 59, and preferably 30 to 54.

[0014] In some embodiments, n is preferably 5, 6, 7, or 8, 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 III is an aliphatic diacid containing 20 to 26 (preferably 20 to 23) carbon atoms, preferably III is an aliphatic diacid containing 20, 21 or 22 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; and / or The insulin parent contains one lysine residue.

[0015] In some embodiments, I 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-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-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-(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, I is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, or, I’ is -HN-(CH2-CH2-O) 10 -CH2-CO, -HN-(CH2-CH2-O) 11 -CH2-CO, -HN-(CH2-CH2-O) 12 -CH2-CO, or -HN-(CH2-CH2-CH2-O)8-CH2-CO, and / or II 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 II is selected from γGlu or βAsp, and / or III is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO-, HOOC-(CH2) 22 -CO- or HOOC-(CH2) 24 -CO-, and preferably III is HOOC-(CH) 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-, and preferably III is HOOC-(CH) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0016] In some embodiments, formula (A) is linked via the C-terminus of I to the amino group of a lysine residue or the N-terminal amino acid residue of the parent insulin, or formula (A') is linked via the C-terminus of I' to the amino group of a lysine residue or the N-terminal amino acid residue of the parent insulin.

[0017] In some embodiments, the acyl moiety is linked to the epsilon amino group of a lysine residue of the insulin parent.

[0018] In some embodiments, the parent insulin lysine residue is located at position B29. In some embodiments, the insulin parent is desB30 human insulin (SEQ ID NO:1 and SEQ ID NO:2, representing the A-chain and B-chain, respectively), A14E, B16H, B25H, desB30 human insulin (SEQ ID NO:3 and SEQ ID NO:4, representing the A-chain and B-chain, respectively), A14E, B16E, B25H, desB30 human insulin (SEQ ID NO:5 and SEQ ID NO:6, representing the A-chain and B-chain, respectively), human insulin (SEQ ID NO:7 and SEQ ID NO:8, representing the A-chain and B-chain, respectively), A21G human insulin (SEQ ID NO:9 and SEQ ID NO:10, representing the A-chain and B-chain, respectively), A21G, desB30 human insulin (SEQ ID NO:11 and SEQ ID NO:12, representing the A-chain and B-chain, respectively), or B28D human insulin (SEQ ID NO:13 and SEQ ID NO:14). No. 14, representing the A and B chains, respectively), and preferably the insulin parent is desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; or A14E, B16E, B25H, desB30 human insulin.

[0019] In some embodiments, the acylated insulin is B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin; B29K(N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-βAsp-6xOEG), d esB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin B29K(N(ε)-eicosandioyl-αAsp-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin;B29K(N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin; B29K(N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin; B29K(N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin; B29K(N(ε) B29K(N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG). , desB30 human insulin; B29K(N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin;B29K(N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αAsp-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu- B29K(N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-βAsp-7xOEG), de sB30 human insulin; B29K(N(ε)-docosandioyl-βAsp-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin;B29K(N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin; B29K(N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-heneicosandioyl-γGlu- desB30 human insulin; B29K(N(ε)-tricosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-tricosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-tricosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-tricosandioyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-tricosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-tetracosandioyl-γGlu-5xOEG), desB30 human insulin sB30 human insulin; B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosanedioyl-γGlu-9xOEG), desB30 human insulin; or B29K (N(ε)-docossanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γ Glu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H , B29K (N(ε)-eicosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin;A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl- A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B2 9K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B 16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B1 6H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30; Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-6xOEG), desB30; Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30; Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A1 4E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-8xOEG), desB30 human insulin Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30; Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30; Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30; Human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tricosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tricosandioyl-γGlu-6xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-tricosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tricosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tetracosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tetracosandioyl-γGlu-6xOEG), desB30 human insulin; A1 4E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docossanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B 16H, B25H, B29K (N(ε)-hexadecandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-5xOEG); desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-6xOEG); desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG); desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG); desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-8x; OEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-8xOEG). A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B 25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A1 4E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; A1 4E, B16E, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A 14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tricosandioyl-γGlu-5xOEG), desB30 human insulin A14E, B16E, B25H, B29K (N(ε)-tricosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tricosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tricosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosandioyl-γGlu-5xOEG), desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-γGlu-9xOEG), desB30 human insulin Insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-12xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-12xPEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-13xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-13xPEG), desB30 human insulin Insulin; B29K(N(ε)-docosandioyl-γGlu-14xPEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-14xPEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-15xPEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-15xPEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-16xPEG), desB30 human insulin; B29K(N B29K(N(ε)-eicosandioyl-γGlu-16xPEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-17xPEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-17xPEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-18xPEG), desB30 human insulin; or B29K(N(ε)-eicosandioyl-γGlu-18xPEG), desB30 human insulin;

[0020] In some embodiments, the acylated insulin is selected from the group consisting of B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin. esB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; A 14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-hexadecandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε) -eicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B A16E, B25H, B29K(N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-12xPEG), desB30 human insulin; or B29K(N(ε)-eicosandioyl-γGlu-12xPEG), desB30 human insulin;

[0021] In some embodiments, the acylated insulin is selected from the group consisting of B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin. B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-hexadecandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

[0022] In another aspect of the present invention, there is provided a pharmaceutical composition comprising an insulin derivative of the present invention as described above, and one or more pharmaceutically acceptable excipients.

[0023] In some embodiments, the pharmaceutical composition comprises at least 1.5 moles of zinc ions / 6 moles of insulin derivative, preferably at least 2.2 moles of zinc ions / 6 moles of insulin derivative, preferably at least 3.5 moles of zinc ions / 6 moles of insulin derivative, preferably at least 4.5 moles of zinc ions / 6 moles of insulin derivative, preferably 4.5-12 moles of zinc ions / 6 moles of insulin derivative, more preferably 4.5-10 moles of zinc ions / 6 moles of insulin derivative, more preferably 4.5-8 moles of zinc ions / 6 moles of insulin derivative, more preferably 4.5-7.5 moles of zinc ions / 6 moles of insulin derivative, more preferably 4.5-7.0 moles of zinc ions / 6 moles of insulin derivative, more preferably 4.5-6.5 moles of zinc ions / 6 moles of insulin derivative; and / or The pH of the drug composition is 6.5 to 8.5, preferably 6.8 to 8.2, preferably 7.0 to 8.2, preferably 7.2 to 7.6, more preferably 7.4 or 7.6.

[0024] In some embodiments, the drug composition further comprises glycerol, phenol, m-cresol, NaCl, and / or NaHPO, preferably, the drug composition further comprises glycerol, phenol, and NaCl, preferably, the drug composition further comprises glycerol, phenol, m-cresol, and NaCl, preferably, the drug composition further comprises glycerol, phenol, NaCl, and NaHPO, more preferably, the drug composition further comprises glycerol, phenol, m-cresol, NaCl, and NaHPO.

[0025] In some embodiments, the glycerol content is no more than about 2.5% (wt / wt), preferably no more than about 2% (wt / wt), 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), more preferably about 1% to about 1.8% (wt / wt), and / or the phenol content is about 16 to 80 mM, preferably about 25 to 75 mM, preferably about 45 to 70 mM, preferably about 45 to 65 mM, and preferably about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 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; and / or the content of m-cresol is about 0 to 35 mM, preferably about 0 to 19 mM, preferably about 0 to 15 mM, and 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; and / or 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, more preferably about 10 to 75 mM, more preferably about 10 to 50 mM, more preferably about 10 to 30 mM, and / or the NaHPO content is about 0 to 75 mM, preferably about 5 to 60 mM, preferably less than about 50 mM, more preferably less than about 25 mM, more preferably less than about 15 mM, and / or The content of the acylated insulin is greater than about 0.3 mM, preferably greater than about 0.6 mM, preferably about 0.3 to 12 mM, preferably about 0.6 to 9.0 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 0.6 to 1.2 mM.

[0026] In some embodiments, the insulin derivatives are selected from the group consisting of B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K( B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

[0027] In another aspect of the present invention, there is provided a pharmaceutical composition comprising about 0.6 to 4.2 mM of the insulin derivative of the present invention, about 1% to about 1.8% (w / w) glycerol, about 45 to 65 mM phenol, about 4.5 to 6.5 moles of zinc ions per 6 moles of insulin derivative, about 10 to 120 mM sodium chloride, and about 0 to 15 mM m-cresol, and having a pH value of about 7.0 to 8.2. Preferably, the insulin derivative is B29K(N(ε)-eicosandioyl-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε) B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

[0028] In another aspect of the present invention, there is provided a pharmaceutical composition comprising about 0.6 mM or 1.2 mM of the insulin derivative of the present invention, 1.7% (w / w) glycerol, about 45 mM phenol, about 10 mM m-cresol, about 6.5 moles of zinc ions / 6 moles of insulin derivative, and about 20 mM sodium chloride, and having a pH value of about 7.0 to 8.0. Preferably, the insulin derivative is B29K(N(ε)-eicosandioyl-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG),desB30 human insulin. insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-12xPEG), desB30 human insulin or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, Preferably, the insulin derivative is B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-12xPEG), desB30 human insulin or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

[0029] In another aspect of the present invention, there is provided a pharmaceutical composition comprising about 0.6 to 4.2 mM of the insulin of the present invention, about 1% to about 2% (preferably about 1.5% to 1.7%) (weight / weight) of glycerol, about 15 mM to 60 mM (preferably about 30 mM to 60 mM, more preferably about 45 mM to 60 mM) of phenol, about 1.5 to 7.0 (preferably about 2.2 to 4.5) moles of zinc ion / 6 moles of insulin derivative, about 10 to 120 mM (preferably about 20 to 50 mM) of sodium chloride, and about 0 to 25 mM (preferably about 0 to 10 mM) of m-cresol, and having a pH value of about 7.0 to 8.2. Preferably, the insulin derivative is B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)- B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

[0030] In another aspect of the present invention, there is provided a pharmaceutical composition comprising about 0.6-4.2 mM of an insulin according to the present invention, about 1.5%-1.7% (w / w) glycerol, about 45-60 mM phenol, about 2.2-4.5 moles of zinc ions / 6 moles of an insulin derivative, about 20 mM sodium chloride, and about 0-10 mM m-cresol, and having a pH value of about 7.0-8.0, preferably the insulin derivative is B29K(N(ε)-eicosandioyl-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

[0031] In some embodiments, the pharmaceutical composition further comprises an insulinotropic GLP-1 compound, preferably 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]acetylamino)ethoxy]ethoxy)acetyl][Aib8, Arg34]GLP-1-(7-37) peptide, N-ε26 The invention further includes an insulinotropic GLP-1 compound selected from -[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.

[0032] In some embodiments, the drug composition has formula (B), i.e., [Acy-(L1) r -(L2) q ]-G1 (B) or a pharmaceutically acceptable salt, amide or ester thereof, where G1 is a GLP-1 analog with Arg at position 34 and Ala or Gly at position 8, corresponding to GLP-1(7-37) (SEQ ID NO:15), and [Acy-(L1) r -(L2) q is a substituent linked to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analog, with the proviso that 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 in form, 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 alkylene glycol-containing amino acid residue, Acy, L1 and L2 are connected by an amide bond, and The order of appearance of L1 and L2 in formula (B) is independently interchangeable.

[0033] (Claim 26) G1 is a [Gly8, Arg34]GLP-1-(7-37) peptide (SEQ ID NO:16) or a [Arg34]GLP-1-(7-37) peptide (SEQ ID NO:17), preferably a [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 in form, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid; 26. The pharmaceutical composition of claim 25.

[0034] In some embodiments, 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-C O-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH 2)2-O-(CH2)2-O-CH2-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(C H2)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-(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-, and / or L1 is selected from γGlu or βAsp, preferably L1 is γGlu, and / or 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-.

[0035] In some embodiments, in formula (B), 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.

[0036] In some embodiments, 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-carboxyheneicosanedioylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanedioylamino]-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-carboxyheneicosanedioylamino)-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-carboxyheneicosanedioylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanedioylamino]-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-carboxyheneicosanedioylamino)-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.

[0037] In some embodiments, 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 -(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-carboxyheneicosanedioylamino)-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-carboxyheneicosanedioylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; Preferably, the insulinotropic GLP-1 compound is N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8, Arg34]GLP-1-(7-37) peptide, or N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanedioylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide.

[0038] The inventors have unexpectedly discovered that not only do pharmaceutical compositions or combined preparations of insulin derivatives (e.g., acylated insulins) and insulin-secreting GLP-1 compounds described in the present invention not reduce the physical stability of the insulin derivatives (e.g., acylated insulins), but also that the combined preparations have superior physical stability to single-drug preparations. Compared with combined preparations of other long-acting insulin derivatives (e.g., insulin degludec and liraglutide), the physical stability of the combined preparations of the present invention is greater than expected. Furthermore, the combined preparations further improve the chemical stability of the insulin derivatives (e.g., acylated insulins) compared with single-drug preparations.

[0039] In some embodiments, the drug composition further comprises a fast-acting insulin. In some embodiments, the fast-acting insulin is Asp B28 Human insulin, Lys B28 Pro B29 Human insulin, Lys B3 Glu B29 Preferably, the fast-acting insulin is selected from one or more of human insulin, human insulin and desB30 human insulin, B28 Human insulin, Lys B28 Pro B29 Human insulin, Lys B3 Glu B29 Human insulin, human insulin or desB30 human insulin.

[0040] In some embodiments, the molar ratio of the insulin derivative to the fast-acting insulin is about 60:3 to about 0.5:3, preferably about 57:3 to about 1:3, preferably about 55:3 to about 1.2:3, preferably about 50:3 to about 1.5:3, preferably about 40:3 to about 1.5:3, preferably about 30:3 to about 1.5:3, preferably about 27:3 to about 1.5:3, preferably about 25:3 to about 1.5:3, preferably about 22:3 to about 1.5:3, preferably about 20:3 to about 1.5:3, preferably about 17:3 to about 1.5:3, preferably about 15:3 to about 1.5:3. 5:3, preferably about 12:3 to about 1.5:3, preferably about 10:3 to about 1.5:3, preferably about 9:3 to about 1.5:3, preferably about 8:3 to about 1.5:3, preferably about 7:3 to about 1.5:3, preferably about 6.9:3 to about 1.5:3, preferably about 6.8:3 to about 1.5:3, preferably about 6.5:3 to about 1.5:3, preferably about 6.3:3 to about 1.5:3, preferably about 6:3 to about 1.5:3, preferably about 5.8:3 to about 1.5:3, preferably about 5.5:3 to about 1.5:3, preferably about 5.3:3 to about 1.5:3, preferably about 5: 3 to about 1.5:3, preferably about 4.8:3 to about 1.5:3, preferably about 4.5:3 to about 1.5:3, preferably about 4.2:3 to about 1.5:3, preferably about 4:3 to about 1.5:3, preferably about 3.9:3 to about 1.5:3, preferably about 3.8:3 to about 1.5:3, preferably about 3.5:3 to about 1.5:3, preferably about 3.2:3 to about 1.5:3, preferably about 3:3 to about 1.5:3, preferably about 2.8:3 to about 1.5:3, preferably about 2.5:3 to about 1.5:3, preferably about 15:3 to about 2:3, preferably about 12:3 to about 2:3, preferably Preferably about 10:3 to about 2:3, preferably about 9:3 to about 2:3, preferably about 8:3 to about 2:3, preferably about 7:3 to about 2:3, preferably about 6.9:3 to about 2:3, preferably about 6.8:3 to about 2:3, preferably about 6.5:3 to about 2:3, preferably about 6.3:3 to about 2:3, preferably about 6:3 to about 2:3, preferably about 5.8:3 to about 2:3, preferably about 5.5:3 to about 2:3, preferably about 5.3:3 to about 2:3, preferably about 5:3 to about 2:3, preferably about 4.8:3 to about 2:3, preferably about 4.5:3 to about 2:3, preferably about 4.2:3 to about 2:3, preferably about 4:3 to about 2:3, preferably about 3.9:3 to about 2:3, preferably about 3.8:3 to about 2:3, preferably about 3.5:3 to about 2:3, preferably about 3.2:3 to about 2:3, preferably about 3:3 to about 2:3, preferably about 15:3 to about 2.4:3, preferably about 12:3 to about 2.4:3, preferably about 10:3 to about 2.4:3, preferably about 9:3 to about 2.4:3, preferably about 8:3 to about 2.4:3, preferably about 7:3 to about 2.4:3, preferably about 6.9:3 to about 2.4:3, preferably about 6.8:3 to about 2.4:3, preferably about 6.5:3 to about 2.4:3, preferably about 6.3:3 to about 2.4:3, preferably about 6:3 to about 2.4:3, The ratio is preferably about 5.8:3 to about 2.4:3, preferably about 5.5:3 to about 2.4:3, preferably about 5.3:3 to about 2.4:3, preferably about 5:3 to about 2.4:3, preferably about 4.8:3 to about 2.4:3, preferably about 4.5:3 to about 2.4:3, preferably about 4.2:3 to about 2.4:3, preferably about 4:3 to about 2.4:3, preferably about 3.9:3 to about 2.4:3, preferably about 3.8:3 to about 2.4:3, preferably about 3.5:3 to about 2.4:3, preferably about 3.2:3 to about 2.4:3, preferably about 3:3 to about 2.4:3, more preferably about 1.5:3, more preferably about 2:3, more preferably about 2.5:3, more preferably about 2.75:3, more preferably about 3:3.

[0041] The inventors have unexpectedly discovered that, after administration, a pharmaceutical composition comprising a dual insulin component of an insulin derivative of the present invention (e.g., an acylated insulin) and insulin aspart has an unexpectedly improved blood glucose lowering effect compared to a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, and that even when the dose ratio of the insulin derivative of the present invention (e.g., an acylated insulin) to insulin aspart is much smaller than the dose ratio of insulin degludec to insulin aspart, an equivalent or greater blood glucose lowering effect can be achieved.

[0042] In some embodiments, the insulin derivative is an insulin derivative of the present invention, preferably B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N( B29K(N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-9xOEG ), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 Human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin;A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin, more preferably the acylated insulin is B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; or B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin;

[0043] In some embodiments, the pharmaceutical composition comprises about 0.09 to 0.36 mM of an insulin derivative and about 0.18 mM of Asp. B28 a pH value of about 7.0 to 8.2, comprising human insulin, about 0.85% to about 2.0% (weight / weight) glycerol, about 15 to 70 mM phenol, about 8 to 14 moles of zinc ions / 6 moles of insulin derivative, about 10 to 120 mM sodium chloride, and about 0 to 15 mM m-cresol, wherein the insulin derivatives are B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

[0044] In some embodiments, the pharmaceutical composition comprises about 0.165-0.18 mM of an insulin derivative and about 0.18 mM of Asp.B28 The insulin derivatives are B29K(N(ε)-eicosanediol-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanediol-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanediol-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanediol-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanediol-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanediol-γGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; or B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin.

[0045] In another embodiment, the insulin derivative or the pharmaceutical composition of the present invention is used as a drug.

[0046] In another embodiment, the above insulin derivative or the above pharmaceutical composition of the present invention is used as a drug for treating or preventing diabetes, hyperglycemia, and / or impaired glucose tolerance.

[0047] In another embodiment, the above insulin derivative or the above pharmaceutical composition of the present invention is used for the treatment or prevention of diabetes, hyperglycemia, and / or impaired glucose tolerance.

[0048] In another aspect, there is provided a use of the insulin derivative of the present invention or the pharmaceutical composition as described above in the preparation of a medicament, preferably the medicament being used for the treatment or prevention of diabetes, hyperglycemia, and / or impaired glucose tolerance.

[0049] In some embodiments, the drug is used to treat diabetes, and the insulin derivative is administered to the same patient every other day or less frequently, and the insulin is not administered to the same patient more frequently for an average period of at least one month, six months, or one year.

[0050] In some embodiments, the drug is used to treat diabetes, and the insulin derivative is administered twice a week or less frequently, and the insulin derivative is not administered more frequently to the same patient for an average period of at least one month, six months, or one year.

[0051] In some embodiments, the drug is used to treat diabetes, and the insulin derivative is administered once a week or less frequently, and the insulin derivative is not administered more frequently to the same patient for an average period of at least one month, six months, or one year.

[0052] The insulin derivatives of the present invention (e.g., acylated insulins) have long pharmacokinetic (hereinafter also referred to as PK) profiles, allowing for twice-weekly, once-weekly or less frequent subcutaneous treatment in diabetic patients.

[0053] In another aspect, the present invention provides a method for treating or preventing diabetes, hyperglycemia, and / or impaired glucose tolerance, said method comprising administering a therapeutically effective amount of the above insulin derivative or the above pharmaceutical composition of the present invention.

[0054] In another aspect, the present invention provides a method for increasing the binding ability of an insulin derivative to an insulin receptor in the presence of albumin, said method comprising: linking an albumin binding residue to naturally occurring insulin or an insulin analogue via a linking group Lin to obtain said insulin derivative, wherein said linking group Lin is a hydrophilic linking group having at least 10, preferably at least 15, preferably at least 25, preferably at least 30, preferably at least 36, preferably 15-100, preferably 25-90, preferably 30-80, preferably 30-59, preferably 30-54 carbon atoms, said albumin binding residue comprising 20-40 carbon atoms, preferably comprising a linear or branched lipophilic group having 20-40 carbon atoms, preferably said albumin binding residue being a fatty acid or aliphatic diacid comprising 20-26 carbon atoms (more preferably a fatty acid or aliphatic diacid comprising 20-24 carbon atoms), with the proviso that in form a hydroxy group has already been removed from the carboxy group of said fatty acid or one of the carboxy groups of said fatty acid or aliphatic diacid, or modifying a naturally occurring insulin or insulin analogue according to formula (A) or formula (A') to obtain said insulin derivative, III-(II) m -(I) n - (A) however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is 5, 6, 7, 8, or 9; I is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is an albumin-binding residue comprising a linear or branched lipophilic group having 20 to 40 carbon atoms, preferably III is a fatty acid or aliphatic diacid comprising 20 to 26 carbon atoms (more preferably a fatty acid or aliphatic diacid comprising 20 to 24 carbon atoms), provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the fatty acid and the aliphatic diacid; III, II and I are connected by an amide bond, and The order of appearance of II and I in formula (A) is independently interchangeable; (A') is III-(II) m -(I') n’ - (A'), however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n' is an integer; I' is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is an albumin-binding residue comprising a linear or branched lipophilic group having 20 to 40 carbon atoms, preferably III is a fatty acid or aliphatic diacid comprising 20 to 26 carbon atoms (more preferably a fatty acid or aliphatic diacid comprising 20 to 24 carbon atoms), provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the fatty acid and the aliphatic diacid; III, II and I' are connected by an amide bond, The order of appearance of II and I' in formula (A') is independently interchangeable, and (I') n’ The total number of carbon atoms is 15 to 100, preferably 20 to 100, preferably 25 to 90, preferably 30 to 80, preferably 30 to 59, preferably 30 to 54.

[0055] In another aspect, the present invention provides a method for improving the potency of an insulin derivative, said method comprising linking an albumin binding residue to naturally occurring insulin or an insulin analogue via a linking group Lin to obtain said insulin derivative, wherein said linking group Lin is a hydrophilic linking group having at least 10, preferably at least 15, preferably at least 25, preferably at least 30, preferably at least 36, preferably 15-100, preferably 25-90, preferably 30-80, preferably 30-59, preferably 30-54 carbon atoms, said albumin binding residue comprising 20-40 carbon atoms, preferably said albumin binding residue comprising a linear or branched lipophilic group having 20-40 carbon atoms, preferably said albumin binding residue being a fatty acid or aliphatic diacid comprising 20-26 carbon atoms (more preferably a fatty acid or aliphatic diacid comprising 20-24 carbon atoms), with the proviso that in form a hydroxy group has already been removed from the carboxy group of the fatty acid or one of the carboxy groups of the fatty acid or the diacid, or modifying a naturally occurring insulin or insulin analogue according to formula (A) or formula (A') to obtain said insulin derivative, III-(II) m -(I) n - (A) however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is 5, 6, 7, 8, or 9; I is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is an albumin-binding residue comprising a linear or branched lipophilic group having 20 to 40 carbon atoms, preferably III is a fatty acid or aliphatic diacid comprising 20 to 26 carbon atoms (more preferably a fatty acid or aliphatic diacid comprising 20 to 24 carbon atoms), provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the fatty acid and the aliphatic diacid; III, II and I are connected by an amide bond, and The order of appearance of II and I in formula (A) is independently interchangeable; (A') is III-(II) m -(I') n’ - (A'), however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n' is an integer; I' is a neutral alkylene glycol-containing amino acid residue, II is an acidic amino acid residue, III is an albumin-binding residue comprising a linear or branched lipophilic group having 20 to 40 carbon atoms, preferably III is a fatty acid or aliphatic diacid comprising 20 to 26 carbon atoms (more preferably a fatty acid or aliphatic diacid comprising 20 to 24 carbon atoms), provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the fatty acid and the aliphatic diacid; III, II and I' are connected by an amide bond, The order of appearance of II and I' in formula (A') is independently interchangeable, and (I') n’ The total number of carbon atoms is 15 to 100, preferably 20 to 100, preferably 25 to 90, preferably 30 to 80, preferably 30 to 59, preferably 30 to 54.

[0056] In some embodiments, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and / or III is an aliphatic diacid containing 20 to 24 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid.

[0057] In some embodiments, the naturally occurring insulin or insulin analog comprises at least one lysine residue, and a linking group Lin, Formula (A) or Formula (A') is linked to the amino group of the lysine residue or the N-terminal amino acid residue of the parent insulin.

[0058] In some embodiments, n is 5, 6, 7, or 8; and / or m is 1, 2, 3, 4, 5 or 6, preferably m is 1, 2, 3 or 4, preferably m is 1 or 2, preferably m is 1, and / or III is an aliphatic diacid containing 20 to 26 (preferably 20 to 23) carbon atoms, preferably III is an aliphatic diacid containing 20, 21 or 22 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; and / or The insulin parent contains one lysine residue.

[0059] In some embodiments, I 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-(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, I is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, or, I’ is -HN-(CH2-CH2-O) 10 -CH2-CO, -HN-(CH2-CH2-O) 11 -CH2-CO, -HN-(CH2-CH2-O) 12 -CH2-CO, or -HN-(CH2-CH2-CH2-O)8-CH2-CO, and / or II 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 II is selected from γGlu or βAsp, and / or III is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO-, HOOC-(CH2) 22 -CO- or HOOC-(CH2) 24 -CO-, and preferably III is HOOC-(CH) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0060] In some embodiments, Formula (A) is linked via the C-terminus of I to the amino group of a lysine residue or N-terminal amino acid residue of the naturally occurring insulin or insulin analog, or Formula (A') is linked via the C-terminus of I' to the amino group of a lysine residue or N-terminal amino acid residue of the naturally occurring insulin or insulin analog.

[0061] In some embodiments, Formula (A) or Formula (A') is linked to the epsilon amino group of a lysine residue in the parent insulin.

[0062] In some embodiments, the lysine residue of the native insulin or insulin analog is located at position B29.

[0063] In some embodiments, the naturally occurring insulin or insulin analog is selected from the group consisting of desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; human insulin, A21G human insulin, A21G, desB30 human insulin; or B28D human insulin, preferably, the parent insulin is desB30 human insulin or A14E, B16H, B25H, desB30 human insulin. [Brief explanation of the drawings]

[0064] [Figure 1a] 1 shows the hypoglycemic effect of the compounds of Examples 1 and 2 of the present invention, insulin degludec, and the vehicle on db / db mice. [Figure 1b] 1a, the AUC of the hypoglycemic effect of the compounds of Examples 1 and 2 of the present invention, insulin degludec, and the vehicle on db / db mice is shown. [Figure 2a] 1 shows the hypoglycemic effect of the compounds of Examples 1 and 2 of the present invention, the compound of Control Example 2, and the solvent on db / db mice. [Figure 2b] Corresponding to FIG. 2a, the AUC of the hypoglycemic effect of the compounds of Examples 1 and 2 of the present invention, the compound of Control Example 2, and the solvent on db / db mice is shown. [Figure 3a] 1 shows the hypoglycemic effect and duration of action of the compounds of Examples 1 to 3 of the present invention and the solvent in db / db mice. [Figure 3b] FIG. 3 corresponds to FIG. 3a and shows the AUC of the hypoglycemic effect of the compounds of Examples 1 to 3 of the present invention and the solvent on db / db mice. [Figure 4a] 1 shows the hypoglycemic effect and duration of action of the compound of Example 2 of the present invention, the compound of Control Example 3, and the solvent in db / db mice. [Figure 4b] FIG. 4A corresponds to FIG. 4A and shows the AUC of the hypoglycemic effect of the compound of Example 2 of the present invention, the compound of Control Example 3, and the solvent on db / db mice. [Figure 5a] 1 shows the hypoglycemic effect and duration of action of the compounds of Control Examples 3 and 4 of the present invention and the solvent in db / db mice. [Figure 5b] FIG. 5A corresponds to FIG. 5A and shows the AUC of the hypoglycemic effect of the compounds of Control Examples 3 and 4 of the present invention and the solvent on db / db mice. [Figure 6a] 1 shows the hypoglycemic effect and duration of action of the compounds of Examples 2, 4 and 5 of the present invention and the solvent in db / db mice. [Figure 6b] FIG. 6A corresponds to FIG. 6A and shows the AUC of the hypoglycemic effect of the compounds of Examples 2, 4 and 5 of the present invention and the solvent on db / db mice. [Figure 7a] 1 shows the hypoglycemic effect of the compound of Example 1 of the present invention and a solvent on streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) rats. [Figure 7b] FIG. 7 corresponds to FIG. 7a and shows the AUC of the hypoglycemic effect of the compound of Example 1 of the present invention and the solvent on STZ-induced type 1 diabetes mellitus (T1DM) rats. [Figure 8a] 1 shows the hypoglycemic effect of the title compounds of Control Example 5, Examples 15 and 16 of the present invention, and the solvent on STZ-induced type 1 diabetes mellitus (T1DM) rats. [Figure 8b] FIG. 8A corresponds to FIG. 8A and shows the AUC of the hypoglycemic effect of the title compounds of Control Example 5, Examples 15 and 16 of the present invention, and the solvent on STZ-induced type 1 diabetes mellitus (T1DM) rats. [Figure 9a] 1 shows the hypoglycemic effect of the compounds of Examples 2 and 4 of the present invention and the solvent on STZ-induced type 1 diabetes mellitus (T1DM) female rats. [Figure 9b] FIG. 9a corresponds to FIG. 9a and shows the AUC of the hypoglycemic effect of the compounds of Examples 2 and 4 of the present invention and the solvent on STZ-induced type 1 diabetes mellitus (T1DM) female rats. [Figure 10a] 1 shows the hypoglycemic effect of the title compounds of Control Example 5, Examples 15 and 16 of the present invention, and the solvent on db / db mice. [Figure 10b]10a, the AUC of the hypoglycemic effect of the title compounds of Control Example 5, Examples 15 and 16 of the present invention, and the solvent on db / db mice is shown. [Figure 11a] 1 shows the hypoglycemic effect of the title compounds of Control Example 5 and Example 16 of the present invention and the solvent on STZ-induced type 1 diabetes mellitus (T1DM) rats. [Figure 11b] 11a, the AUC of the hypoglycemic effect of the title compounds of Control Example 5 and Example 16 of the present invention and the solvent on STZ-induced type 1 diabetes mellitus (T1DM) rats is shown. [Figure 12a] This shows the blood glucose lowering effects of insulin aspart, a pharmaceutical composition containing a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition containing a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent in STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 12b] Corresponding to Figure 12a, the AUC of the blood glucose lowering effect of insulin aspart, a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent is shown in STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 13a] This shows the blood glucose lowering effects of insulin aspart, a pharmaceutical composition containing a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition containing a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent in STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 13b] Corresponding to Figure 13a, the AUC of the blood glucose lowering effect of insulin aspart, a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent is shown in STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 14a]Figure 1 shows the blood glucose levels of mice before the fourth administration of insulin aspart, a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and solvent to STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 14b] Figure 1 shows the blood glucose levels of mice before the eighth administration of insulin aspart, a pharmaceutical composition containing a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition containing a dual insulin component of an acylated insulin of the present invention and insulin aspart, and solvent to STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 14c] Figure 1 shows the blood glucose levels of mice before the 10th administration of insulin aspart, a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and solvent to STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 15a] Figure 1 shows the blood glucose levels of mice 1 hour after the fourth administration of insulin aspart, a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and solvent to STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 15b] Figure 1 shows the blood glucose levels of mice 1 hour after the eighth administration of insulin aspart, a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and solvent to STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 15c]Figure 1 shows the blood glucose levels of mice 1 hour after the 10th administration of insulin aspart, a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and solvent to STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 16] This shows the HbA1c-lowering effects of insulin aspart, a pharmaceutical composition containing a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition containing a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent in STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 17a] 1 shows the hypoglycemic effect of the compound of Example 4 of the present invention, insulin degludec, and a vehicle on STZ-induced type 1 diabetes mellitus (T1DM) rats. [Figure 17b] 17a, which shows the AUC of the hypoglycemic effect of the compound of Example 4 of the present invention, insulin degludec, and the vehicle on STZ-induced type 1 diabetic (T1DM) rats. [Figure 18a] This shows the blood glucose lowering effects of a pharmaceutical composition containing a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition containing a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent on STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 18b] Corresponding to Figure 18a, the AUC of the hypoglycemic effect of a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent on STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice is shown. [Figure 19]This shows the HbA1c-lowering effects of a pharmaceutical composition containing a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition containing a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent in STZ-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice. [Figure 20a] 1 shows the hypoglycemic effect in db / db mice of a pharmaceutical composition containing a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition containing a dual insulin component of an acylated insulin of the present invention and insulin aspart, and a solvent. [Figure 20b] Corresponding to Figure 20a, the AUC of the hypoglycemic effect in db / db mice of a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and the solvent is shown. [Figure 21a] 1 shows random blood glucose levels in db / db mice after injection of a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and vehicle. [Figure 21b] Corresponding to Figure 21a, the AUC of random blood glucose in db / db mice after injection of a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and vehicle is shown. [Figure 21c] 1 shows fasting blood glucose levels in db / db mice after injection of a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and vehicle. [Figure 21d]Corresponding to Figure 21c, the AUC of fasting blood glucose in db / db mice after injection of a pharmaceutical composition comprising a dual insulin component of insulin degludec and insulin aspart, a pharmaceutical composition comprising a dual insulin component of an acylated insulin of the present invention and insulin aspart, and vehicle is shown. [Figure 22] 1 shows the receptor binding ability of the compound of Example 2 of the present invention and the control compound 2 in the presence of 2% HSA and 0% HSA. DETAILED DESCRIPTION OF THE INVENTION

[0065] definition Here, the term insulin includes naturally occurring insulin, such as human insulin, as well as insulin analogues and insulin derivatives thereof.

[0066] The term insulin analogue formally includes polypeptides having a molecular structure derivable from that of naturally occurring insulin (e.g., human insulin) by deficiency 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.

[0067] Here, the term "insulin derivative" refers to a naturally occurring insulin or insulin analogue that has been chemically modified, the modification may be, for example, the introduction of a side chain at one or more positions of the insulin skeleton, or the oxidation or reduction of an amino acid residue on the insulin, or the conversion of a free carboxy group to an ester group, or the acylation of a free amino or hydroxy group. The acylated insulin of the present invention belongs to the insulin derivatives.

[0068] 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, the portion of an insulin derivative or acylated insulin that is not linked to a side chain or has no 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 analog.

[0069] Here, the term "amino acid residue" includes amino acids in which a hydrogen atom has been removed from an amino group, and / or a hydroxy group has been removed from a carboxy group, and / or a hydrogen atom has been removed from a sulfhydryl group. Amino acid residues may be referred to imprecisely as amino acids.

[0070] Unless otherwise specified, all amino acids referred to herein are L-amino acids. The term "albumin-binding residue" refers to a residue capable of non-covalently binding to human serum albumin. Albumin-binding residues linked to insulin typically have a binding affinity for human serum albumin of, for example, less than about 10 μM, or even less than about 1 μM. Albumin binding can be measured by surface plasmon resonance as described in J. Biol. Chem. 277(38), 35035-35042, (2002).

[0071] As used herein, a "hydrophilic linking group" is a linking group that separates the parent insulin from the albumin binding residue by a chemical moiety that contains at least six non-hydrogen atoms, 30-50% of which are N or O.

[0072] "Lipophilic" refers to the group's ability to dissolve in fats, oils, lipids, and lipophilic non-polar solvents (e.g., hexane or toluene). Lipophilic groups include, but are not limited to, fats, fatty acids, aliphatic diacids, and the like, and generally have a "lipid tail." The lipid tail present in these lipophilic groups may be saturated or unsaturated, depending on whether the lipid tail contains a double bond. Lipid tails may include a variety of lengths, such as tails having 7 to 12 carbons (e.g., C 7-12 Alkyl group or C 7-12 alkenyl groups), tails with 13 to 22 carbons (e.g., C 13-22 Alkyl group or C 13-22 alkenyl groups), or tails with 23 to 30 carbons (e.g., C 23-30 Alkyl group or C 23-30 alkenyl group).

[0073] Here, the term alkylene glycol includes oligo / polyalkylene glycol moieties and monoalkylene glycol moieties. Monoalkylene glycols and polyalkylene glycols 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 moiety may be monodisperse (having a clearly defined length / molecular weight) or polydisperse (having an ill-defined length / average molecular weight). The monoalkylene glycol moiety includes -OCH2CHO-, -OCH2CH2CHO-, or -OCH2CH2CH2CHO-, each containing a different group at its end.

[0074] The term "fatty acid" includes straight- or branched-chain aliphatic carboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non-limiting examples of fatty acids are, for example, myristic acid, palmitic acid, stearic acid, and eicosanoic acid.

[0075] Here, 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.

[0076] As used herein, fast-acting insulin includes fast-acting naturally occurring insulins, insulin analogs, and insulin derivatives. Fast-acting insulins generally begin to act within, for example, 1-20 minutes, peak after about 1 hour, and continue to act for 3-5 hours.

[0077] The term "basal insulin" refers to insulin that has a longer duration of action than regular or normal human insulin.

[0078] As used herein, the term "chemical stability" refers to the insulin derivatives of the present invention being sufficiently chemically stable in the desired formulation; i.e., only amounts of chemical degradation products are formed that do not impair the shelf life of the final drug product. Chemical degradation products include deamidation products, isoaspartic acid ester formation, dimer formation, racemization products, products resulting from dehydration processes, etc. Chemical stability can be measured by HPLC analysis of aged samples or formulations.

[0079] In this application, the term "binding ability to insulin receptor" refers to the interaction between insulin and insulin receptor, and the magnitude or strength of such interaction can be measured, for example, by surface plasmon resonance (SPR). For example, when measuring by SPR, when a solution containing insulin flows through a chip coated with insulin receptor, the interaction between insulin and insulin receptor changes the deflection angle of SPR, and such a change is usually expressed as a relative response value. Generally, a larger relative response value indicates a higher binding ability to insulin receptor.

[0080] High physical stability means that the fibrillation tendency is less than 50% of that of human insulin. Fibrillation can be described by the lag time before fibrils start to form under given conditions.

[0081] A polypeptide having insulin receptor and IGF-1 receptor affinity refers to a polypeptide that can interact with the insulin receptor and the human IGF-1 receptor in suitable binding assays, which are well known in the art.

[0082] As used herein, "drug efficacy" or "efficacy" refers to the ability of a drug or active compound to produce a certain action or effect (e.g., blood glucose lowering). For example, when administered at the same dose, the insulin derivatives of the present invention produce a higher blood glucose lowering effect or effect than insulin degludec or other existing insulin derivatives.

[0083] The term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other conditions that cause hyperglycemia. The term is used to describe a metabolic disorder 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 builds up in the blood.

[0084] Type 1 diabetes, also known as 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 known as 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.

[0085] As used herein, the term "GLP-1 analog" or "analog of GLP-1" 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. Specifically, the sequence of GLP-1(7-37) is set forth in SEQ ID NO:15 in the Sequence Listing. The peptide having the sequence set forth in SEQ ID NO:15 may be referred to as "native" GLP-1 or "native" GLP-1(7-37).

[0086] In the sequence listing, the first amino acid residue (histidine) of SEQ ID NO:15 is numbered 1. However, hereinafter, in accordance with established convention in the art, the histidine residue will be numbered 7, and subsequent amino acid residues will be numbered accordingly, culminating in glycine numbered 37. Thus, in general, the amino acid residue numbering or position numbering of the GLP-1(7-37) sequence referred to in this application is the sequence starting with His at position 7 and ending with Gly at position 37.

[0087] The [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:15), respectively. The [Arg34]GLP-1-(7-37) peptide is a GLP-1 analog having Arg at position corresponding to position 34 of GLP-1(7-37) (SEQ ID NO:15). Specifically, the amino acid sequences of the [Gly8, Arg34]GLP-1-(7-37) peptide and the [Arg34]GLP-1-(7-37) peptide are set forth in SEQ ID NO:16 and SEQ ID NO:17, respectively, in the Sequence Listing.

[0088] 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, which may also be referred to as side chains, have been covalently attached to said peptide.

[0089] In this application, insulin or GLP-1 compounds are named according to the following principles: they are named according to mutations and modifications (e.g., acylation) of human insulin or natural GLP-1(7-37). The acyl moiety is named according to the IUPAC nomenclature and, in other cases, according to the peptide nomenclature. For example, the following acyl moieties are named:

[0090] [ka]

[0091] For example, they can be named "eicosandioyl-γGlu-OEG-OEG," "eicosandioyl-γGlu-2xOEG," or "eicosandioyl-gGlu-2xOEG," or "19-carboxynonadecanoyl-γGlu-OEG-OEG," where OEG stands for the abbreviation for the group —NH(CH)O(CH)OCHCO— (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 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].

[0092] For example, the insulin of Control Example 2 of the present invention (having the sequence / structure shown below) is referred to as "B29K(N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin," "B29K(N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin," or "B29K(N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin," indicating that the amino acid K at position B29 in human insulin has been modified by acylation at the epsilon nitrogen of the lysine residue of B29 (referred to as N(ε) or (N(ε))) with the residue eicosandioyl-γGlu-2xOEG, and that the amino acid T at position B30 in human insulin is missing. Also, for example, the insulin of Control Example 5 (having the sequence / structure shown below) is referred to as "B29K(N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin," indicating that the amino acid K at position B29 in human insulin has been modified by acylation at the epsilon nitrogen of the lysine residue of B29 (referred to as N(ε) or (N(ε))) with the residue eicosandioyl-γGlu-2xOEG, and that the amino acid T at position B30 in human insulin is missing. ) is called "A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-gGlu-2xOEG), desB30 human insulin" or "A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin" and indicates that 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, the amino acid K at position B29 in human insulin has been modified at the epsilon nitrogen (referred to as N(ε)) of the lysine residue at B29 by acylation with the residue eicosandioyl-gGlu-2xOEG, and the amino acid T at position B30 in human insulin is missing.

[0093] [ka]

[0094] In this application, "nxPEG" refers to -NH(CH2CH2O) n The term "12xPEG" refers to the group -NH(CHCHO) 12 This represents the group CH2CO-.

[0095] Insulin is a polypeptide hormone secreted by β-cells in the pancreas and consists of two polypeptide chains, A and B, which are linked by two interchain disulfide bonds. The A chain is also characterized by having one intrachain disulfide bond.

[0096] There are three main methods for preparing human insulin in microorganisms. Two involve Escherichia coli. One involves expressing a fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, IL, pp. 729-739). Another uses a signal peptide to allow secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). The third method uses Saccharomyces cerevisiae, where the insulin precursor is secreted into the culture medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art is replete with methods for expressing insulin precursors in E. coli or Saccharomyces cerevisiae, see for example US Pat. No. 5,962,267, WO95 / 16708, EP0055945, EP0163529, EP0347845 and EP0741188.

[0097] The construction, expression, processing, and purification of insulin analog vectors can be carried out using techniques known to those skilled in the art. For example, the insulin analogs can be prepared by expressing a DNA sequence encoding the target insulin analog in a suitable host cell using known techniques, such as those disclosed in U.S. Pat. No. 6,500,645. For example, insulin analogs may be prepared using the method reported in the following publication: Glendorf T, Sørensen 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. In this publication, mutations are introduced into an insulin-encoding vector by overlap extension PCR. Insulin analogs are expressed in the Saccharomyces cerevisiae strain MT663 as pre-insulin-like fusion proteins with an Ala-Ala-Lys small C-peptide. The single-chain precursor is enzymatically converted to the double-chain desB30 analog by the hydrolytic A. lyticus endoprotease.

[0098] The isolated insulin analogs can be acylated at the desired position by acylation methods known in the art, and examples of such insulin analogs are described, for example, in Chinese patent applications with publication numbers CN1029977C, CN1043719A, and CN1148984A.

[0099] The nucleic acid sequence encoding each insulin analog polypeptide can be prepared synthetically by established standard methods, such as those described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869 or Matthes et al. (1984) EMBO Journal 3:801-805.

[0100] The term "excipient" broadly refers to any ingredient other than the active therapeutic ingredient. An excipient may be an inactive, non-active, and / or non-pharmaceutically active substance.

[0101] Excipients may be used for a variety of purposes depending on the drug composition, e.g., as carriers, solvents, diluents, tableting aids, and / or to improve administration and / or absorption of an active agent. Examples of excipients include, but are not limited to, diluents, buffers, preservatives, tension adjusting agents (also called tonicity agents or isotonicity agents), chelating agents, surfactants, protease inhibitors, wetting agents, emulsifiers, antioxidants, bulking agents, metal ions, oily solvents, proteins and / or zwitterions, and stabilizers.

[0102] Drug compositions of pharmaceutically active ingredients and various excipients are known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th Edition (1995) and any subsequent versions).

[0103] It is assumed that, for the patient's convenience, the patient prefers that the time interval (time delay) between administration of an acylated insulin of this invention and the next administration of an acylated insulin of this invention be the same or approximately the same length in days. It may further be expected that the patient prefers to administer the acylated insulin once a week, i.e., on the same day of the week, such as every Sunday. Averaging over a period of one month, six months, or one year, this amounts to administering the acylated insulin every sixth day, and no more frequently. For some patients, it may be necessary to administer the acylated insulin 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 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 every third day, or approximately every third day, and no more frequently, averaging over a period of one month, six months, or one year. Still other patients may find it advantageous to administer acylated insulin twice a week, for example, with about 3-4 days between each administration, when averaged over a period of one month, six months, or one year. For some patients, it may be necessary to administer acylated insulin every other day, or about every other day, when averaged over a period of one month, six months, or one year, and no more frequently. For other patients, it may be necessary to administer acylated insulin every other day, or about every other day, when averaged over a period of one month, six months, or one year, and no more frequently. For some patients, it may be necessary to administer acylated insulin every seventh day, or about every seventh day, when averaged over a period of one month, six months, or one year, and no more frequently. Still other patients may not administer acylated insulin at exactly the same length of time intervals (number of days) every week, month, or year. Averaging over a period of one month, six months, or one year, some patients may be able to administer acylated insulin at time intervals of every 5 to 7 days, and no more frequently, as the case may be.Other patients may optionally administer acylated insulin at time intervals of every 4 to 6 days, and no more frequently, averaged over a period of one month, six months, or one year. Further, other patients may optionally administer acylated insulin at time intervals of every 3 to 7 days, and no more frequently, averaged over a period of one month, six months, or one year.

[0104] The primary target diseases and conditions of this 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 effects of insulin have clinical relevance or benefit, such as prediabetes, impaired glucose tolerance, metabolic syndrome, obesity, cachexia, in vivo β-cell damage / death, hyperphagia, and inflammation. All of these types of conditions are known or believed to benefit from a stable metabolic state in subjects suffering from the disease or condition. In any event, any method of treatment involving the administration of insulin can be modified by practicing the teachings of this invention, meaning that such treatments include the administration of long-acting insulins as provided herein. [Example]

[0105] The following examples are offered by way of illustration and not by way of limitation. The abbreviations used in this application are as follows:

[0106] OEG is the amino acid residue -NH(CH2)2O(CH2)2OCH2CO-. OSu is succinimid-1-yloxy-2,5-dioxo-pyrrolidin-1-yloxy.

[0107] OtBu is oxy-tert-butyl. HCl is hydrogen chloride.

[0108] γGlu or gGlu is a γL-glutamyl group. NHS is N-hydroxysuccinimide.

[0109] DCC is dicyclohexylcarbodiimide. AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0110] OH is the hydroxyl radical. CH3CN is acetonitrile.

[0111] Gly is glycine. Arg is arginine.

[0112] TFA is trifluoroacetic acid. HbA1c is glycosylated hemoglobin.

[0113] AUC is the area under the time-blood glucose curve. RU is a response unit.

[0114] The following examples and general methods are directed to intermediate compounds and final products as determined by the specification and synthetic schemes. The following examples illustrate the preparation of compounds of the present invention in detail, but the chemical reactions described are disclosed with general applicability to the preparation of compounds of the present invention. In some cases, the above reactions may not be applicable to all compounds within the scope of the present invention as described above. Those skilled in the art can easily identify compounds for which this occurs. In these cases, the above reactions can be successfully carried out by conventional modifications known to those skilled in the art, i.e., by appropriate protection of interfering groups, by changing to other conventional reagents, or by conventional modification of reaction conditions. In all preparation methods, all raw materials are known or can be easily prepared using known raw materials. All temperatures are given in degrees Celsius, and unless otherwise specified, when referring to yields, all parts and percentages are given by weight, and when referring to solvents and eluents, all parts are given by volume.

[0115] Example 1 B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin (compound 1)

[0116] [ka]

[0117] 1. Synthesis of des(B30) human insulin des(B30) human insulin is patented in China CN1056618C It is prepared according to the method described in Example 11 of the present invention.

[0118] 2. Preparation of Target Insulin DesB30 human insulin (5 g, 0.876 mmol) was dissolved in 100 mM aqueous NaHPO (150 mL), acetonitrile (100 mL) was added, and the pH was adjusted to 10–12.5 with 1N NaOH. tert-Butyleicosandioyl-γGlu-(5xOEG-OSu)-OtBu (1.36 g, 0.964 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 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 approximately 30 mL and poured into ice-cold n-heptane (300 mL). The precipitated product was isolated by filtration and washed twice with n-heptane. After drying in vacuo, it was purified by ion exchange chromatography (Resource Q, 0.25% to 1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5) and reverse-phase chromatography (acetonitrile, water, TFA). The purified fractions were combined, the pH was adjusted to 5.2 with 1 N HCl, and the precipitate was isolated and lyophilized to give the title compound 1.

[0119] LC-MS (electrospray): m / z = 1377.53 [M+5H] 5+ 3. Preparation of the intermediate tert-butyleicosandioyl-γGlu-(5xOEG-OSu)-OtBu 3.1 tert-Butyl eicosanoidioyl-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, triethylamine (13.95 mL) was added, and the resulting cloudy mixture was stirred at room temperature. DCC (11.39 g, 55.19 mmol) was added and stirred overnight. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 24.12 g (97% yield) of tert-butyl eicosanedioyl-OSu.

[0120] LC-MS(Scie×100API): m / z=496.36(M+1) + 3.2 tert-Butyl eicosanoidioyl-γGlu-OtBu tert-Butyleicosandioyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred. H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water were added in this order. 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, and the mixture was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 27.27 g (96% yield) of tert-butyleicosandioyl-γGlu-OtBu was obtained.

[0121] LC-MS(Scie×100API): m / z=584.44(M+1) + 3.3 tert-Butyl eicosanoidioyl-γGlu-(OSu)-OtBu Under nitrogen gas protection, tert-butyleicosandioyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added, and the mixture was stirred for 10 min. NHS (5.38 g, 50.17 mmol) and DCC (10.60 g, 51.38 mmol) were then added. The mixture was stirred overnight at room temperature. The filtrate was filtered and concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and then separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure, methyl tert-butyl ether was added, stirred for 30 minutes, suction filtered, and the filter cake was dried overnight in vacuo to obtain 25.76 g (yield 81%) of tert-butyleicosandioyl-γGlu-(OSu)-OtBu.

[0122] LC-MS(Scie×100API): m / z=681.46(M+1) + 3.4 tert-Butyleicosanedioyl-γGlu-(2xOEG-OH)-OtBu tert-Butyleicosandioyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred. 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added sequentially and heated to obtain a clear solution. The solution was stirred at room temperature for 4 h. 10% aqueous citric acid (200 mL) was then added, and the lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 30.75 g (93% yield) of tert-butyleicosandioyl-γGlu-(2xOEG-OH)-OtBu was obtained.

[0123] LC-MS(Scie×100API): m / z=874.59(M+1) + 3.5 tert-Butyleicosanedioyl-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyleicosandioyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added, and the mixture was stirred for 10 min. NHS (4.05 g, 35.18 mmol) and DCC (7.98 g, 38.70 mmol) were 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 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 31.09 g (91% yield) of tert-butyleicosandioyl-γGlu-(2xOEG-OSu)-OtBu.

[0124] LC-MS(Scie×100API): m / z=971.61(M+1) + 3.6 tert-Butyl eicosanoidioyl-γGlu-(5xOEG-OH)-OtBu tert-Butyleicosandioyl-γGlu-(2xOEG-OSu)-OtBu (31.09 g, 32.01 mmol) was dissolved in dichloromethane (350 mL) and stirred. 3xAEEA (14.52 g, 32.01 mmol), triethylamine (8.90 mL), and water (25 mL) were added sequentially and heated to obtain a clear solution. The solution was stirred at room temperature for 4 h. 10% aqueous citric acid (200 mL) was then added, and the lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 38.99 g (93% yield) of tert-butyleicosandioyl-γGlu-(5xOEG-OH)-OtBu was obtained.

[0125] LC-MS(Scie×100API): m / z=1309.81(M+1)+ 3.7 tert-Butyl eicosanoidioyl-γGlu-(5xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyleicosandioyl-γGlu-(5xOEG-OH)-OtBu (38.99 g, 29.77 mmol) was dissolved in dichloromethane (400 mL), triethylamine (8.28 mL) was added, and the mixture was stirred for 10 min. NHS (3.43 g, 29.77 mmol) and DCC (6.76 g, 32.75 mmol) were 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 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 38.11 g (91% yield) of tert-butyleicosandioyl-γGlu-(5xOEG-OSu)-OtBu.

[0126] LC-MS(Scie×100API): m / z=1406.83(M+1) + Example 2: B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin (compound 2)

[0127] [ka]

[0128] Compound 2 was prepared following a procedure similar to that of Example 1, Part 2. LC-MS (electrospray): m / z=1406.28 [M+5H] 5+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(6xOEG-OSu)-OtBu was prepared according to steps similar to those in Example 1, Part 3.

[0129] LC-MS(Scie×100API): m / z=1551.90(M+1) + Example 3: B29K(N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin (compound 3)

[0130] [ka]

[0131] Compound 3 was prepared following a procedure similar to that of Example 1, Part 2. LC-MS (electrospray): m / z = 1464.30 [M+5H] 5+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(8xOEG-OSu)-OtBu was prepared according to steps similar to those in Example 1, Part 3.

[0132] LC-MS(Scie×100API): m / z=1814.02(M+1) + Example 4: B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin (compound 4)

[0133] [ka]

[0134] Compound 4 was prepared following a procedure similar to that of Example 1, Part 2. LC-MS (electrospray): m / z = 1411.88 [M+5H] 5+ Intermediates tert-Butyldocosandioyl-γGlu-(6xOEG-OSu)-OtBu was prepared according to steps similar to those in Example 1, Part 3.

[0135] LC-MS(Scie×100API): m / z=1579.94(M+1) + Example 5: B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin (compound 5)

[0136] [ka]

[0137] Compound 5 was prepared following a procedure similar to that of Example 1, Part 2. LC-MS (electrospray): m / z = 1469.91 [M+5H] 5+ Intermediates tert-Butyl docosanol gamma Glu-(8xOEG-OSu)-OtBu was prepared according to steps similar to those in Example 1, Part 3.

[0138] LC-MS(Scie×100API): m / z=1870.08(M+1) + Control Example 1 B29K(N(ε)-hexadecandioyl-γGlu), desB30 human insulin (insulin degludec, reference compound 1) The control compound, insulin degludec, was prepared according to Example 4 of patent CN105820233A.

[0139] Control Example 2 B29K (N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin (control compound 2)

[0140] [ka]

[0141] Control Compound 2 was prepared following steps similar to those in Example 1, Part 2. LC-MS (electrospray): m / z = 1290.22 [M+5H] 5+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(2xOEG-OSu)-OtBu was prepared according to steps similar to those in Example 1, Part 3.

[0142] LC-MS(Scie×100API): m / z=971.61(M+1) + Control Example 3 B29K (N(ε)-octadecandioyl-γGlu-2xOEG), desB30 human insulin (control compound 3)

[0143] [ka]

[0144] Control compound 3 was prepared following similar steps as in Example 1, Part 2. LC-MS (electrospray): m / z = 1284.61 [M+5H] 5+ Control Example 4 B29K(N(ε)-octadecandioyl-γGlu-6xOEG), desB30 human insulin (control compound 4)

[0145] [ka]

[0146] Control compound 4 was prepared following similar steps as in Example 1, Part 2. LC-MS (electrospray): m / z = 1400.68 [M+5H] 5+ Example 6 Pharmacodynamic studies in db / db mice This study aims to confirm the blood glucose (BG) regulating effect of the acylated insulin of the present invention in cases of diabetes.

[0147] The acylated insulins of Examples 1-5 and the control compounds of Control Examples 1-4 were tested in a single-dose study in an obese diabetic mouse model (db / db mice). The hypoglycemic effects of the acylated insulins were tested at different doses of 9 U / kg or 10 U / kg.

[0148] Eight- to nine-week-old male db / db (BKS / Lepr) mice were housed in appropriately designed breeding boxes in a barrier environment, with free access to standard chow and purified water. 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, the mice were used in the experiments.

[0149] Before the start of the experiment, basal blood glucose was assessed at time -1 / 1 h (9:30 AM) and mice were weighed. Mice were randomly assigned to vehicle or treatment groups based on blood glucose and body weight, and treated with subcutaneous injections of either vehicle or acylated insulin (9 U / kg or 10 U / kg). The vehicle contained 19.6 mg / mL glycerol, 1.5 mg / mL phenol, and 1.72 mg / mL m-cresol, with a zinc ion concentration of 55 μg / mL and a pH of 7.6.

[0150] The acylated insulin was dissolved in a solvent to a dose concentration of 1.8 or 2 U / mL, and the dose volume was 5 mL / kg (i.e., 50 μL / 10 g body weight). A single subcutaneous injection was administered into the dorsal neck. The acylated insulin was administered at approximately 10:30 AM (time 0). During the administration period, the animals were fasted but allowed water access. Blood glucose levels were assessed in mice 3, 6, 9, 12, and 15 hours after administration. To simulate feeding, an oral glucose tolerance test (OGTT) was initiated after blood glucose detection at 15 hours. Blood glucose levels were measured 30, 60, 120, and 180 minutes after intragastric administration of a glucose solution (100 mg / mL, 10 mL / kg). Three consecutive OGTT experiments were performed. Preliminary results indicated that the efficacy of the test compound was nearly eliminated in the final OGTT, and the experiment was terminated after 30 hours of blood glucose assessment.

[0151] The rats' tails were cleaned with an alcohol swab, and a drop of blood was collected from the tail with a disposable blood collection needle and measured using a blood glucose meter and attached test strips (Roche). A blood glucose-time dose-response curve was constructed for each single dose of acylated insulin.

[0152] To describe the effect of the acylated insulins of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve. A smaller AUC value indicates a better blood glucose lowering effect and better efficacy.

[0153] Test results: The blood glucose lowering effects of the acylated insulin of the present invention and the control compound in db / db mice are shown in Figures 1a to 6b and Table 1. Specifically, Figures 1a and 1b show that the hypoglycemic effect of the acylated insulins of the present invention, such as Compound 1 and Compound 2, in db / db mice is significantly superior to that of insulin degludec, and the effective duration of action is longer than that of insulin degludec.

[0154] Figures 2a and 2b show that the hypoglycemic effect of the acylated insulins of the present invention, such as Compound 1 and Compound 2, on db / db mice was significantly superior to that of the control Compound 2, and the efficacy of Compound 1 and Compound 2 of the present invention was increased by 39.5% and 45.1%, respectively, within 0 to 16.5 hours of administration compared to the control Compound 2, as shown in Table 1.

[0155] [Table 1]

[0156] Percentage increase in efficacy relative to Control Compound 2 = [(AUC(test compound) - AUC(solvent)) / ((AUC(Control Compound 2) - AUC(solvent)) - 1] * 100%, where test compound refers to an acylated insulin of the present invention.

[0157] Figures 3a-3b show that Compounds 1, 2 and 3 of the present invention all have excellent efficacy and remain effective in db / db mice when monitored for up to 30 hours, demonstrating a significantly extended duration of hypoglycemic action.

[0158] 4a to 5b show that the hypoglycemic effect of the acylated insulin of the present invention, such as Compound 2, on db / db mice was significantly superior to that of Control Compounds 3 and 4.

[0159] Figures 6a-6b show that Compound 4, Compound 5 and Compound 2 of the present invention all have excellent efficacy and remain effective in db / db mice when monitored for up to 41 hours, demonstrating a significantly prolonged duration of hypoglycemic action.

[0160] Example 7 Pharmacodynamic study in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) rats Male Wistar rats, 8 weeks old and weighing 180–220 g, were housed in suitable enclosures (5 rats / box) in a barrier environment. Standard chow and purified water were available ad libitum. Environmental conditions were controlled at 40–60% relative humidity and 22–24°C. After a 4-day acclimation period and a 12-h fast, rats were intraperitoneally injected with 60 mg / kg of streptozotocin (Sigma) solution (10 mg / mL in 0.1 M citrate buffer). After administration, glucose (20%) was appropriately supplemented in the drinking water to prevent sudden hypoglycemia, and supplementation was discontinued 12 h later. Four days after streptozotocin administration, random blood glucose monitoring was performed. Rats with blood glucose levels of 20 mmol / L or higher were selected as T1DM model rats for subsequent studies.

[0161] Before the start of the experiment, basal blood glucose was assessed at time -1 / 1 h (9:30 AM) and rats were weighed. Rats were randomly assigned to either the vehicle or treatment group based on blood glucose and body weight. They were then treated with either a subcutaneous injection of the vehicle or 3 U / kg of acylated insulin. The vehicle contained 19.6 mg / mL glycerol, 1.5 mg / mL phenol, and 1.72 mg / mL m-cresol, with a zinc ion concentration of 55 μg / mL and a pH of 7.6.

[0162] The acylated insulin was dissolved in a solvent to a dose concentration of 1.5 U / mL, with a dose volume of 2 mL / kg (i.e., 0.2 mL / 100 g body weight). A single subcutaneous dose was administered subcutaneously in the dorsal neck. The acylated insulin was administered at approximately 9:30 AM (time 0), and the rats' blood glucose levels were assessed 2 and 4 hours after administration. An oral glucose tolerance test (OGTT) was performed once each at 4 and 7 hours (as described below).

[0163] Oral glucose tolerance test (OGTT) Detection time: Blood was collected from the tip of the tail at predetermined time points to measure fasting blood glucose levels (0 min), and then a glucose solution (100 mg / mL or 200 mg / mL, 10 mL / kg) was administered intragastrically. Blood glucose was then measured 30, 60, 120, and 180 min after the glucose load.

[0164] The rat's tail was cleaned with an alcohol swab, and a drop of blood was collected from the tail with a disposable blood collection needle, and the blood glucose level was measured using a blood glucose meter (Roche) and the attached test strip.

[0165] A blood glucose-time dose-response curve was constructed for each single dose of acylated insulin. To describe the effect of acylated insulin on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each single dose-response curve.

[0166] 7a to 7b show that the acylated insulin of the present invention has an excellent blood glucose lowering effect even in type 1 diabetic (T1DM) rats, ie, has an excellent drug efficacy.

[0167] Example 8 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 6)

[0168] [ka]

[0169] 1.N-ε 26 Preparation of -[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 prepared using a standard recombinant protein expression method (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 NaHPO aqueous solution (150 mL), acetonitrile (100 mL) was added, and the pH was adjusted to pH 10-12.5 with 1 N NaOH. tert-Butyleicosandioyl-γGlu(2xOEG-OSu)-OtBu (1.59 g, 1.63 mmol) was dissolved in acetonitrile (50 mL) and slowly added to the [Gly8, Arg34]GLP-1-(7-37) peptide solution, maintaining the pH 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 isolated 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 approximately 30 mL and poured into ice-cold n-heptane (300 mL). The precipitated product was isolated by filtration and washed twice with n-heptane. After drying in vacuo, 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) and reverse-phase chromatography (acetonitrile, water, TFA). The purified fractions were combined, the pH was adjusted to 5.2 with 1N HCl, and the precipitate was isolated and lyophilized to give the title compound.

[0170] LC-MS (electrospray): m / z = 1028.79 [M+4H]4+ 2. Preparation of the intermediate tert-butyleicosandioyl-γGlu-(2xOEG-OSu)-OtBu 2.1 tert-Butyl eicosanoidioyl-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, and the resulting cloudy mixture was stirred at room temperature. DCC (11.39 g, 55.19 mmol) was added, and the mixture was further stirred overnight. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 24.12 g (97% yield) of tert-butyl eicosanedioyl-OSu.

[0171] LC-MS(Scie×100API): m / z=496.36(M+1) + 2.2 tert-Butyl eicosanoidioyl-γGlu-OtBu tert-Butyleicosandioyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred. H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water (25 mL) were added in this order and heated to obtain a clear solution. The solution was stirred at room temperature for 4 h. Next, 10% aqueous citric acid (200 mL) was added, and the mixture was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 27.27 g (96% yield) of tert-butyleicosandioyl-γGlu-OtBu was obtained.

[0172] LC-MS(Scie×100API): m / z=584.44(M+1) + 2.3 tert-Butyl eicosanoidioyl-γGlu(OSu)-OtBu Under nitrogen gas protection, tert-butyleicosandioyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added, and the mixture was stirred for 10 min. NHS (5.38 g, 50.17 mmol) and DCC (10.60 g, 51.38 mmol) were 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, and the layers were separated. Saturated brine was added to the upper organic layer and washed with water. After separation, the upper organic layer 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, and suction filtered. The filter cake was dried overnight in vacuo to obtain 25.76 g (yield 81%) of tert-butyleicosandioyl-γGlu-(OSu)-OtBu.

[0173] LC-MS(Scie×100API): m / z=681.46(M+1) + 2.4 tert-Butyleicosanedioyl-γGlu-(2xOEG-OH)-OtBu tert-Butyleicosandioyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred. 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added sequentially and heated to obtain a clear solution. The solution was stirred at room temperature for 4 h. 10% aqueous citric acid (200 mL) was then added, and the layers were separated. The lower organic layer was washed with saturated brine. After separation, the lower organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 30.75 g (93% yield) of tert-butyleicosandioyl-γGlu-(2xOEG-OH)-OtBu was obtained.

[0174] LC-MS(Scie×100API): m / z=874.59(M+1) + 2.5 tert-Butyleicosanedioyl-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyleicosandioyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added, and the mixture was stirred for 10 min. NHS (4.05 g, 35.18 mmol) and DCC (7.98 g, 38.70 mmol) were 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 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 31.09 g (91% yield) of tert-butyleicosandioyl-γGlu-(2xOEG-OSu)-OtBu.

[0175] LC-MS(Scie×100API): m / z=971.61(M+1) + Example 9 N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8, Arg34]GLP-1-(7-37) peptide (compound 7)

[0176] [ka]

[0177] Following steps similar to those in Example 8, Part 1, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8, Arg34]GLP-1-(7-37) peptide was prepared.

[0178] LC-MS (electrospray): m / z = 992.52 [M+4H]4+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(OEG-OSu)-OtBu was prepared following steps similar to those in Example 8, Part 2.

[0179] LC-MS(Scie×100API): m / z=826.54(M+1) + Example 10 N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide (compound 8)

[0180] [ka]

[0181] Following steps similar to those in Example 8, Part 1, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide was prepared.

[0182] LC-MS (electrospray): m / z = 956.25 [M+4H] 4+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(OSu)-OtBu was prepared following steps similar to those in Example 8, Part 2.

[0183] LC-MS(Scie×100API): m / z=681.46(M+1) + Example 11 N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide (compound 9)

[0184] [ka]

[0185] Following steps similar to those in Example 8, Part 1, N-ε 26-(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide was prepared.

[0186] LC-MS (electrospray): m / z = 959.75 [M+4H] 4+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(OSu)-OtBu was prepared following steps similar to those in Example 8, Part 2.

[0187] LC-MS(Scie×100API): m / z=681.46(M+1) + Example 12 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 10)

[0188] [ka]

[0189] Following steps similar to those in Example 8, Part 1, 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.

[0190] LC-MS (electrospray): m / z = 1021.78 [M+4H] 4+ Example 13 N-ε 26 -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide (compound 11)

[0191] [ka]

[0192] Following steps similar to those in Example 8, Part 1, N-ε26 -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide was prepared.

[0193] LC-MS (electrospray): m / z = 949.24 [M+4H] 4+ Intermediates tert-Butyloctadecanediol-γGlu-(OSu)-OtBu was prepared following steps similar to those in Example 8, Part 2.

[0194] LC-MS(Scie×100API): m / z=653.43 (M+1) + Example 14: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanedioylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (compound 12)

[0195] [ka]

[0196] Following steps similar to those in Example 8, Part 1, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanedioylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide was prepared.

[0197] LC-MS (electrospray): m / z = 1035.80 [M+4H] 4+ Intermediates tert-Butyldocosandioyl-γGlu-(2xOEG-OSu)-OtBu was prepared following steps similar to those in Example 8, Part 2.

[0198] LC-MS(Scie×100API): m / z=999.64(M+1) + Control Example 5 A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin (control compound 5)

[0199] [ka]

[0200] 1. Preparation of A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-2xOEG), and desB30 human insulin A14E, B16H, B25H, and desB30 human insulins were prepared by standard methods for preparing insulin analogs (see Glendorf T, Sørensen 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). A14E, B16H, B25H, and desB30 human insulins (5 g, 0.888 mmol) were dissolved in 150 mL of 100 mM NaHPO aqueous solution, acetonitrile (100 mL) was added, and the pH was adjusted to 10-12.5 with 1 N NaOH. tert-Butyleicosandioyl-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 between 10 and 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 mixture of trifluoroacetic acid (60 mL) and dichloromethane (60 mL) and stirred at room temperature for 30 min. The mixture was concentrated to approximately 30 mL and poured into ice-cold n-heptane (300 mL). The precipitated product was separated by filtration and washed twice with n-heptane. After drying under 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) and reverse phase chromatography (acetonitrile, water, TFA). The purified fractions were combined, the pH was adjusted to 5.2 with 1N HCl, and the precipitate was isolated and lyophilized to obtain reference compound 5.

[0201] LC-MS (electrospray): m / z = 1063.6852 [M+6H] 6+ 2. Preparation of the intermediate tert-butyleicosandioyl-γGlu-(2xOEG-OSu)-OtBu was carried out following steps similar to those in Example 1, Part 3.

[0202] 2.1 tert-Butyl eicosanoidioyl-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, triethylamine (13.95 mL) was added, and the resulting cloudy mixture was stirred at room temperature. DCC (11.39 g, 55.19 mmol) was added and stirred overnight. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 24.12 g (97% yield) of tert-butyl eicosanedioyl-OSu.

[0203] LC-MS(Scie×100API): m / z=496.36(M+1) + 2.2 tert-Butyl eicosanoidioyl-γGlu-OtBu tert-Butyleicosandioyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred. H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water were added in this order. 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, and the mixture was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 27.27 g (96% yield) of tert-butyleicosandioyl-γGlu-OtBu was obtained.

[0204] LC-MS(Scie×100API): m / z=584.44(M+1) + 2.3 tert-Butyl eicosanoidioyl-γGlu-(OSu)-OtBu Under nitrogen gas protection, tert-butyleicosandioyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added, and the mixture was stirred for 10 min. NHS (5.38 g, 50.17 mmol) and DCC (10.60 g, 51.38 mmol) were added. The mixture was stirred overnight at room temperature. The filtrate was filtered and concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and then separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure, methyl tert-butyl ether was added, stirred for 30 minutes, suction filtered, and the filter cake was dried overnight in vacuo to obtain 25.76 g (yield 81%) of tert-butyleicosandioyl-γGlu-(OSu)-OtBu.

[0205] LC-MS(Scie×100API): m / z=681.46(M+1) + 2.4 tert-Butyleicosanedioyl-γGlu-(2xOEG-OH)-OtBu tert-Butyleicosandioyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred. 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added sequentially and heated to obtain a clear solution. The solution was stirred at room temperature for 4 h. 10% aqueous citric acid (200 mL) was then added, and the lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 30.75 g (93% yield) of tert-butyleicosandioyl-γGlu-(2xOEG-OH)-OtBu was obtained.

[0206] LC-MS(Scie×100API): m / z=874.59(M+1) + 2.5 tert-Butyleicosanedioyl-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyleicosandioyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added, and the mixture was stirred for 10 min. NHS (4.05 g, 35.18 mmol) and DCC (7.98 g, 38.70 mmol) were 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 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 31.09 g (91% yield) of tert-butyleicosandioyl-γGlu-(2xOEG-OSu)-OtBu.

[0207] LC-MS(Scie×100API): m / z=971.61(M+1) + Example 15 A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin (compound 13)

[0208] [ka]

[0209] Compounds A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin were prepared following a procedure similar to that of Control Example 5, Part 1.

[0210] LC-MS (electrospray): m / z = 1160.3997 [M+6H] 6+ The preparation of the intermediate tert-butyleicosandioyl-γGlu-(6xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2.

[0211] LC-MS(Scie×100API): m / z=1551.90(M+1) + Example 16 A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin (compound 14)

[0212] [ka]

[0213] Compounds A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin were prepared following a procedure similar to that of Control Example 5, Part 1.

[0214] LC-MS (electrospray): m / z = 1165.0674 [M+6H] 6+ The preparation of the intermediate tert-butyldocosandioyl-γGlu-(6xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2.

[0215] LC-MS(Scie×100API): m / z=1579.94(M+1) + Example 17 With reference to the experimental procedures similar to those in Example 7, a pharmacodynamic study was conducted in streptozotocin (STZ)-induced type 1 diabetic (T1DM) rats.

[0216] Before the start of the experiment on the day, basal blood glucose was evaluated at time -1 hour (9:30 AM) and rats were weighed. According to random blood glucose and body weight, rats were assigned to a vehicle group or a treatment group, and treated by subcutaneous injection of the vehicle or the title compounds of Control Example 5, Examples 15 and 16 (Control Compound 5, Compound 13 and Compound 14) at a dose of 33.5 U / kg. The vehicle contained phenol 5.65 mg / mL, glycerol 15 mg / mL, disodium hydrogen phosphate 0.708 mg / mL, and sodium chloride 0.585 mg / mL, and the pH of the vehicle was 7.6.

[0217] The acylated insulin was dissolved in the solvent to a concentration of 33.5 U / mL, with a dose volume of 1 mL / kg (i.e., 0.1 mL / 100 g body weight). A single subcutaneous (sc) dose was administered subcutaneously in the dorsal neck. The acylated insulin was administered between approximately 9:30 and 10:00 AM (time 0), and blood glucose levels were monitored at 3, 6, 9, 24, 48, 72, 96, and 120 h after administration.

[0218] A blood glucose-time dose-response curve was constructed for each single dose of acylated insulin (control compound 5, compound 14, and compound 13). To explain the effect of the acylated insulin on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each single dose-response curve. A smaller AUC value indicates a better blood glucose-lowering effect and better drug efficacy.

[0219] Figures 8a and 8b show that the acylated insulins of the present invention have unexpectedly increased efficacy. For example, compared with the compound of Control Example 5, the title compounds of Examples 15 and 16, Compounds 13 and 14, both have superior hypoglycemic effects in STZ-induced type 1 diabetic (T1DM) rats, i.e., superior efficacy.

[0220] Example 18 A pharmacodynamic study was conducted in streptozotocin (STZ)-induced type 1 diabetic (T1DM) female rats using experimental procedures similar to those in Example 17, except that the acylated insulins used were the title compounds of Examples 2 and 4 (Compound 2 and Compound 4), and the administered dose was 67 U / kg.

[0221] The experimental results, as shown in Figures 9a and 9b, indicate that the acylated insulin compounds 2 and 4 of the present invention have excellent hypoglycemic effects even in female rats with type 1 diabetes mellitus (T1DM), i.e., they have excellent pharmacological efficacy.

[0222] Example 19 Pharmacodynamic studies in db / db mice With reference to the experimental procedures similar to those in Example 6, the control compound 5 and the title compounds of Examples 15 and 16 (i.e., control compound 5, compound 13, and compound 14) were tested in a single-dose study in an obese diabetic mouse model (db / db mice). The hypoglycemic effects of the above acylated insulins were tested at a dose of 9 U / kg.

[0223] Eight- to nine-week-old male db / db (BKS / Lepr) mice were housed in appropriately designed breeding boxes in a barrier environment, with free access to standard chow and purified water. 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, the mice were used in the experiments.

[0224] Before the start of the experiment, basal blood glucose was assessed at time -1 / 1 h (9:30 AM) and mice were weighed. Mice were randomly assigned to vehicle or treatment groups according to blood glucose and body weight, and treated with either vehicle or 9 U / kg acylated insulin subcutaneously. The vehicle used contained phenol 5.65 mg / mL, glycerol 15 mg / mL, disodium hydrogen phosphate 0.708 mg / mL, and sodium chloride 0.585 mg / mL, with a pH of 7.6.

[0225] The acylated insulin was dissolved in a solvent to a dose concentration of 1.8 U / mL, and the dose volume was 5 mL / kg (i.e., 50 μL / 10 g body weight). A single subcutaneous injection was administered in the dorsal neck. Acylated insulin was administered at approximately 10:30 AM (time 0). Animals were fasted but allowed access to water during the dosing period, and blood glucose levels were assessed in mice 3, 6, 9, and 21.5 h after dosing. To simulate feeding, an oral glucose tolerance test (OGTT) was initiated after blood glucose measurement at 21.5 h. Blood glucose was measured 30, 60, 120, and 360 min after intragastric administration of a 100 mg / mL glucose solution (7.5 mL / kg). After the 360-min blood glucose measurement in the first OGTT, a second OGTT experiment was initiated. Blood glucose was measured 30, 90, 210, and 360 min after intragastric administration of a 50 mg / mL glucose solution (10 mL / kg). After the 360-min blood glucose measurement in the second OGTT, a third OGTT experiment was initiated. Blood glucose was measured 30, 60, and 120 min after intragastric administration of a 50 mg / mL glucose solution (10 mL / kg). In the final OGTT, the efficacy of the test compound had not yet disappeared, and the experiment was terminated after 36 h of blood glucose assessment.

[0226] The rats' tails were cleaned with an alcohol swab, and a drop of blood was collected from the tail using a disposable blood collection needle. Blood glucose levels were measured using a blood glucose meter and attached test strips (Roche). A blood glucose-time dose-response curve was constructed for each single dose of acylated insulin. To describe the effect of acylated insulin on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each single dose-response curve. A smaller AUC value indicates a better blood glucose-lowering effect and better drug efficacy.

[0227] 10a-10b show that, compared with the control compound 5, the acylated insulin compounds 14 and 13 of the present invention have significantly improved hypoglycemic effects in type 2 diabetic db / db mice.

[0228] Example 20 Pharmacodynamic study in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) rats Eight-week-old SD rats (half female and half male) weighing 180–220 g were housed in appropriate breeding boxes (5 rats / box) in a barrier environment. They were provided with standard chow and purified water ad libitum. The environmental conditions were controlled at 40–60% relative humidity and 22–24°C. After a 4-day acclimation period and a 12-h fast, the rats were intraperitoneally injected with 60 mg / kg streptozotocin (Sigma) solution (10 mg / mL in 0.1 M citrate buffer). After 3 days of streptozotocin administration, random blood glucose monitoring was performed, and rats with blood glucose levels of 20 mmol / L or higher were selected as T1DM model rats for subsequent studies.

[0229] The test was started 14 days after modeling. Before the start of the experiment, basal blood glucose was evaluated at time -1 / 1 hour (9:30 AM) and rats' weights were measured. Rats were randomly assigned to vehicle or treatment groups according to blood glucose and weight, and treated by subcutaneous injection of vehicle or the title compounds of Control Example 5 and Example 16 (i.e., Control Compound 5 and Compound 14), respectively, at a dose of 25 U / kg. The vehicle contained phenol 5.65 mg / mL, glycerol 15 mg / mL, disodium hydrogen phosphate 0.708 mg / mL, and sodium chloride 0.585 mg / mL, and the pH of the vehicle was 7.6.

[0230] The acylated insulin was dissolved in a solvent to a concentration of 25 U / mL, with a dose volume of 1 mL / kg (i.e., 0.1 mL / 100 g body weight). SD rats were administered subcutaneously in the dorsal neck, four times every four days. The rats were allowed to eat ad libitum throughout the study. Acylated insulin was administered between approximately 9:30 and 10:00 AM (time 0). Blood glucose levels were monitored 3, 6, 9, 24, 48, 72, and 96 h after the first dose, followed by 6 h and once every 24 h after each dose.

[0231] A blood glucose-time dose-response curve was constructed for each single dose of acylated insulin. To describe the effect of acylated insulin on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each single dose-response curve.

[0232] As shown in Figures 11a-11b, compared with the control compound 5, the acylated insulin of the present invention has an unexpectedly increased hypoglycemic effect in type 1 diabetic (T1DM) rats after administration, and the hypoglycemic effect of compound 14 is significantly superior to that of the control compound 5.

[0233] Example 21 Pharmacodynamic study in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice This study aims to confirm the blood glucose (BG) regulating effect of a composition containing the acylated insulin of the present invention and insulin aspart in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice.

[0234] Male C57 / 6J mice (purchased from Weiting Lihua) aged 4-6 weeks were housed in a barrier environment in a suitable breeding box, with free access to standard chow and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22-24°C. After an acclimation period of 1-2 weeks, the mice were used in the experiments.

[0235] After the acclimation period, the animals were fasted for 12 hours and then intraperitoneally injected with 150 mg / kg of streptozotocin (Sigma) solution (10 mg / mL in 0.1 M citrate buffer). After 3 days of streptozotocin administration, random blood glucose detection was performed, and mice with blood glucose levels above 20 mmol / L were selected as T1DM model mice for subsequent testing.

[0236] Before the start of the experiment on the day, random blood glucose levels of the mice were detected and the weights of the mice were measured. According to random blood glucose and body weight, the mice were assigned to a vehicle group or a treatment group, with 8 mice per group, for a total of 5 groups. Each group was subcutaneously injected with the vehicle, or with insulin aspart (0.36 U / kg), or with a pharmaceutical composition containing 0.84 U / kg of insulin degludec and 0.36 U / kg of insulin aspart, or with two pharmaceutical compositions containing Compound 4, the title compound of Example 4 of the present invention, and insulin aspart, respectively. When the two pharmaceutical compositions were injected, the injection doses of Compound 4 were 0.82 U / kg and 0.64 U / kg, respectively, and the injection doses of insulin aspart were both 0.36 U / kg. The solvent contained 19.6 mg / mL of glycerol, 1.5 mg / mL of phenol, and 1.72 mg / mL of m-cresol. The zinc ion concentration was 55 μg / mL, and the pH value of the solvent was 7.6.

[0237] A premix of Compound 4 and insulin aspart was dissolved in a solvent to a dose concentration of 0.072 U / mL (based on the insulin aspart concentration in the premix), with a dose volume of 5 mL / kg (i.e., 50 μL / 10 g body weight). A single subcutaneous injection was administered subcutaneously (SC) into the dorsal neck. Drug administration occurred at approximately 4:00 PM (time 0). Animals were fasted but allowed access to water during the administration period. Blood glucose was assessed in mice 0.5, 1, 2, 3, 6, and 15 hours after administration.

[0238] The rat's tail was cleaned with an alcohol swab, and a drop of blood was collected from the tail using a disposable blood collection needle. Blood glucose levels were measured using a blood glucose meter and attached test strips (Roche). A blood glucose-time dose-response curve was also constructed. To explain the effect of the premixed insulin of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve. A smaller AUC value indicates a better blood glucose-lowering effect and better efficacy.

[0239] Figures 12a and 12b show that after administration of a pharmaceutical composition comprising the acylated insulin of the present invention and insulin aspart, it has an unexpectedly increased hypoglycemic effect in type 1 diabetes mellitus (T1DM) mice compared to a pharmaceutical composition comprising insulin degludec and insulin aspart, and can achieve an equivalent or greater hypoglycemic effect even when the dose ratio of compound 4 to insulin aspart is smaller than the dose ratio of insulin degludec to insulin aspart.

[0240] Example 22 Following procedures similar to those in Example 21, the blood glucose (BG) regulating effect of a composition containing an acylated insulin of the present invention and insulin aspart in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice was tested.

[0241] Before the start of the experiment on the day, random blood glucose levels of the mice were detected and the weights of the mice were measured. According to random blood glucose and body weight, mice were assigned to a vehicle group or a treatment group, with 8 mice per group, for a total of 7 groups. Each group received subcutaneous injections of vehicle, insulin aspart (3 U / kg), a pharmaceutical composition containing insulin degludec 7 U / kg and insulin aspart 3 U / kg, or four pharmaceutical compositions containing compound 4, the title compound of Example 4 of the present invention, and insulin aspart. When the four pharmaceutical compositions were injected, the injection doses of compound 4 were 6.79 U / kg, 5.34 U / kg, 3.84 U / kg, and 2.39 U / kg, respectively, and the injection dose of insulin aspart was 3 U / kg. The solvent contained 19.6 mg / mL of glycerol, 1.5 mg / mL of phenol, and 1.72 mg / mL of m-cresol. The zinc ion concentration was 55 μg / mL, and the pH value of the solvent was 7.6.

[0242] A premix of compound 4 and insulin aspart was dissolved in a solvent to a dose concentration of 0.6 U / mL (based on the insulin aspart concentration in the premix), and the dose volume was 5 mL / kg (i.e., 50 μL / 10 g body weight). Subcutaneous administration (SC) was performed in the dorsal neck. Drugs were administered daily at approximately 5:00 PM (time 0) for 10 consecutive days. Mice were allowed to eat ad libitum during the administration period. Random blood glucose levels were assessed before the fourth, eighth, and tenth doses (time 0) and 1 hour after the fourth, eighth, and tenth doses. Random blood glucose levels were also assessed before the eighth dose (time 0) and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 16, and 24 hours after the dose. After the final dose, mice were fasted for 1 hour, and blood was collected via the orbit to determine the percentage of glycated hemoglobin (Hb1Ac) in whole blood.

[0243] The rats' tails were cleaned with an alcohol swab, and a drop of blood was collected from the tail using a disposable blood collection needle. Blood glucose levels were measured using a blood glucose meter and attached test strips (Roche). A blood glucose-time dose-response curve was also prepared after the eighth administration. To explain the effect of the premixed insulin of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve after the eighth administration. A smaller AUC value indicates a better blood glucose-lowering effect and better efficacy.

[0244] Figures 13a to 16 show that after administration of a composition containing an acylated insulin of the present invention and insulin aspart, it has an unexpectedly increased hypoglycemic effect and a superior cumulative hypoglycemic effect in type 1 diabetic (T1DM) mice compared to a pharmaceutical composition containing insulin degludec and insulin aspart.

[0245] Specifically, Figures 13a and 13b show that after administration of a composition comprising the acylated insulin of the present invention and insulin aspart, it has an unexpectedly increased hypoglycemic effect in type 1 diabetic (T1DM) mice compared to a pharmaceutical composition comprising insulin degludec and insulin aspart, and can achieve a better hypoglycemic effect even when the dose ratio of compound 4 to insulin aspart is much smaller than the dose ratio of insulin degludec to insulin aspart.

[0246] Figures 14a to 14c show the blood glucose status of mice in each administration group before the fourth, eighth, and tenth administrations (0 h), respectively, and reveal that the pharmaceutical composition containing the acylated insulin of the present invention and insulin aspart has superior efficacy and a superior cumulative blood glucose lowering effect compared to the pharmaceutical composition containing insulin degludec and insulin aspart.

[0247] Figures 15a to 15c show the blood glucose status of the mice 1 hour after the fourth, eighth, and tenth administrations, respectively, and reveal that the pharmaceutical composition containing the acylated insulin of the present invention and insulin aspart has superior efficacy and a superior cumulative blood glucose lowering effect compared to the pharmaceutical composition containing insulin degludec and insulin aspart.

[0248] Figure 16 shows that after administration, the composition comprising the acylated insulin of the present invention and insulin aspart has a better Hb1Ac lowering effect than the pharmaceutical composition comprising insulin degludec and insulin aspart, and can still achieve a better Hb1Ac lowering effect even when the dose ratio of compound 4 to insulin aspart is much smaller than the dose ratio of insulin degludec to insulin aspart.

[0249] Example 23 This experiment was designed to determine the chemical stability of the acylated insulin formulations of this invention.

[0250] Acylated insulin preparations Compound 4, the title compound of Example 4, was dissolved in 0.1% NaOH solution to a final concentration of 4.8 mM (pH value of approximately 10-11), and phenol, m-cresol, zinc acetate, glycerol, and sodium chloride were added in that order according to the amounts of each component in the table below to produce an acylated insulin preparation with a final insulin concentration of 1.2 mM (200 U / mL or 8.46 mg / mL), where the Zn content is expressed as Zn / 6 moles of acylated insulin (abbreviated as "Zn / 6ins").

[0251] In this example, the chemical stability of the formulation may be shown by the change in high molecular weight protein (HMWP) relative to day 0 after 14 and 20 days of storage at 25°C and 37°C, and also by the change in the amount of related substances after 14 and 20 days of storage at 25°C and 37°C.

[0252] Measurement of high molecular weight proteins (HMWPs) By high performance liquid chromatography (HPLC) High molecular weight proteins (HMWPs) The content was measured using a Waters Xbride BEH 200A (7.8 x 300 mm), 5 μm column with a column temperature of 30°C and a sample cell temperature of 5°C, using a mobile phase containing 600 mL of 0.1% arginine solution, 150 mL of glacial acetic acid, and 250 mL of acetonitrile at a flow rate of 0.5 mL / min. The detection wavelength was 276 nm, and the sample injection volume was 10 μL. Table 2 shows the increase in HMWP concentration on days 14 and 20 compared to day 0 at 25°C and 37°C.

[0253] [Table 2]

[0254] The table above shows that the amount of HMWP in the acylated insulin preparations of this invention increases very slowly over time, demonstrating the excellent chemical stability of all the acylated insulin preparations. In particular, the increase in HMWP content is slower in the 6.5Zn / 6ins preparation than in the 5.5Zn / 6ins preparation.

[0255] Measurement of the amount of related substances The content of insulin-related substances was measured by high-performance liquid chromatography (HPLC) using a Waters Kromasil 300A-5μm-C8 (4.6*250mm) column at a flow rate of 1.0mL / min, with the column temperature at 40℃ and the sample cell temperature at room temperature. Elution was performed with the following mobile phase:

[0256] Phase A contained 0.1 M anhydrous sodium sulfate, 0.1 M sodium dihydrogen phosphate dihydrate, 10% acetonitrile (v / v), and the pH value was adjusted to 5.0 with NaOH.

[0257] Phase B was 50% acetonitrile (v / v). Gradient: Linear change from 45% / 55% A / B to 35% / 65% A / B from 0 to 45 min, linear change to 20% / 80% A / B from 45 to 50 min, isocratic gradient to 20% / 80% A / B from 50 to 60 min, linear change to 45% / 55% A / B from 60 to 60.1 min, isocratic gradient to 45% / 55% A / B from 60.1 to 70 min.

[0258] Table 3 shows the increase in the amount of related substances on days 14 and 20 relative to day 0 at 37°C.

[0259] [Table 3]

[0260] From the above table, it can be seen that the amount of insulin-related substances in the acylated insulin preparations of the present invention also increases very slowly over time, demonstrating that the acylated insulin preparations are very stable.

[0261] Example 24 The purpose of this experiment was to determine the chemical stability of the acylated insulin formulations of the present invention. The acylated insulin formulations in Tables 4 to 6 were prepared using the amounts of each component listed in Tables 4 to 6, following a process similar to that of Example 23. The changes in HMWP and related substances were also measured following a process similar to that of Example 23. Tables 4 to 6 below show the changes in HMWP and related substances in acylated insulin formulations using different formulation methods.

[0262] [Table 4]

[0263] [Table 5]

[0264] [Table 6]

[0265] The above table shows that the amounts of HMWP and related substances in the acylated insulin preparations of this invention both increased slowly over time, and that the increases in HMWP and related substances were particularly slow as the Zn ion content increased or NaHPO was added, demonstrating the excellent chemical stability of the acylated insulin preparations of this invention.

[0266] Example 25 The purpose of this experiment was to determine the chemical stability of the acylated insulin formulations of the present invention. The acylated insulin formulations in Table 8 were prepared using the amounts of each component in Table 7, following a process similar to that in Example 23. The changes in HMWP and related substances were also measured following a process similar to that in Example 26. The table below shows the changes in HMWP and related substances for acylated insulin formulations using different formulation methods.

[0267] [Table 7]

[0268] The above table shows that the amount of HMWP and related substances in the acylated insulin preparations of this invention both increase slowly over time, demonstrating the excellent chemical stability of all acylated insulin preparations of this invention.

[0269] Example 26 Pharmacodynamic study in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) rats Eight-week-old SD rats (half female and half male) weighing 170–250 g were housed in appropriate breeding boxes (4 rats / box) in a barrier environment. They were provided with standard chow and purified water ad libitum. The environmental conditions were controlled at 40–70% relative humidity and 22–26°C. After a 4-day acclimation period and a 12-h fast, the rats were intraperitoneally injected with 60 mg / kg streptozotocin (Sigma) solution (10 mg / mL in 0.1 M citrate buffer). After 4 and 8 days of streptozotocin administration, random blood glucose monitoring was performed. Rats with blood glucose levels of 20 mmol / L or higher were selected as T1DM model rats for subsequent studies.

[0270] The study began 8 days after modeling. One day before administration, basal blood glucose levels were monitored and rat weights were measured. Rats were randomly assigned to a vehicle group or treatment group according to blood glucose levels and body weights, and treated with subcutaneous injections of either the vehicle or insulin degludec (50 U / kg) or Compound 4 (25 U / kg or 40 U / kg), respectively. The vehicle contained 60 mM phenol, 15 mg / mL glycerol, 10 mM m-cresol, and 0.585 mg / mL sodium chloride, with a pH of 7.4.

[0271] The acylated insulin was dissolved in a solvent to a concentration of 25 U / mL or 40 U / mL, with a dose volume of 1 mL / kg (i.e., 0.1 mL / 100 g body weight). SD rats were administered subcutaneously in the dorsal neck, once every other day, for a total of 11 doses. The rats were allowed to eat ad libitum throughout the study. Acylated insulin was administered between approximately 9:30 and 10:30 AM. Blood glucose levels were monitored 3, 4, 5, 6, 24, and 48 h after the first dose, followed by one dose at 4, 24, and 48 h after each dose.

[0272] A blood glucose-time dose-response curve was constructed for each single dose of acylated insulin. To describe the effect of acylated insulin on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each single dose-response curve.

[0273] As shown in Figures 17a-17b, compared with insulin degludec, the acylated insulin of the present invention has an unexpectedly increased hypoglycemic effect in type 1 diabetic (T1DM) rats after administration, and the hypoglycemic effect of compound 4 is significantly superior to that of insulin degludec.

[0274] Example 27 Following procedures similar to those in Example 21, the blood glucose (BG) regulating effect of a composition containing an acylated insulin of the present invention and insulin aspart in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) C57 / 6J mice was tested.

[0275] Before the start of the experiment on the day, random blood glucose levels of the mice were detected and the weights of the mice were measured. Mice were randomly assigned to vehicle or treatment groups according to blood glucose and body weight, with 8 groups in total, each consisting of 9 mice (5 males and 4 females). Each group was subcutaneously injected with either the vehicle, a pharmaceutical composition containing 7 U / kg of insulin degludec and 3 U / kg of insulin aspart, or one of six pharmaceutical compositions containing Compound 4, the title compound of Example 4 of the present invention, and insulin aspart. When the six pharmaceutical compositions were injected, the injection doses of Compound 4 were 1.49 U / kg, 1.99 U / kg, 2.45 U / kg, 2.85 U / kg, 3.43 U / kg, and 3.92 U / kg, respectively, and the injection dose of insulin aspart was 3 U / kg. The solvent contained 60 mM phenol, 10 mM m-cresol, 15 mg / mL glycerol, and 15 mM NaHPO, and the pH of the solvent was 7.6.

[0276] A premix of compound 4 and insulin aspart was dissolved in a solvent to a dose concentration of 0.6 U / mL (based on the insulin aspart concentration in the premix), and the dose volume was 5 mL / kg (i.e., 50 μL / 10 g body weight). Subcutaneous administration (SC) was performed in the dorsal neck. Drugs were administered daily at approximately 4:00 PM (time 0) for 15 consecutive days. Mice were allowed to eat ad libitum during the administration period. Random blood glucose measurements were performed on the mice before (0 h) and 1 hour after the first, second, fifth, eighth, and 15th doses, and at 0.5, 1, 2, 4, 6, 16, 20, and 24 hours after the second, fifth, eighth, and 15th doses. After the final dose, mice were fasted for 2 h, and blood was collected via the orbit to determine the percentage of glycated hemoglobin (Hb1Ac) in whole blood.

[0277] The rats' tails were cleaned with an alcohol swab, and a drop of blood was collected from the tail using a disposable blood collection needle. Blood glucose levels were measured using a blood glucose meter and attached test strips (Roche). A blood glucose-time dose-response curve was also prepared after the 15th administration. To explain the effect of the premixed insulin of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve after the 15th administration. A smaller AUC value indicates a better blood glucose-lowering effect and better efficacy.

[0278] Figures 18a and 18b show that after administration of a composition comprising the acylated insulin of the present invention and insulin aspart, it has an unexpectedly increased hypoglycemic effect in type 1 diabetic (T1DM) mice compared to a pharmaceutical composition comprising insulin degludec and insulin aspart, and can achieve a better hypoglycemic effect even when the dose ratio of compound 4 to insulin aspart is much smaller than the dose ratio of insulin degludec to insulin aspart.

[0279] Figure 19 shows that the composition comprising the acylated insulin of the present invention and insulin aspart has a better Hb1Ac lowering effect after administration than the pharmaceutical composition comprising insulin degludec and insulin aspart, and can achieve a better Hb1Ac lowering effect even when the dose ratio of compound 4 to insulin aspart is much smaller than the dose ratio of insulin degludec to insulin aspart.

[0280] Example 28 Pharmacodynamic studies in db / db mice This study aims to confirm the regulatory effect of a combination comprising an acylated insulin of the present invention and insulin aspart on blood glucose (BG) in an obese diabetic mouse model (db / db mice) in the case of diabetes.

[0281] Eight- to nine-week-old male db / db (BKS / Lepr) mice were housed in appropriately designed breeding boxes in a barrier environment, with free access to standard chow and purified water. 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, the mice were used in the experiments.

[0282] Before the start of the experiment on the day, the random blood glucose of mice was detected, and the weight of mice was measured.According to the random blood glucose and weight, mice were assigned to vehicle group or treatment group, total 5 groups, each group has 8 mice, each group is treated as follows: vehicle is subcutaneously injected, or a pharmaceutical composition containing insulin degludec 7U / kg and insulin aspart 3U / kg is subcutaneously injected, or three pharmaceutical compositions containing compound 4, the title compound of Example 4 of the present invention, and insulin aspart are subcutaneously injected, where when the above three pharmaceutical compositions are injected, the injection dose of compound 4 is 2.0U / kg, 2.4U / kg, 3.84U / kg, respectively, and the injection dose of insulin aspart is 3U / kg, where the solvent contains phenol 60mM, m-cresol 10mM, glycerol 15mg / mL, Na2HPO4 15mM, and pH value is 7.6.

[0283] The acylated insulin was dissolved in a solvent to a dose concentration of 0.6 U / mL, and the dose volume was 5 mL / kg (i.e., 50 μL / 10 g body weight). Four subcutaneous injections were administered in the dorsal neck. Acylated insulin was administered at approximately 9:30 AM (time 0). Animals were fasted but allowed access to water during the dosing period, and blood glucose levels were assessed in mice at 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours after dosing.

[0284] The rats' tails were cleaned with an alcohol swab, and a drop of blood was collected from the tail with a disposable blood collection needle and measured using a blood glucose meter and attached test strips (Roche). A blood glucose-time dose-response curve was constructed for each single dose of acylated insulin.

[0285] To describe the effect of the acylated insulins of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve. A smaller AUC value indicates a better blood glucose lowering effect and better efficacy.

[0286] Figures 20a and 20b show that after administration of a composition comprising an acylated insulin of the present invention and insulin aspart, it has an unexpectedly increased blood glucose lowering effect in an obese diabetic mouse model (db / db mice) compared to a pharmaceutical composition comprising insulin degludec and insulin aspart, and can achieve a better blood glucose lowering effect even when the dose ratio of compound 4 to insulin aspart is much smaller than the dose ratio of insulin degludec to insulin aspart.

[0287] Example 29 This experiment was designed to determine the chemical stability of the acylated insulin formulations of this invention.

[0288] Acylated insulin preparations Compound 4, the title compound of Example 4, was dissolved in 0.03% NaOH solution to a concentration of 2.4 mM, and the pH was adjusted to 7.4 with 4% NaOH solution. Phenol, m-cresol, glycerol, and sodium chloride were mixed according to the amounts of each component in the table below and added to the solution of Compound 4 to adjust the pH to 7.4. Then, zinc acetate was added to the solution of Compound 4 in an average of three portions according to the amounts in the table below to adjust the pH to the final value. An acylated insulin preparation with a final insulin concentration of 1.2 mM (200 U / mL or 8.46 mg / mL) was prepared, where the Zn content is expressed as Zn / 6 moles of acylated insulin (abbreviated as "Zn / 6ins").

[0289] 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 14 and 21 days of storage at 25°C and 37°C, and by the change in the amount of related substances after 21 days of storage at 37°C.

[0290] Measurement of high molecular weight proteins (HMWPs) The high molecular weight protein (HMWP) content was measured by high-performance liquid chromatography (HPLC) using a Shodex™ PROTEIN KW-802.5 (8.0 mm ID x 300 mmL) column at a flow rate of 0.5 mL / min with a mobile phase containing 3 L of 0.1% arginine solution, 750 mL of glacial acetic acid, and 1250 mL of acetonitrile, at a column temperature of 30°C and a sample cell temperature of 5°C. The detection wavelength was 276 nm, and the sample injection volume was 10 μL. Table 8 shows the increase in HMWP on days 14 and 21 relative to day 0 at 25°C and 37°C.

[0291] [Table 8]

[0292] The above table shows that the amount of HMWP in the acylated insulin preparations of this invention increases very slowly over time within the above pH range, demonstrating that the acylated insulin preparations of this invention have excellent chemical stability across the above pH range.

[0293] 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 Waters Kromasil 100A-3.5μm-C8 (4.6*250mm) column at a flow rate of 1.0mL / min with an elution phase at a column temperature of 40℃ and a sample cell temperature of 10℃. Elution was performed with the following mobile phase:

[0294] Phase A contained 0.1 M anhydrous sodium sulfate, 0.1 M sodium dihydrogen phosphate dihydrate, 10% acetonitrile (v / v), and the pH value was adjusted to 3.0 with concentrated phosphoric acid.

[0295] Phase B was 60% acetonitrile (v / v). Gradient: isocratic gradient of 41.3% / 58.7% A / B from 0 to 40 min, linear change to 0% / 100% A / B from 40 to 50 min, linear change to 41.3% / 58.7% A / B from 50 to 51 min, isocratic gradient to 41.3% / 58.7% A / B from 51 to 65 min. Table 9 shows the increase in related substances on day 21 relative to day 0 at 37°C.

[0296] [Table 9]

[0297] The above table shows that the amount of related substances in the acylated insulin preparations of this invention changes very slowly over time in the above pH range, demonstrating that all of the above acylated insulin preparations of this invention have excellent chemical stability.

[0298] Example 30 The purpose of this experiment was to determine the chemical stability of the acylated insulin formulations of the present invention. The acylated insulin formulations in Tables 10 and 11 were prepared using the amounts of each component in Tables 10 and 11, following a process similar to that of Example 29. The changes in HMWP and related substances were also measured following a process similar to that of Example 29. Tables 10 and 11 below show the changes in HMWP and related substances for acylated insulin formulations using different formulation methods.

[0299] [Table 10]

[0300] [Table 11]

[0301] The above table shows that the amount of HMWP and related substances in the acylated insulin preparations of this invention both increase slowly over time, demonstrating the excellent chemical stability of all acylated insulin preparations of this invention.

[0302] Example 31 The purpose of this experiment was to determine the chemical stability of the combined preparation of acylated insulin and insulin aspart of the present invention.

[0303] For this combined formulation of acylated insulin and insulin aspart, combinations 1 to 5 were prepared according to the amounts of each component listed in Table 12. Compound 4 is designated as acylated insulin with a Zn content of Zn / 6 moles (abbreviated as "Zn / 6ins").

[0304] [Table 12]

[0305] In this example, the chemical stability of the formulation can be demonstrated by the change in high molecular weight protein (HMWP) relative to day 0 after 14 and 28 days of storage at 37°C.

[0306] Measurement of high molecular weight proteins (HMWPs) The high molecular weight protein (HMWP) content was measured by high-performance liquid chromatography (HPLC) using a Tskgel G2000sWXL, 5 μm (7.8 × 300 mm) column at a flow rate of 0.5 mL / min with a mobile phase containing 400 mL of isopropyl alcohol, 300 mL of glacial acetic acid, and 300 mL of water, at a column temperature of 30°C and a sample cell temperature of 10°C. The detection wavelength was 276 nm, and the sample injection volume was 10 μL. Table 13 shows the increase in HMWP on days 14 and 28 relative to day 0 at 37°C.

[0307] [Table 13]

[0308] The above table shows that the amount of HMWP in the combined preparations of the acylated insulin and insulin aspart of the present invention increases very slowly over time, demonstrating that all of the combined preparations have excellent chemical stability.

[0309] Example 32 The purpose of this experiment was to determine the chemical stability of the combined preparation of acylated insulin and insulin aspart of the present invention.

[0310] Combinations 6 to 10 were prepared using the amounts of each component listed in Table 14 below, and the changes in HMWP were measured using a procedure similar to that in Example 31. Table 15 below shows the changes in HMWP of acylated insulin preparations with different formulation methods.

[0311] [Table 14]

[0312] [Table 15]

[0313] The above table shows that the amount of HMWP in the combined preparations of the acylated insulin and insulin aspart of the present invention increases very slowly over time, demonstrating that all of the combined preparations have excellent chemical stability.

[0314] Example 33 The purpose of this experiment was to determine the chemical stability of the combined preparation of acylated insulin and insulin aspart of the present invention.

[0315] Combinations 11 and 12 were prepared using the amounts of each ingredient in Table 16 below, and the change in HMWP was measured following a procedure similar to that in Example 31. Table 17 below shows the change in HMWP of acylated insulin preparations with different formulation methods.

[0316] [Table 16]

[0317] [Table 17]

[0318] The above table shows that the amount of HMWP in the combined preparations of the acylated insulin and insulin aspart of the present invention increases very slowly over time, demonstrating that all of the combined preparations have excellent chemical stability.

[0319] Example 34 Pharmacodynamic studies in db / db mice This study aims to confirm the regulatory effect of a composition comprising an acylated insulin of the present invention and insulin aspart on blood glucose (BG) in an obese diabetic mouse model (db / db mice) in the case of diabetes.

[0320] Experimental db / db mice were obtained using a similar experimental procedure to that of Example 28. Before the start of the experiment on the day, the mice's random blood glucose levels were measured and their weights were measured. According to the random blood glucose levels and weights, the mice were assigned to a vehicle group or a treatment group, with a total of three groups, each with five mice. Each group was treated by subcutaneous injection of the vehicle, or a pharmaceutical composition containing 9.3 U / kg of insulin degludec and 4 U / kg of insulin aspart, or a pharmaceutical composition containing Compound 4, the title compound of Example 4 of the present invention, and insulin aspart, with the proviso that when the pharmaceutical composition was injected, the injection dose of Compound 4 was 3.7 U / kg and the injection dose of insulin aspart was 4 U / kg. The solvent contained 55 mM phenol, 10 mM m-cresol, 8.5 mg / mL glycerol, and 60 mM NaCl, with a pH of 7.6.

[0321] The above acylated insulin and insulin aspart injection solutions were dissolved in solvent to the corresponding administration concentrations, and the administration volume was 5 mL / kg (i.e., 50 μL / 10 g body weight). They were administered subcutaneously (SC) once daily. Animals were allowed free access to food and water during the administration period. Random blood glucose levels were assessed at 0.5, 1, 2, 3, 4, 6, and 8 hours after administration on Day 21 of continuous administration, and fasting blood glucose levels were assessed at 0.5, 1, 2, 3, 4, 6, 8, and 10 hours after administration on Day 18 of continuous administration.

[0322] The rats' tails were cleaned with an alcohol swab, and blood samples were collected from the tails using a disposable blood collection needle and measured using a blood glucose meter and attached test strips (Roche). Blood glucose-time dose-response curves were constructed for each single-dose injection of acylated insulin and insulin aspart.

[0323] To evaluate the effect of the acylated insulin and insulin aspart compositions of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve. A smaller AUC value indicates a better blood glucose-lowering effect and better efficacy.

[0324] Figures 21a to 21d show that after administration of a composition comprising an acylated insulin of the present invention and insulin aspart, it has an unexpectedly increased blood glucose lowering effect in an obese diabetic mouse model (db / db mice) compared to a pharmaceutical composition comprising insulin degludec and insulin aspart, and even when the dose ratio of compound 4 to insulin aspart is much smaller than the dose ratio of insulin degludec to insulin aspart, it can achieve a better blood glucose lowering effect and have a longer duration of blood glucose lowering.

[0325] Example 35 Pharmacokinetics This example is intended to illustrate the in vivo pharmacokinetic properties of the compounds of the present invention.

[0326] Pharmacokinetics in SD rats Twenty-four SD rats were divided into groups of six (half female and half male) into three groups: a low-dose group (Compound 4 low dose group), a medium-dose group (Compound 4 medium dose group), and a high-dose group (Compound 4 high dose group) administered 2, 6, and 18 U / kg of the title compound of Example 4 by subcutaneous injection, and an insulin degludec group administered 14 U / kg of degludec. Blood samples were taken from the low-, medium-, and high-dose groups of Compound 4 and the insulin degludec group before administration (0 min), and 0.5, 1.5, 4, 6, 8, 24, 48, and 72 hours after administration to measure blood drug concentrations. Pharmacokinetic parameters C were calculated using a non-compartmental model in WinNonLin v8.0 software. max , T max , T 1 / 2 , AUC 0~t , Vd, Cl, and MRT were calculated, and the test results are summarized in Table 18.

[0327] [Table 18]

[0328] C max = peak concentration, T max = time to peak, T 1 / 2 = terminal elimination half-life, AUC0~t =Area under the plasma concentration-time curve from 0 to t time, AUC INF = area under the plasma concentration-time curve from the time of administration to infinity, Vd = apparent volume of distribution, Cl = clearance, MRT = mean residence time Pharmacokinetics in Beagles Thirty-six dogs, six per group (half male and half female), were divided into low, medium, and high dose groups of Compound 4 administered subcutaneously at 0.3, 0.6, and 1.2 U / kg, respectively; an intravenous Compound 4 group administered intravenously at 0.6 U / kg; and an insulin degludec group administered subcutaneously at 0.6 U / kg and intravenously at 0.6 U / kg. In the compound 4 intravenous group and insulin degludec intravenous group, blood samples were taken before administration and 2 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 30 h, 36 h, and 48 h after administration to measure blood drug concentrations. In the compound 4 low-dose group, high-dose group, and insulin degludec subcutaneous injection group, blood samples were taken before administration and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 30 h, 36 h, and 48 h after administration to measure blood drug concentrations. In the compound 4 medium-dose group, blood samples were taken before administration and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 30 h, 36 h, 48 h, and 72 h (final dose) after the first and final administration to measure blood drug concentrations. The pharmacokinetic parameter C was calculated using a non-compartmental model in WinNonLin v8.0 software. max , T max , T 1 / 2 , AUC 0~t , MRT was calculated and the test results are summarized in Table 19.

[0329] [Table 19]

[0330] As can be seen from the above experimental results, the acylated insulin derivative compound 4 of the present invention exhibits a long half-life and a more stable blood glucose lowering effect in both rats and beagle dogs.

[0331] Example 36 B29K (N(ε)-docosandioyl-γGlu-OEG), desB30 human insulin (compound 20)

[0332] [ka]

[0333] Compound B29K (N(ε)-docosandioyl-γGlu-OEG), desB30 human insulin, was prepared following a procedure similar to that described in Example 1, part 2.

[0334] LC-MS (electrospray): m / z = 1266.8122 [M+5H] 5+ Intermediates tert-Butyldocosandioyl-γGlu-(OEG-OSu)-OtBu was prepared according to steps similar to those in Example 1, Part 3.

[0335] LC-MS(Scie×100API): m / z=854.57(M+1) + Example 37 B29K(N(ε)-docosandioyl-γGlu-12xPEG), desB30 human insulin (compound 21)

[0336] [ka]

[0337] Compound B29K (N(ε)-docosandioyl-γGlu-12xPEG), desB30 human insulin, was prepared following steps similar to those in Example 1, part 2.

[0338] LC-MS (electrospray): m / z = 1354.8667 [M+5H] 5+ Intermediates tert-Butyldocosandioyl-γGlu-(12xPEG-OSu)-OtBu was prepared according to steps similar to those in Example 1, Part 3.

[0339] LC-MS(Scie×100API): m / z=1294.83 (M+1) + Example 38 Receptor binding ability of the insulin derivatives of the present invention This test aims to demonstrate the ability of the insulin derivatives of the present invention to bind to the insulin receptor.

[0340] Using surface plasmon resonance (SPR) analysis, the binding ability of Compound 2 of the present invention and Control Compound 2 to the his-tagged insulin receptor A extracellular domain (IRA, Sino Biological) was tested in the absence and presence of 2% human serum albumin (HSA), respectively.

[0341] The samples of compound 2 and control compound 2 were diluted with running buffer (Cytiva) or running buffer containing 2.0% HSA so that the injection concentration of both samples was 400 nM. An NTA sensor chip (Cytiva) was selected, and SPR analysis was performed on a Biacore T200 (Cytiva) at 25 °C. 0.5 M NiCl (Cytiva) was injected at a flow rate of 10 μL / min for 60 s, followed by washing with HBS-EP buffer (Cytiva). 3 μg / mL IRA receptor was injected at a flow rate of 5 μL / min for 180 s to allow the IRA receptor to bind to the chip surface. Then, a sample of the insulin derivative to be tested was injected at a flow rate of 30 μL / min for 60 s, followed by a 60 s dissociation period. After each injection, the chip was regenerated by injecting 350 mM EDTA (Cytiva) for 60 seconds at a flow rate of 10 μL / min, and finally washed with HBS-P buffer (Cytiva) to detect the next sample. The response value 4 seconds before sample dissociation was selected as the receptor binding strength test result, and the test was repeated three times for each sample.

[0342] Figure 22 shows the receptor binding ability of Compound 2 and Control Compound 2 in the presence of 2% HSA (simulating physiological conditions) compared to the case of 0% HSA. As can be seen from Figure 22, in the presence of 2% HSA, Compound 2 has significantly improved receptor binding ability compared to Control Compound 2, and the effect of albumin on the receptor binding ability of Compound 2 of the present invention is significantly reduced compared to Control Compound 2.

[0343] This demonstrates that in the presence of albumin, insulin derivatives of the present invention, such as compound 2, have a receptor-binding ability that is significantly improved beyond expectations compared to control compound 2, i.e., the effect of albumin on the receptor-binding ability of the insulin derivatives of the present invention is significantly reduced compared to control compound 2.

[0344] Although the present invention has been described with reference to the above embodiments, it should be understood that the above embodiments are merely for illustrative and explanatory purposes and are not intended to limit the present invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and that many further variations and modifications can be made based on the teachings of the present invention, and that all of these variations and modifications are included within the scope of the claims of the present invention. The scope of the claims of the present invention is limited by the scope of the appended claims and their equivalents.

[0345] array SEQ ID NO.1: DesB30 human insulin A chain: Gly Ile Val Glu Gln Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO.2: DesB30 human insulin B chain: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys SEQ ID NO.3: A14E, B16H, B25H, desB30 human insulin A chain: Gly Ile Val Glu Gln Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO.4: 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.5: A14E, B16E, B25H, desB30 human insulin A chain: Gly Ile Val Glu Gln Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO.6: 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.7: Human insulin A chain: Gly Ile Val Glu Gln Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO.8: Human insulin B chain: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr SEQ ID NO.9: A21G human insulin A chain: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly SEQ ID NO.10: A21G human insulin B chain: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr SEQ ID NO.11: A21G, desB30 human insulin A chain: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly SEQ ID NO.12: A21G, desB30 human insulin B chain: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys SEQ ID NO.13: B28D human insulin A chain: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn SEQ ID NO.14: B28D human insulin B chain: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr SEQ ID NO.15: GLP-1-(7-37)ペプチド 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 SEQ ID NO.16: [Gly8、Arg34]GLP-1-(7-37)ペプチド 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 SEQ ID NO.17: [Arg34]GLP-1-(7-37)ペプチド 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. 1. An insulin derivative comprising a parent insulin, an albumin binding residue, and a linking group Lin, wherein the albumin binding residue is linked to the parent insulin via the linking group Lin; the linking group Lin comprises 5 to 9 neutral, alkylene glycol-containing amino acid residues; Here, the neutral amino acid residue containing alkylene glycol 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 the albumin binding residue is a fatty acid or aliphatic diacid containing 20 to 26 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the fatty acid and the carboxy group of the aliphatic diacid; the insulin parent is selected from the group consisting of: desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; human insulin; A21G human insulin; A21G, desB30 human insulin; and B28D human insulin; The insulin parent contains at least one lysine residue located at position B29, and An insulin derivative, wherein the albumin binding residue is bound via the linking group Lin to the amino group of the lysine residue located at position B29 or to the N-terminal amino acid residue of the insulin parent molecule.

2. 2. The insulin derivative of claim 1, further comprising one or more linking groups II, wherein the linking group II is an acidic amino acid residue selected from the group consisting of γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp, and α-D-Asp, and the linking group II is attached between the albumin binding residue and the linking group Lin and / or between the linking group Lin and the insulin parent.

3. an acylated insulin, the insulin parent of which contains at least one lysine residue at position B29 and is selected from the group consisting of desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; human insulin; A21G human insulin; A21G, desB30 human insulin; and B28D human insulin; The acyl moiety of the acylated insulin is linked to the lysine residue located at position B29 of the parent insulin or to the amino group of the N-terminal amino acid residue, and the acyl moiety has the formula (A), i.e., III-(II) ｍ -(I) ｎ - (A) however, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n is 5, 6, 7, 8, or 9; I is a neutral, alkylene glycol-containing amino acid residue; The neutral alkylene glycol-containing amino acid residue 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[[ID=z35]] ２ ), ４ -O-CH ２ -CO-; and II is an acidic amino acid residue selected from the group consisting of γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp and α-D-Asp; III is a fatty acid or aliphatic diacid containing 20 to 26 carbon atoms, provided that, in form, a hydroxy group has been removed from one of the carboxy groups of the fatty acid and the carboxy group of the aliphatic diacid; III, II, and I are connected by an amide bond, and The order of appearance of II and I in formula (A) is independently interchangeable; The formula (A) is linked via the C-terminus of I to the lysine residue at position B29 of the insulin parent molecule or to the amino group of the N-terminal amino acid residue. The insulin derivative according to claim 1.

4. an acylated insulin, the insulin parent of which contains at least one lysine residue at position B29 and is selected from the group consisting of desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; human insulin; A21G human insulin; A21G, desB30 human insulin; and B28D human insulin, wherein The acyl moiety of the acylated insulin is linked to the lysine residue at position B29 of the parent insulin or to the amino group of the N-terminal amino acid residue, and the acyl moiety has the formula (A), i.e., III-(II) ｍ -(I) ｎ - (A) however, m is 0 or an integer from 1 to 10, and n is 5, 6, 7, 8, or 9; I is a neutral amino acid residue containing alkylene glycol, and the alkylene glycol-containing amino acid residue 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 ' II is an acidic amino acid residue selected from the group consisting of γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp and α-D-Asp; III is an aliphatic diacid containing 20 to 26 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; III, II, and I are connected by an amide bond, and The order of appearance of II and I in formula (A) is independently interchangeable; The formula (A) is linked via the C-terminus of I to the lysine residue at position B29 of the insulin parent molecule or to the amino group of the N-terminal amino acid residue. The insulin derivative according to claim 3.

5. 5. An insulin derivative according to any one of claims 1, 3 and 4, wherein the fatty acid or aliphatic diacid has 20 to 24 carbon atoms.

6. n is 5, 6, 7 or 8; and / or m is 1, 2, 3 or 4; and / or III is an aliphatic diacid having 20, 21 or 22 carbon atoms, provided that, in form, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid; and / or the insulin parent contains one lysine residue; 5. The insulin derivative according to claim 3 or 4.

7. The I is -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-, and / or wherein II is an amino acid residue selected from the group consisting of γGlu and βAsp; and / or The above III is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) １９ -CO-, HOOC-(CH ２ ) ２０ -CO-, HOOC-(CH ２ ) ２１ -CO-, HOOC-(CH ２ ) ２２ -CO-, or HOOC-(CH ２ ) ２４ -CO-, 5. The insulin derivative according to claim 3 or 4.

8. 8. The insulin derivative of any one of claims 3 to 7, wherein the acyl group moiety is linked to the ε-amino group of a lysine residue of the parent insulin.

9. 9. The insulin derivative according to any one of claims 1 to 8, wherein the parent insulin is desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; or A14E, B16E, B25H, desB30 human insulin.

10. The acylated insulin may be: B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-5xOEG-γGlu), desB30 human insulin; B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin; B29K (N(ε)-eicosanedioyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αAsp-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin; B29K (N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin; B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-βAsp-8xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αAsp-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-βAsp-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin; B29K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-trichosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-trichosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-trichosanedioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-trichosanedioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-tetracosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-Eicosanediol-γGlu-9xOEG), desB30 human insulin; B29K (N(ε)-docosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanediol-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanediol-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanediol-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanediol-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanediol-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanediol-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanediol-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-trichosanedioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-trichosanedioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-trichosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-trichosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-hexadecandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-trichosanedioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-trichosanedioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-trichosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-trichosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-12xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-12xPEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-13xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-13xPEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-14xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-14xPEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-15xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-15xPEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-16xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-16xPEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-17xPEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-17xPEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-18xPEG), desB30 human insulin; and B29K (N(ε)-eicosandioyl-γGlu-18xPEG), desB30 human insulin 5. The insulin derivative according to claim 3 or 4, selected from the group consisting of:

11. A pharmaceutical composition comprising an insulin derivative according to any one of claims 1 to 10 and one or more pharmaceutically acceptable excipients.

12. containing at least 1.5 moles of zinc ions per 6 moles of insulin derivative, and / or the pH is 6.5 to 8.5, Glycerol, phenol, m-cresol, NaCl, and / or Na ２ HPO ４ further comprising the glycerol content does not exceed 2.5% (w / w), and / or the phenol content is between 16 and 80 mM, and / or the m-cresol content is 0 to 35 mM, and / or the NaCl content is between 0 and 150 mM; and / or the content of said insulin derivative is greater than 0.3 mM; The pharmaceutical composition of claim 11.

13. The insulin derivatives include B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; The pharmaceutical composition according to claim 11 or 12,

14. 11. A pharmaceutical composition comprising 0.6-4.2 mM of an insulin derivative according to any one of claims 1-10, 1%-1.8% (w / w) glycerol, 45-65 mM phenol, 4.5-6.5 moles of zinc ions / 6 moles of insulin derivative, 10-120 mM sodium chloride, and 0-15 mM m-cresol, and having a pH value of 7.0-8.

2.

15. 11. A pharmaceutical composition comprising 0.6 mM to 1.2 mM of an insulin derivative according to any one of claims 1 to 10, 1.7% (w / w) glycerol, 45 mM phenol, 10 mM m-cresol, 6.5 moles of zinc ions / 6 moles of insulin derivative, and 20 mM sodium chloride, with a pH value of 7.0 to 8.

0.

16. 11. A pharmaceutical composition comprising 0.6-4.2 mM of an insulin derivative according to any one of claims 1-10, 1%-2% (w / w) of glycerol, 15 mM-60 mM of phenol, 1.5-7.0 moles of zinc ions / 6 moles of insulin derivative, 10-120 mM of sodium chloride, and 0-25 mM of m-cresol, and having a pH value of 7.0-8.

2.

17. 11. A pharmaceutical composition comprising 1.2-1.5 mM of an insulin derivative according to any one of claims 1-10, 1.5%-1.7% (w / w) of glycerol, 45-60 mM of phenol, 2.2-4.5 moles of zinc ions / 6 moles of insulin derivative, 20 mM of sodium chloride, and 0-10 mM of m-cresol, and having a pH value of 7.0-8.

0.

18. The insulin derivatives include B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or The pharmaceutical composition according to any one of claims 14 to 17, which is A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin.

19. The pharmaceutical composition according to any one of claims 11 to 18, further comprising a fast-acting insulin.

20. The fast-acting insulin is Asp Ｂ２８ Human insulin, Lys Ｂ２８ Pro Ｂ２９ Human insulin, Lys Ｂ３ Glu Ｂ２９ 20. The pharmaceutical composition of claim 19, selected from one or more of human insulin, human insulin and desB30 human insulin.

21. The pharmaceutical composition according to claim 19 or 20, wherein the molar ratio of said insulin derivative to said fast-acting insulin is 60:3 to 0.5:3, preferably 7:3 to 1.5:3, preferably 6.9:3 to 1.5:3, preferably 6.8:3 to 1.5:3, preferably 6.5:3 to 1.5:3, preferably 7:3 to 2:3, preferably 6.9:3 to 2:3, preferably 6.8:3 to 2:3, preferably 3:3 to 2:3, preferably 3:

3.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the insulin derivative is the insulin derivative according to claim 10.

23. The insulin derivative is B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; 23. The pharmaceutical composition of claim 22.

24. 24. The pharmaceutical composition of claim 23, wherein the insulin derivative is B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin or B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin.

25. 0.09-0.36 mM insulin derivative and 0.18 mM Asp Ｂ２８ 1. A method for preparing an insulin derivative comprising: human insulin, 0.85% to 2.0% (w / w) glycerol, 15 to 70 mM phenol, 8 to 14 moles of zinc ions / 6 moles of insulin derivative, 10 to 120 mM sodium chloride, and 0 to 15 mM m-cresol, and having a pH value of 7.0 to 8.2, wherein the insulin derivative is B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, The pharmaceutical composition according to any one of claims 19 to 22.

26. 0.165-0.18 mM insulin derivative and 0.18 mM Asp Ｂ２８ 1.5%-1.7% (w / w) glycerol, 20 mM-30 mM phenol, 9-12 moles of zinc ions / 6 moles of insulin derivative, 20 mM-75 mM sodium chloride, and 10 mM-15 mM m-cresol, at a pH of 7.0-8.2, said insulin derivative comprising: B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin; or B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; The pharmaceutical composition according to any one of claims 19 to 25.

27. An insulin derivative according to any one of claims 1 to 10 or a pharmaceutical composition according to any one of claims 11 to 26 for use in the treatment or prevention of diabetes, hyperglycemia, and / or impaired glucose tolerance, preferably wherein the diabetes is type 1 diabetes or type 2 diabetes.

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