Pharmaceutical compositions of insulin derivatives

Abstract

To develop stable compositions of insulin derivatives comprising one or more aryl boron moieties, such as aryl boronic acids or aryl boroxols.SOLUTION: The present invention relates to a composition comprising: an insulin derivative comprising at least one aryl boronic acid or aryl boroxol; and methionine, as well as the use hereof in the treatment of diabetes, including diabetes of Type 1 and diabetes of Type 2.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to novel pharmaceutical compositions of insulin derivatives containing at least one aryl boron moiety, and to the use of such compositions for the treatment or prevention of medical conditions associated with diabetes.

[0002] Incorporation by reference of sequence listing Sequence Listing This application is submitted with a Sequence Listing in electronic format, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Recently, insulin derivatives that exhibit glucose-sensitive insulin activity based on the inclusion of one or more arylboronic acid or aryl boroxol moieties have been published (Patent Documents 1, 2, 3, 4, 5).

[0004] Aryl boron compounds such as aryl boronic acids may have low stability in aqueous solutions at near neutral pH. The CB bond can be hydrolyzed to give a phenyl residue and free boronic acid, Ph-H+B(OH)3, or the compound can be oxidized to give a phenol residue plus free boronic acid, Ph-OH+B(OH)3. It is therefore desirable to develop stable compositions of insulin derivatives that contain one or more aryl boron moieties, such as aryl boronic acids or aryl boroxoles. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2020 / 201041 [Patent Document 2] International Publication No. 2019 / 092125 [Patent Document 3] International Publication No. 2022 / 109078 [Patent Document 4] International Publication No. 2021 / 202802 [Patent Document 5] International Publication No. 2023 / 225534 Summary of the Invention

[0006] In its broadest aspect, the present invention relates to a liquid pharmaceutical composition of an insulin derivative, the insulin derivative comprising one or more aryl boron moieties, such as aryl boronic acid or aryl boroxol moieties. Surprisingly, it has been found that the addition of methionine results in a more stable composition. It has also been found that the addition of a sugar alcohol (polyol), such as mannitol, results in a more stable composition. Furthermore, it has been found that the addition of both methionine and mannitol results in a surprisingly stable composition. In a further aspect, the composition comprises methionine and mannitol. In one aspect, the composition is stable. In one aspect, the composition is more stable than when the composition does not contain methionine. In one aspect, the composition is more stable than when the composition does not contain mannitol. In one aspect, the composition is more stable than when the composition does not contain methionine and mannitol.

[0007] The present invention may also solve further problems that become apparent from the disclosure of the exemplary embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In one aspect, the composition according to the invention comprises: a) an insulin derivative containing at least one aryl boron moiety, such as an aryl boronic acid or an aryl boroxole; b) methionine.

[0009] In one aspect, the composition according to the invention comprises: a) an insulin derivative containing at least one aryl boron moiety, such as an aryl boronic acid or an aryl boroxole; b) a sugar alcohol, such as mannitol or sorbitol.

[0010] In one aspect, the composition according to the invention comprises: a) an insulin derivative containing at least one aryl boron moiety, such as an aryl boronic acid or an aryl boroxole; b) methionine, b) a sugar alcohol, such as mannitol or sorbitol.

[0011] In one aspect, the composition according to the invention comprises: a) an insulin derivative containing at least one aryl boron moiety, such as an aryl boronic acid or an aryl boroxole; b) glycerol, or sugar alcohols such as mannitol or sorbitol, either alone or in combination.

[0012] In one aspect, the composition according to the invention comprises: a) an insulin derivative containing at least one arylboronic acid or arylboroxole; b) methionine, b) glycerol, or sugar alcohols such as mannitol or sorbitol, either alone or in combination.

[0013] Insulin derivatives As used herein, the term "insulin derivative" refers to modified insulin peptides, where the modification is in the form of covalent attachment of a chemical moiety, also commonly referred to as a side chain or modifying group. In one aspect, the modification is in the form of covalent attachment of at least one modifying group comprising at least one arylboronic acid or arylboroxole moiety. In one embodiment, each modifying group comprises at least two arylboronic acid or arylboroxole moieties. In one embodiment, each modifying group comprises at least two arylboroxole moieties.

[0014] Examples of such insulin derivatives are disclosed in WO 2020 / 201041, WO 2019 / 092125, WO 2022 / 109078, WO 2021 / 202802, and WO 2023 / 225534, which are incorporated by reference herein.

[0015] In some embodiments, the insulin derivative may be present in the composition in its fully or partially ionized form, e.g., one or more carboxylic acid groups (-COOH) may be deprotonated to a carboxylate group (-COO-) and / or one or more amino groups (-NH2) may be protonated to a -NH3+ group. In some embodiments, the insulin derivative is added to the composition in the form of a salt. In some embodiments, the insulin derivative is added to the composition in the form of a sodium salt.

[0016] Arylboronic Acids and Arylboroxoles The compositions of the invention include insulin derivatives that contain at least one aryl boron moiety, which may be an aryl boronic acid or an aryl boroxole.

[0017] As used herein, the terms "aryl boron moiety," "aryl boronic acid," and "aryl boroxole" include substituted or unsubstituted variants. An example of an aryl boronic acid is phenyl boronic acid (Formula 1). An example of an aryl boroxole is benzoboroxole, also known as benzoxaborole (Formula 2). [ka] Chemical formula 1. Phenylboronic acid [ka] Chemical formula 2. Benzoboroxole

[0018] The aryl boron moiety may be part of a larger chemical moiety, such as formula 4, where R1 is CF3, F, or H, R2 is H or CH3, and R3 is H or CH3. [ka] Chemical formula 4.

[0019] The arylboronic acid- or arylboroxol-containing moiety is covalently attached to the insulin peptide via a chemical handle and, optionally, a linker, which may be a chemical linker or a peptide extension of the insulin peptide, or a combination thereof.

[0020] Insulin Peptides As used herein, the term "insulin peptide" refers to a peptide that is either human insulin or a human insulin analog. In one embodiment, the term "insulin peptide" as used herein refers to a peptide that is either human insulin or an analog thereof that has insulin activity, i.e., activates the insulin receptor.

[0021] Human insulin As used herein, the term "human insulin" refers to the human insulin hormone, whose structure and properties are well known. Human insulin has two polypeptide chains, designated the A chain and the B chain. The A chain is a peptide of 21 amino acids, and the B chain is a peptide of 30 amino acids, and the two chains are connected by disulfide bridges, the first bridge being between the cysteine ​​at position 7 of the A chain and the cysteine ​​at position 7 of the B chain, and the second bridge being between the cysteine ​​at position 20 of the A chain and the cysteine ​​at position 19 of the B chain. The third bridge is between the cysteine ​​at position 6 of the A chain and the cysteine ​​at position 11 of the A chain. The human insulin A chain has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1), while the B chain has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2).

[0022] Insulin analogues As used herein, the term "insulin analog" refers to a modified human insulin in which one or more amino acid residues of insulin have been replaced by other amino acid residues and / or one or more amino acid residues have been deleted from insulin and / or one or more amino acid residues have been added and / or inserted into insulin. As used herein, the term "insulin analog" refers to an insulin analog that exhibits insulin activity, i.e., binds to and activates the insulin receptor.

[0023] Modifications within the insulin molecule are represented by stating the chain (A or B), the position, and the one-letter or three-letter code of the amino acid residue replacing the natural amino acid residue. As used herein, terms such as "A1", "A2", and "A3" refer to amino acids at positions 1, 2, and 3, respectively (counting from the N-terminus) of the A chain of insulin. Similarly, terms such as B1, B2, and B3 refer to amino acids at positions 1, 2, and 3, respectively (counting from the N-terminus) of the B chain of insulin. Using the one-letter code of amino acids, the term B29K specifies that the amino acid at position B29 is K. Using the three-letter code of amino acids, the corresponding expression is B29Lys. "desB30" refers to an insulin analog lacking the B30 amino acid.

[0024] In certain embodiments, an analog "has" or "contains" a particular change. In other certain embodiments, an analog "consists of" a change. When the terms "consisting of" or "consisting of" are used in reference to an analog, for example, where an analog consists of or consists of a particular group of amino acid mutations, it will be understood that the particular amino acid mutations are the only amino acid mutations in the analog. In contrast, an analog that "contains" a particular group of amino acid mutations may have additional mutations.

[0025] Examples of insulin analogues include: desB30 human insulin (A chain of SEQ ID NO:1 and B chain of SEQ ID NO:3).

[0026] antioxidants Antioxidants can be used to stabilize pharmaceutical proteins in liquid formulations from oxidation. A commonly known antioxidant is ascorbic acid (vitamin C). The addition of ascorbic acid to compositions of insulin derivatives containing at least one arylboronic acid or arylboroxole resulted in a significant decrease in the stability of the insulin derivatives in the formulation.

[0027] Methionine L-methionine is a proteinogenic amino acid. Methionine residues in polypeptides or proteins are susceptible to oxidation, and methionine has been used as a stabilizer in formulations of polypeptides or proteins that contain methionine residues. The amount added should be sufficient to inhibit oxidation of the methionine residues to an acceptable level for the amount of methionine sulfoxide. Generally, this can be accomplished by adding methionine to the composition such that the ratio of added methionine to methionine residues ranges from about 1:1 to about 1000:1, such as about 10:1 to about 100:1. Human insulin does not contain any methionine residues, and therefore methionine is not a common excipient in insulin formulations, although methionine has been reported to have antioxidant properties.

[0028] It has surprisingly been found that the addition of methionine to compositions of insulin derivatives comprising at least one arylboronic acid or arylboroxole results in increased stability of the insulin derivative in the formulation.

[0029] As used herein, the term "methionine" includes L-methionine, D-methionine, and the racemic form of methionine.

[0030] Isotonic agent The tonicity agent has the effect of maintaining the appropriate osmotic pressure when the solution of insulin derivative is injected into the body. Commonly used tonicity agents include salts (e.g., sodium chloride), sugars (including monosaccharides, disaccharides or polysaccharides or water-soluble glucans, such as fructose, glucose, mannose, lactose, sucrose, trehalose, dextran), sugar alcohols (e.g., mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, etc.), amino acids (e.g., L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), alditols (e.g., glycerol (glycerin), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol), polyethylene glycols (e.g., PEG400), or mixtures thereof. Tonicity agents are also commonly called isotonicity agents. The use of isotonicity agents in pharmaceutical compositions is well known to those skilled in the art. For convenience, reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0031] Glycerol and sugar alcohols as stabilizers Surprisingly, it has been found that the addition of glycerol, which is commonly used as an isotonicity agent, to compositions of insulin derivatives containing at least one arylboronic acid or arylboroxole leads to an increase in the stability of the insulin derivative in the formulation. Even more surprisingly, it has been found that the addition of the sugar alcohol mannitol in a much smaller amount than when used as an isotonicity agent to compositions of insulin derivatives containing at least one arylboronic acid or arylboroxole leads to an increase in the stability of the insulin derivative in the formulation. A similar effect was found for the sugar alcohol sorbitol. In comparison, the addition of the sugars sucrose and trehalose did not affect the stability of the insulin derivative in the formulation. It is therefore very surprising that mannitol, sorbitol, and / or glycerol act as stabilizers for compositions of insulin derivatives containing at least one arylboronic acid or arylboroxole.

[0032] As used herein, the term "mannitol" includes D-mannitol, L-mannitol and the racemic form of mannitol.

[0033] zinc Zinc is routinely used in therapeutic insulin preparations to promote hexamer formation as a precaution against degradation during storage. 2+ It is driven by interactions between the Zn residue and insulin HisB10 residue. 2+ is usually added in the form of a zinc salt such as zinc chloride or zinc acetate.

[0034] The tested compositions comprising the insulin derivative of Example 1 were more stable in the presence of zinc than in its absence. Surprisingly, the tested compositions comprising the insulin derivative of Example 2 were found to be more stable in the absence of zinc than in its presence.

[0035] Preservatives In a further aspect of the present invention, the formulation further comprises a pharma- ceutically acceptable preservative. The preservative can be selected from preservatives suitable for use in pharmaceutical preparations. In a further embodiment of the present invention, the preservative is selected from phenolic preservatives, such as phenol and m-cresol, or a mixture thereof. In a further embodiment of the present invention, the preservative is selected from the group consisting of phenol and m-cresol, or a mixture thereof. The use of preservatives in pharmaceutical compositions is well known to those skilled in the art. For convenience, reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0036] Additional excipients The composition of the present invention may also include additional excipients. The term "excipient" refers broadly to any ingredient other than the active therapeutic ingredient. An excipient may be a non-active, inactive, and / or non-pharmaceutical active ingredient. An excipient may serve various purposes, for example, as a carrier, vehicle, diluent, and / or function to improve the administration and / or absorption of an active ingredient. Non-limiting examples of excipients are solvents, diluents, buffers, preservatives, tonicity adjusters, chelating agents, and stabilizers. The formulation of pharmaceutically active ingredients with various excipients is known in the art (see, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st Edition (2005) and any subsequent editions)).

[0037] The composition of the present invention is in the form of a liquid formulation, i.e. an aqueous formulation that contains water.The liquid formulation may be a solution or a suspension.The composition of the present invention may be for parenteral administration, for example, by subcutaneous, intramuscular, intraperitoneal or intravenous injection.

[0038] LIST OF EMBODIMENTS The present invention is further illustrated by the following non-limiting embodiments. 1. A liquid pharmaceutical composition comprising: a) an insulin derivative containing at least one aryl boron moiety; b) methionine. 2. A liquid pharmaceutical composition comprising: a) an insulin derivative containing at least one arylboronic acid or arylboroxole; b) methionine. 3. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is within the range of 5 mM to 300 mM. 4. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is within the range of 5 mM to 60 mM. 5. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is within the range of 5 mM to 45 mM. 6. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is within the range of 5 mM to 30 mM. 7. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is within the range of 7 mM to 20 mM. 8. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is 15 mM. 9. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is 16 mM. 10. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is 30 mM. 11. The liquid pharmaceutical composition according to any one of embodiments 1 to 2, wherein the concentration of methionine is 45 mM. 12. The liquid pharmaceutical composition according to any one of embodiments 1 to 11, further comprising one or more sugar alcohols. 13. The liquid pharmaceutical composition according to any one of embodiments 1 to 12, further comprising a sugar alcohol. 14. The liquid pharmaceutical composition according to any one of embodiments 1 to 13, wherein the total concentration of sugar alcohol in the composition is in the range of 1 mM to 300 mM. 15. The liquid pharmaceutical composition according to any one of embodiments 1 to 14, wherein the total concentration of sugar alcohol in the composition is in the range of 2 mM to 300 mM. 16. The liquid pharmaceutical composition according to any one of embodiments 1 to 15, wherein the total concentration of sugar alcohol in the composition is in the range of 5 mM to 300 mM. 17. The liquid pharmaceutical composition according to any one of embodiments 1 to 16, wherein the total concentration of sugar alcohol in the composition is in the range of 10 mM to 300 mM. 18. The liquid pharmaceutical composition according to any one of embodiments 1 to 17, wherein the total concentration of sugar alcohol in the composition is in the range of 20 mM to 40 mM. 19. The liquid pharmaceutical composition according to any one of embodiments 1 to 18, further comprising mannitol. 20. The liquid pharmaceutical composition according to any one of embodiments 1 to 19, wherein the concentration of mannitol in the composition is in the range of 10 mM to 300 mM. 21. The liquid pharmaceutical composition according to any one of embodiments 1 to 20, wherein the concentration of mannitol in the composition is in the range of 20 mM to 40 mM. 22. The liquid pharmaceutical composition according to any one of embodiments 1 to 21, wherein the concentration of mannitol in the composition is in the range of 40 mM to 80 mM. 23. The liquid pharmaceutical composition according to any one of embodiments 1 to 22, wherein the concentration of mannitol in the composition is 20 mM. 24. The liquid pharmaceutical composition according to any one of embodiments 1 to 23, wherein the concentration of mannitol in the composition is 30 mM. 25. The liquid pharmaceutical composition according to any one of embodiments 1 to 24, wherein the concentration of mannitol in the composition is 40 mM. 26. The liquid pharmaceutical composition according to any one of embodiments 1 to 25, wherein the concentration of mannitol in the composition is 80 mM. 27. The liquid pharmaceutical composition according to any one of embodiments 1-26, further comprising a sugar alcohol or glycerol, either alone or in combination. 28. The liquid pharmaceutical composition according to any one of embodiments 1 to 27, further comprising glycerol and a sugar alcohol. 29. The liquid pharmaceutical composition according to any one of embodiments 1-28, further comprising mannitol, sorbitol or glycerol, either alone or in combination. 30. The liquid pharmaceutical composition according to any one of embodiments 1-29, further comprising mannitol and glycerol. 31. The liquid pharmaceutical composition according to any one of embodiments 1 to 30, wherein the concentration of glycerol in the composition is in the range of 10 mM to 600 mM. 32. The liquid pharmaceutical composition according to any one of embodiments 1 to 31, wherein the concentration of glycerol in the composition is in the range of 10 mM to 300 mM. 33. A liquid pharmaceutical composition according to any one of embodiments 1 to 32, wherein the concentration of glycerol in the composition is in the range of 100 mM to 300 mM. 34. The liquid pharmaceutical composition according to any one of embodiments 1 to 33, wherein the concentration of glycerol in the composition is in the range of 100 mM to 230 mM. 35. The liquid pharmaceutical composition according to any one of embodiments 1 to 34, wherein the concentration of glycerol in the composition is within the range of 150 mM to 230 mM. 36. The liquid pharmaceutical composition according to any one of embodiments 1-35, wherein the concentration of glycerol in the composition is in the range of 152 mM to 217 mM. 37. A liquid pharmaceutical composition according to any one of embodiments 1-36, wherein the concentration of glycerol in the composition is in the range of 185 mM to 217 mM. 38. The liquid pharmaceutical composition according to any one of embodiments 1-37, wherein the concentration of glycerol in the composition is 152 mM. 39. The liquid pharmaceutical composition according to any one of embodiments 1 to 38, wherein the concentration of glycerol in the composition is 165 mM. 40. The liquid pharmaceutical composition according to any one of embodiments 1-39, wherein the concentration of glycerol in the composition is 174 mM. 41. The liquid pharmaceutical composition according to any one of embodiments 1 to 40, wherein the concentration of glycerol in the composition is 185 mM. 42. The liquid pharmaceutical composition according to any one of embodiments 1-41, wherein the concentration of glycerol in the composition is 195 mM. 43. The liquid pharmaceutical composition according to any one of embodiments 1-42, wherein the concentration of glycerol in the composition is 217 mM. 44. The liquid pharmaceutical composition according to any one of embodiments 1 to 43, having a pH in the range of 5.0 to 8.0. 45. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the pH is within the range of 6.4 to 7.4. 46. ​​The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the pH is 6.6. 47. A liquid pharmaceutical composition according to any one of embodiments 1 to 46, wherein the concentration of methionine is in the range of 5 mM to 45 mM, the concentration of mannitol in the composition is in the range of 40 mM to 80 mM, and the concentration of glycerol in the composition is in the range of 152 mM to 217 mM. 48. The liquid pharmaceutical composition according to any one of embodiments 1 to 47, further comprising a phenolic preservative or a mixture of phenolic preservatives. 49. The liquid pharmaceutical composition according to any one of embodiments 1-48, wherein the phenolic preservative is phenol. 50. The liquid pharmaceutical composition according to any one of embodiments 1-49, wherein the phenolic preservative is phenol at a concentration of 60 mM. 51. The liquid pharmaceutical composition according to any one of embodiments 1-50, wherein the phenolic preservative is m-cresol. 52. The liquid pharmaceutical composition according to any one of embodiments 1-51, wherein the phenolic preservative is m-cresol at a concentration of 32 mM. 53. The liquid pharmaceutical composition according to any one of embodiments 1-52, wherein the mixture of phenolic preservatives is phenol and m-cresol. 54. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the mixture of phenolic preservatives is phenol and m-cresol, the concentration of phenol being in the range of 10-60 mM and the concentration of meta-cresol being in the range of 10-40 mM. 55. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the mixture of phenolic preservatives is phenol and m-cresol, the concentration of phenol being in the range of 16-32 mM and the concentration of meta-cresol being in the range of 16-32 mM. 56. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the mixture of phenolic preservatives is phenol and m-cresol, the concentration of phenol being 16 mM and the concentration of meta-cresol being 16 mM. 57. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the mixture of phenolic preservatives is phenol and m-cresol, the concentration of phenol being 21 mM and the concentration of meta-cresol being 21 mM. 58. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the mixture of phenolic preservatives is phenol and m-cresol, the concentration of phenol being 25 mM and the concentration of meta-cresol being 25 mM. 59. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the mixture of phenolic preservatives is phenol and m-cresol, the concentration of phenol being 16 mM and the concentration of meta-cresol being 32 mM. 60. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the mixture of phenolic preservatives is phenol and m-cresol, the concentration of phenol being 60 mM and the concentration of meta-cresol being 16 mM. 61. The liquid pharmaceutical composition according to any one of embodiments 1-60, wherein the insulin derivative comprises at least four aryl boron moieties. 62. The liquid pharmaceutical composition according to any one of embodiments 1-61, wherein the insulin derivative contains 4-20 aryl boron moieties. 63. The liquid pharmaceutical composition according to any one of embodiments 1-62, wherein the insulin derivative contains 4 to 16 aryl boron moieties. 64. The liquid pharmaceutical composition according to any one of embodiments 1-63, wherein the insulin derivative contains 4 to 12 aryl boron moieties. 65. The liquid pharmaceutical composition according to any one of embodiments 1-64, wherein the insulin derivative contains 4 to 8 aryl boron moieties. 66. The liquid pharmaceutical composition according to any one of embodiments 1-65, wherein the insulin derivative contains 4-6 aryl boron moieties. 67. The liquid pharmaceutical composition according to any one of embodiments 1-66, wherein the arylboronic acid or arylboroxole is an arylboronic acid. 68. The liquid pharmaceutical composition according to any one of embodiments 1-67, wherein the arylboronic acid or arylboroxole is an arylboroxole. 69. The liquid pharmaceutical composition according to any one of embodiments 1-68, wherein the insulin derivative comprises one or more modifying groups each comprising two arylboroxoles. 70. The liquid pharmaceutical composition according to any one of embodiments 1-69, wherein the insulin derivative comprises 1-6 modifying groups, each comprising two arylboroxoles. 71. The insulin derivative is optionally substituted. [ka] 71. The liquid pharmaceutical composition according to any one of embodiments 1 to 70, comprising at least one of: 72. The insulin derivative is optionally substituted. [ka] 72. The liquid pharmaceutical composition according to any one of embodiments 1 to 71, comprising at least one of: 73. An insulin derivative, [ka] wherein the phenyl ring is optionally substituted with one or more substituents selected from F, Cl, CHF2, and CF3. 74. Insulin derivatives: [ka] (wherein R1 is H, F, Cl, CHF2, or CF3). 75. Insulin derivatives are [ka] (wherein R1 is H or CF3). 76. Insulin derivatives are [ka] 76. The liquid pharmaceutical composition according to any one of embodiments 1 to 75, comprising at least one of: 77. Insulin derivatives are [ka] (wherein R1 is H, F, Cl, CHF2, or CF3, R2 is H or CH3, and R3 is H or CH3). 78. Insulin derivatives are [ka] (wherein R1 is H, F, Cl, CHF2, or CF3, R2 is H or CH3, and R3 is H or CH3). 79. The liquid pharmaceutical composition according to any one of embodiments 77-78, wherein R1 is H or CF3. 80. The liquid pharmaceutical composition according to any one of embodiments 77-78, wherein R2 is H and R3 is H. 81. The liquid pharmaceutical composition according to any one of embodiments 77-78, wherein R2 is H and R3 is CH3. 82. The liquid pharmaceutical composition according to any one of embodiments 77-78, wherein R2 is CH3 and R3 is H. 83. The liquid pharmaceutical composition according to any one of embodiments 1-82, wherein the insulin derivative comprises at least one of the following: [ka] 84. Insulin derivatives are covalently attached to amino acid residues within the insulin backbone and / or peptide-extended insulin backbone. [ka] 84. The liquid pharmaceutical composition according to any one of embodiments 1 to 83, comprising at least one of: 85. The liquid pharmaceutical composition according to any one of embodiments 1-84, wherein the insulin derivative comprises at least one of the following: [ka] 86. Insulin derivatives are covalently attached to amino acid residues within the insulin backbone and / or peptide-extended insulin backbone. [ka] 86. The liquid pharmaceutical composition according to any one of embodiments 1 to 85, comprising at least one of: 87. An insulin derivative, [ka] 87. The liquid pharmaceutical composition according to any one of embodiments 1 to 86, which is (the insulin derivative of Example 1), or a pharma- ceutically acceptable salt thereof. 88. The liquid pharmaceutical composition according to any one of embodiments 1 to 87, wherein the insulin derivative is the insulin derivative of Example 1. 89. An insulin derivative, [ka] 89. The liquid pharmaceutical composition according to any one of the preceding embodiments, which is (the insulin derivative of Example 2), or a pharma- ceutically acceptable salt thereof. 90. The liquid pharmaceutical composition according to any one of embodiments 1-89, wherein the insulin derivative is the insulin derivative of Example 2. 91. The composition comprises: The insulin derivative of Example 2, Glycerol, Mannitol, methionine. 92. The composition comprises: The insulin derivative of Example 2, Phenol and Metacresol, Glycerol, Mannitol, methionine. 93. The composition comprising: The insulin derivative of Example 2, Phenol and Metacresol, Glycerol, Mannitol, methionine. 94. The composition comprises: 0.6 to 4.8 mM of the insulin derivative of Example 2, 15-45 mM methionine; 140-250 mM glycerol; 20-80 mM mannitol. 95. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the composition comprises 30-45 mM methionine. 96. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the composition comprises 40-80 mM mannitol. 97. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein the composition comprises 152-217 mM glycerol. 98. The liquid pharmaceutical composition of any one of embodiments 1-97, wherein the composition is substantially free of zinc. 99. The composition comprises: 0.6 to 4.8 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol. 100. A composition comprising: 0.6 to 4.8 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1 to 99, wherein the pH is 6.6. 101. A composition comprising: 0.6 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1 to 100, wherein the pH is 6.6. 102. The composition, 1.2 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-101, wherein the pH is 6.6. 103. A composition comprising: 2.4 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1 to 102, wherein the pH is 6.6. 104. The composition, 3.6 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-103, wherein the pH is 6.6. 105. A composition comprising: 4.8 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-104, wherein the pH is 6.6. 106. The composition, 0.6 to 4.8 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-105, wherein the pH is 6.6. 107. The composition comprises: 0.6 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-106, wherein the pH is 6.6. 108. The composition, 1.2 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-107, wherein the pH is 6.6. 109. The composition comprises: 2.4 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-108, wherein the pH is 6.6. 110. The composition, 3.6 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1-109, wherein the pH is 6.6. 111. The composition, 4.8 mM of the insulin derivative of Example 2, 30 mM methionine, 152 mM glycerol, 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of embodiments 1 to 110, wherein the pH is 6.6. 112. The composition, 0.6 to 4.8 mM of the insulin derivative of Example 2, 16 mM phenol, 16 mM metacresol, 185 mM glycerol, 40 mM mannitol, 30 mM methionine, The liquid pharmaceutical composition according to any one of embodiments 1 to 111, wherein the pH is 6.6. 113. The composition, 0.6 to 4.8 mM of the insulin derivative of Example 2, 16 mM phenol, 16 mM metacresol, 185 mM glycerol, 40 mM mannitol, 30 mM methionine; The liquid pharmaceutical composition according to any one of embodiments 1 to 112, wherein the pH is 6.6. 114. The liquid pharmaceutical composition according to any one of embodiments 1-113, wherein the composition is substantially free of zinc. 115. The liquid pharmaceutical composition according to any one of embodiments 1-114, wherein the composition is substantially free of zinc ions. 116. The liquid pharmaceutical composition according to any one of embodiments 1 to 115, wherein when the insulin derivative is the insulin derivative of Example 2, the composition is substantially free of zinc. 117. The liquid pharmaceutical composition according to any one of the preceding embodiments, wherein when the insulin derivative is the insulin derivative of Example 2, the composition is substantially free of zinc ions. 118. A liquid pharmaceutical composition according to any one of embodiments 1 to 117 for use as a medicament. 119. A liquid pharmaceutical composition according to any one of embodiments 1 to 117 for use in the prevention or treatment of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome). 120. A liquid pharmaceutical composition according to any one of embodiments 1 to 117 for use in the prevention or treatment of diabetes. 121. A liquid pharmaceutical composition according to any one of embodiments 1 to 117 for use in the prevention or treatment of type 1 diabetes and type 2 diabetes. 122. Use of a liquid pharmaceutical composition according to any one of embodiments 1 to 117 for the manufacture of a medicament for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome). 123. A method for treating or preventing diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome), comprising administering to a subject in need thereof a therapeutically effective amount of a liquid pharmaceutical composition according to any one of embodiments 1-117. 124. A method for treating diabetes in a subject, comprising administering to said subject a liquid pharmaceutical composition described in any one of embodiments 1 to 117.

[0039] The invention is further defined by the following second list of embodiments. 1. A liquid pharmaceutical composition comprising: a) an insulin derivative containing at least one arylboronic acid or arylboroxole; b) methionine. 2. The liquid pharmaceutical composition of embodiment 1, wherein the concentration of methionine is within the range of 5 mM to 300 mM. 3. The liquid pharmaceutical composition according to embodiment 1 or 2, further comprising glycerol, or a sugar alcohol such as mannitol or sorbitol, either alone or in combination. 4. The liquid pharmaceutical composition according to any one of embodiments 1 to 3, further comprising mannitol. 5. The liquid pharmaceutical composition according to any one of embodiments 1 to 4, further comprising mannitol and glycerol. 6. The liquid pharmaceutical composition according to any one of embodiments 1 to 5, wherein the concentration of mannitol in the composition is within the range of 10 mM to 300 mM. 7. The liquid pharmaceutical composition according to any one of embodiments 1 to 6, wherein the concentration of glycerol in the composition is within the range of 10 mM to 600 mM. 8. The liquid pharmaceutical composition according to any one of embodiments 1 to 7, wherein the pH is within the range of 6.4 to 7.4. 9. The liquid pharmaceutical composition according to any one of embodiments 1 to 8, further comprising a phenolic preservative or a mixture of phenolic preservatives. 10. The liquid pharmaceutical composition according to any one of embodiments 1-9, wherein the arylboronic acid or arylboroxole is an arylboroxole. 11. The liquid pharmaceutical composition according to any one of embodiments 1 to 10, wherein the insulin derivative comprising at least one arylboronic acid or arylboroxole is the insulin derivative of Example 1 or the insulin derivative of Example 2. 12. The composition comprising: The insulin derivative of Example 2, Phenol and Metacresol, Glycerol, Mannitol, methionine. 13. The composition comprising: 0.6 to 4.8 mM of the insulin derivative of Example 2, 16 mM phenol, 16 mM metacresol, 185 mM glycerol, 40 mM mannitol, 30 mM methionine; The liquid pharmaceutical composition according to any one of embodiments 1 to 12, wherein the pH is 6.6. 14. A liquid pharmaceutical composition according to any one of embodiments 1 to 13 for use as a medicament. 15. A liquid pharmaceutical composition according to any one of embodiments 1 to 14 for use in the prevention or treatment of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome). EXAMPLES

[0040] material and method The mannitol used in the following examples is D-mannitol.

[0041] The sorbitol used in the following examples is D-sorbitol.

[0042] The methionine used in the following examples is L-methionine.

[0043] The ascorbic acid (sodium ascorbate) used in the following examples is L-ascorbic acid (sodium L-ascorbate).

[0044] List of Abbreviations ACN Acetonitrile ALP achromobacter lyticus protease BEH Ethylene Bridged Hybrid (Ethylene Bridged Hybrid Particle Technology) C18 Octadecanyl (HPLC column) CV column volume Dap 2,3-diaminopropionic acid DCC N,N'-dicyclohexylcarbodiimide DIC N,N-Diisopropylcarbodiimide DMF N,N-Dimethylformamide HATU 1-((Dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo- [4,5-b]pyridine 3-oxide hexafluorophosphate HCl Hydrochloric acid HPLC High Performance Liquid Chromatography hrs time LCMS Liquid Chromatography Mass Spectrometry min MeCN Acetonitrile mm millimeters mM millimolar NaCl Sodium chloride NaOH Sodium hydroxide NBS N-Bromosuccinimide NHS N-hydroxysuccinmide NMR nuclear magnetic resonance qs required amount (quantum satis) RP-HPLC Reversed-phase high-performance liquid chromatography tBu tert-butyl TFA Trifluoroacetic acid UPLC Ultra High Performance Liquid Chromatography UV ultraviolet light Å Angstrom

[0045] Example 1: Preparation of the insulin derivative of Example 1 [ka] The insulin derivative of Example 1 is disclosed in WO 2020 / 201041 (Example 280) and was prepared as described in WO 2020 / 201041 (the insulin A chain has the sequence of SEQ ID NO: 1 and the extended B chain has the sequence of SEQ ID NO: 4).

[0046] Example 2: Preparation of the insulin derivative of Example 2 [ka]

[0047] Expression of insulin variants in yeast and transformation with ALP etc. The insulin analog GKPE-(GEQP)4-GEQGGKPEGGGSGGGGSGGGGS-B1 desB30 human insulin (insulin A chain has the sequence of SEQ ID NO: 1 and extended B chain has the sequence of SEQ ID NO: 5) was expressed in yeast using well-known techniques, for example as disclosed in WO 2017 / 032798. More specifically, the insulin analog was expressed as a single chain precursor, isolated by ion exchange capture, and cleaved into the two-chain insulin analog by treatment with ALP as described below.

[0048] GKPE-(GEQP)4-GEQG-GKPEGGGSGGGGSGGGGS-B1 desB30 human insulin means desB30 human insulin extended from B1 with GKPE-GEQPGEQPGEQPGEQP-GEQG-GKPEGGGSGGGGSGGGGS (sequence number 6) (the C-terminal S is connected to B1F).

[0049] Precursor capture on SP Sepharose BB: Yeast supernatant was loaded onto a column packed with SP Sepharose BB at a flow rate of 10-20 CV / h. A wash with 0.1 M citric acid, pH 3.5, and a wash with 40% EtOH were performed. Analogs were eluted with 0.2 M sodium acetate pH 5.5 / 35% EtOH.

[0050] ALP digestion: The solution of single-chain precursor was adjusted to pH 9 and ALP enzyme was added at 1:100 (w / w). The reaction was monitored by UPLC. In preparation for RP-HPLC purification, the ALP cleavage pool was adjusted to pH 2.5 and diluted 2-fold.

[0051] RP-HPLC purification: Purification was carried out by RP-HPLC C18 as follows: Column: 15um C18 50×250mm 200Å Buffer: A: 0.2% formic acid, 5% EtOH, B: 0.2% formic acid, 50% EtOH Gradient: 20–55% B buffer. Gradient: 20CV Flow rate 20CV / hour Loading g approx. 5g / L resin Fractions were analyzed by UPLC, pooled and lyophilized.

[0052] Preparation of (S)-4-((S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)-5-(tert-butoxy)-5-oxopentanoic acid (building block 1) [ka] NBS (34.0 g, 191 mmol) was added to a solution of 3-trifluoromethyl-4-methylbenzoic acid (39.0 g, 191 mmol) in concentrated sulfuric acid (400 mL) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then poured into ice water (2 L). The resulting precipitate was filtered off, washed with water (500 mL), dissolved in ethyl acetate (400 mL), dried over anhydrous sodium sulfate, filtered and evaporated to give 3-bromo-4-methyl-5-trifluoromethylbenzoic acid as a white solid. Yield: 53.4 g (98%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.71 (bs, 1H); 8.35 (d, J=0.4 Hz, 1H); 8.15 (d, J=0.9 Hz, 1H); 2.56 (s, 3H).

[0053] Concentrated sulfuric acid (24 mL) was added to a solution of 3-bromo-4-methyl-5-trifluoromethylbenzoic acid (35.0 g, 124 mmol) in methanol (500 mL) and the reaction mixture was stirred at reflux for 4 h and at room temperature for 16 h. The reaction mixture was then evaporated under reduced pressure, dissolved in diethyl ether (250 mL), washed with water (2×100 mL) and a mixture of a saturated solution of potassium carbonate (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated to give methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate as a white solid. Yield: 35.3 g (96%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 8.36 (d, J = 1.1 Hz, 1H); 8.13 (d, J = 1.1 Hz, 1H); 3.90 (s, 3H); 2.55 (d, J = 1.3 Hz, 3H).

[0054] A suspension of NBS (31.7 g, 178 mmol) and methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate (35.3 g, 119 mmol) in water (300 mL) was stirred at 80° C. under a 100 W bulb for 6 h. The reaction mixture was extracted with diethyl ether (2×200 mL). The organic layer was washed with brine (150 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated to give methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate as a yellow solid. Yield: 44.0 g (98%). 1 H NMR spectrum (300 MHz, CDCl3, δH): 8.47 (d, J = 1.5 Hz, 1H); 8.31 (d, J = 1.3 Hz, 1H); 4.75 (s, 2H); 3.98 (s, 3H).

[0055] A solution of methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate (44.0 g, 117 mmol) and potassium acetate (22.9 g, 234 mmol) in MeCN (0.5 L) was stirred at 75° C. overnight. The suspension was filtered through filter paper and evaporated. The crude product was dissolved in dichloromethane and filtered again. Evaporation gave methyl 4-(acetoxymethyl)-3-bromo-5-(trifluoromethyl)benzoate as a white solid. Yield: 37.9 g (91%). 1 H NMR spectrum (300 MHz, CDCl3, δH): 8.49 (d, J = 1.3 Hz, 1H); 8.34 (d, J = 1.3 Hz, 1H); 5.37 (s, 2H); 3.99 (s, 3H); 2.11 (s, 3H).

[0056] A solution of methyl 4-(acetoxymethyl)-3-bromo-5-(trifluoromethyl)benzoate (37.9 g, 107 mmol), bis(pinacolato)diboron (29.8 g, 117 mmol), potassium acetate (31.4 g, 294 mmol), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.57 g, 1.92 mmol) in dry tetrahydrofuran (500 mL) was stirred at 75° C. under an argon atmosphere for 13 days. The reaction mixture was then cooled to room temperature, filtered, and evaporated. The crude product was filtered through a silica gel column (Silicagel, 0.063-0.200 mm, eluent: cyclohexane / ethyl acetate 8:1) to give methyl 4-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)benzoate. Yield: 31.1 g (72%). 1 H NMR spectrum (300 MHz, CDCl3, δH): 8.65 (s, 1H); 8.43 (s, 1H); 5.48 (s, 2H); 3.97 (s, 3H); 2.05 (s, 3H); 1.36 (s, 12H).

[0057] A solution of methyl 4-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)benzoate (31.0 g, 77.1 mmol) and sodium hydroxide (15.4 g, 386 mmol) in water (300 mL) was stirred at room temperature for 3 h. Then a solution of hydrochloric acid (35 mL) in water (100 mL) was added to lower the pH to 1. The reaction mixture was stirred overnight. The precipitate was filtered off and dried to give 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid as a white solid. Yield: 16.6 g (86%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.47 (bs, 1H); 9.66 (s, 1H); 8.62 (s, 1H); 8.24 (s, 1H); 5.22 (s, 2H).

[0058] A solution of pentafluorophenol (7.48 g, 40.7 mmol), 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (10.0 mg, 40.7 mmol), and N,N'-dicyclohexylcarbodiimide (DCC, 8.37 mg, 40.7 mmol) in MeCN (0.5 L) was stirred at room temperature overnight. The reaction mixture was filtered, evaporated, dissolved in MeCN, refiltered, and evaporated to give pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate as a white solid. Yield: 16.7g (100%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

[0059] 2-Chlorotrityl chloride resin 100-200 mesh 1.5 mmol / g (3, 4.47 g, 6.71 mmol) was left in anhydrous dichloromethane (30 mL) for 30 min to swell. A solution of (2S)-5-(tert-butoxy)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-5-oxopentanoic acid (Fmoc-Glu-OtBu, 1.90 g, 4.47 mmol) and N,N-diisopropylethylamine (2.96 mL, 17.0 mmol) in anhydrous dichloromethane (30 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (1.56 mL, 8.95 mmol) in a methanol / dichloromethane mixture (4:1, 2 × 5 min, 2 × 40 mL). The resin was then washed with DMF (2×30 mL), dichloromethane (2×40 mL) and DMF (3×40 mL). The Fmoc group was removed by treatment with 20% piperidine in DMF (1×5 min, 1×20 min, 2×40 mL). The resin was washed with DMF (3×40 mL), 2-propanol (2×40 mL) and dichloromethane (3×40 mL). A solution of (S)-2,3-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Dap(Fmoc)-OH, 3.68 g, 6.71 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 2.55 g, 6.71 mmol), and 2,4,6-trimethylpyridine (1.60 mL, 12.1 mmol) in DMF (40 mL) was added to the resin and the mixture was shaken for 2 h. The resin was filtered and washed with DMF (2 x 40 mL), dichloromethane (2 x 40 mL), and DMF (2 x 40 mL). The Fmoc group was removed by treatment with 20% piperidine in DMF (1×5 min, 1×30 min, 2×40 mL). The resin was washed with DMF (3×40 mL), 2-propanol (2×40 mL) and dichloromethane (3×40 mL).A solution of pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (5.53 g, 13.4 mmol) and triethylamine (4.99 mL, 35.8 mmol) in DMF (40 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and washed with DMF (6×40 mL) and dichloromethane (10×50 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (60 mL) for 16 h. The resin was filtered off and washed with dichloromethane (4×50 mL). The crude product (4) was dried in vacuum, extracted with ethyl acetate (2×70 mL) and 1 M aqueous potassium hydrogen sulfate (50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered and the solvent was evaporated. The crude product was then triturated in diethyl ether (20 mL) to give (S)-4-((S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)-5-(tert-butoxy)-5-oxopentanoic acid (building block 1) as a beige solid. Yield: 1.89 g (57%). 1 H NMR spectrum (300 MHz, AcOD-d4, δH): 8.50 (s, 1H); 8.46 (s, 1H); 8.29 (s, 1H); 8.26 (s, 1H); 5.28 (d, J = 2.6 Hz, 4H); 5.20 (t, J = 5.9 Hz, 1H); 4.55 (dd, J = 8.5 and 5.2 Hz, 1H); 4.08 (dd, J = 6.0 and 2.1 Hz, 2H); 2.57-2.42 (m, 2H); 2.34-2.16 (m, 1H); 2.17-2.08 (m, 1H); 1.47 (s, 9H). LC-MS: 746.3 (M+H)+.

[0060] Preparation of insulin derivatives Building block 1 was activated with NHS / DIC in THF and the tBu protecting group was removed by treatment with TFA, which was then evaporated prior to conjugation with insulin.

[0061] GKPE-(GEQP)4-GEQGGKPEGGGSGGGGSGGGGS-B1 desB30 human insulin (536 mg, 0.057 mmol) was dissolved in 0.1 M Na2HPO4 (5.6 mL) and DMSO (2.4 mL) and the pH was adjusted to 10.8 with 1.0 M aqueous NaOH. Building block 1 (147 mg, 0.19 mmol) was NHS-activated and tBu-deprotected as described above, then dissolved in DMF (0.5 mL) and added dropwise to the given insulin solution over 10 min while the pH was kept near 10.8 by dropwise addition of 0.1 M NaOH. LCMS shows the formation of the desired product. The mixture was diluted with MeCN and water and the pH was adjusted to 3.8 by dropwise addition of 1 M HCl. The product was purified by reversed-phase HPLC (RP-HPLC) on a C18 column using 0.1% TFA in water as buffer A and 0.1% TFA in MeCN as buffer B. The product was isolated by lyophilization. LCMS found 1894.1 [M+6H-5×water] 6+ , calculated value 1894.1;1623.6[M+7H-5×Water] 7+ Calculated value 1623.6; 1420.8 [M+8H-5×water] 8+ , calculated value 1420.8; and 1263.0 [M+9H-5×water] 9+ , calculated value 1263.0(C 480 H 666 B6F 18 N 122 O 169 (For S6).

[0062] Example 3: Effect of the antioxidants methionine and sodium ascorbate on stability In this example, the stabilizing effect of two antioxidants, methionine and sodium ascorbate, was investigated. Chemical stability can be measured in terms of purity, the content of intact insulin derivative, determined at time zero and at specific temperatures and storage times. The formulations tested are shown in Table 1.

[0063] Preparation process Each formulation was prepared from the following stock solutions: 2 mM insulin derivative of Example 1, 100 mM sodium phosphate, 500 mM phenol, 160 mM metacresol, 1 M NaCl, 2.17 M glycerol, 10 mM zinc acetate, 1.34 M methionine, 1.01 M sodium ascorbate, water for injection.

[0064] The formulations were sterilized by filtration through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as needed and adding water to reach the final desired volume. After filtration, the formulations were filled into HPLC vials containing 50 μl for time zero analysis and into 3 ml drug cartridges containing 0.5 ml of formulation for storage stability analysis.

[0065] Methods and assay experiments Cartridges were stored at 37° C. for 4 weeks and analyzed for purity, which was compared to the results of analysis of a time point zero sample.

[0066] The purity (as percentage) of the insulin derivative of Example 1 was determined using a UPLC equipped with a Waters column BEH C18, 2.1×150 mm, 1.7 μm, 130 Å, using 0.1% TFA in milliQ water (buffer A) and 0.09% TFA in 90% ACN (buffer B) with 9% acetonitrile as wash buffer, with gradient elution over a run time of about 64 min. The column temperature was 35° C., the injection volume was 1 μl, while the flow rate was set at 0.3 ml / min. The chromatography was analyzed by UV detection (215 nm) and the purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0067] result The purity (as a percentage) at time zero and after 4 weeks of storage at 37° C. for formulations 1-5 is shown in Table 1. Purity loss is also provided as the difference (in percentage points) between the purity after 4 weeks at 37° C. and the purity at time zero.

[0068] For formulation 1 without antioxidants, the purity loss after 4 weeks at 37° C. was 8.7 percentage points. Surprisingly, it was found that the addition of methionine improved the stability of the insulin formulation, while sodium ascorbate resulted in a purity loss of over 50 percentage points after 4 weeks. [Table 1]

[0069] Example 4: Effect of glycerol, sugar alcohols, and sugars on stability In this example, the stabilizing effect of glycerol, sugar alcohols, and sugars on the formulation of the insulin derivative of Example 1 was investigated. The formulations tested are shown in Table 2.

[0070] Preparation process Each formulation was prepared from the following stock solutions: 2 mM insulin derivative of Example 1, 100 mM sodium phosphate, 500 mM phenol, 160 mM metacresol, 1 M NaCl, 2.17 M glycerol, 10 mM zinc acetate, 200 mM sorbitol, 300 mM mannitol, 200 mM sucrose, 200 mM trehalose, water for injection.

[0071] The formulations were sterilized by filtration through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as needed and adding water to reach the final desired volume. After filtration, the formulations were filled into HPLC vials containing 50 μl for time zero analysis and into 3 ml drug cartridges containing 0.5 ml of formulation for storage stability.

[0072] Methods and assay experiments The cartridges were stored at 37° C. for one week and analyzed for purity. Additionally, the cartridges were stored at 45° C. for an additional 6 days to further accelerate degradation and analyzed for purity.

[0073] The purity (as percentage) of the insulin derivative of Example 1 was determined using a UPLC equipped with a Waters column BEH C18, 2.1×150 mm, 1.7 μm, 130 Å, using 0.1% TFA in milliQ water (buffer A) and 0.09% TFA in 90% ACN (buffer B) with 9% acetonitrile as wash buffer, with gradient elution over a run time of about 70 min. The column temperature was 50° C., the injection volume was 2 μl, while the flow rate was set at 0.3 ml / min. The chromatography was analyzed by UV detection (215 nm) and the purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0074] result The purity (as a percentage) for formulations 6-15 at time zero and after one week storage at 37° C. and after one week at 37° C. plus an additional six days storage at 45° C. is shown in Tables 2a and 2b. Purity loss is also provided as the difference (in percentage points) between the purity after storage and the purity at time zero.

[0075] The purity loss after 1 week at 37° C. plus an additional 6 days at 45° C. for formulation 6, which had no glycerol, sugar alcohol, or sugar in the formulation, was 12.3 percentage points.

[0076] Tonicity agents are commonly used in pharmaceutical formulations intended for subcutaneous injection to ensure proper osmolality of the formulation. Surprisingly, it was found that the stability was significantly improved by adding the tonicity agent glycerol at a concentration of 174 mM (Formulation 7).

[0077] Even more surprisingly, formulation 8, which contained only 20 mM of the sugar alcohol mannitol, showed even better stability, with a purity loss of 5.0 percentage points after 1 week at 37° C. plus an additional 6 days at 45° C.

[0078] Surprisingly, the combination of 174 mM glycerol and 20 mM mannitol (formulation 10) can further improve stability. [Table 2]

[0079] The addition of 174 mM glycerol and 20 mM sugar alcohol sorbitol (formulation 13) resulted in increased stability compared to 174 mM glycerol alone (formulation 7), however the stabilizing effect was not as good as the formulation containing 174 mM glycerol and 20 mM mannitol (formulation 10).

[0080] Conversely, the addition of 20 mM of the sugars sucrose (formulation 14) or trehalose (formulation 15) did not affect stability, as can be seen by comparison with formulation 7. [Table 3]

[0081] Variation of the mannitol content (see Table 2b) showed that mannitol has a stabilizing effect over a wide concentration range.

[0082] Example 5: Effect of Methionine in Combination with Mannitol on Stability In this example, the stabilizing effect of the combination of methionine and mannitol acids was investigated. In addition, the stability at various pH levels was determined. Chemical stability can be measured in terms of purity, the content of intact insulin derivatives determined at time zero and at specific temperatures and storage times. The compositions tested are shown in Table 3.

[0083] Preparation process Each formulation was prepared from the following stock solutions: 5 mM insulin derivative of Example 1, 100 mM sodium phosphate, 500 mM phenol, 160 mM metacresol, 1 M sodium chloride, 2.17 M glycerol, 10 mM zinc acetate, 335 mM methionine, 400 mM mannitol, water for injection.

[0084] The formulation was sterilized by filtering through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as necessary and adding water for injection to reach the final desired volume. After filtration, the formulation was filled into 3 ml size drug cartridges containing 0.5 ml of the formulation storage stability.

[0085] Methods and assay experiments Cartridges were stored at 45° C. for 1 week and analyzed for purity, which was compared to the results of an analysis of a time point zero sample.

[0086] The purity (as percentage) of the insulin derivative of Example 1 was determined using a UPLC equipped with a Waters column BEH C18, 2.1×150 mm, 1.7 μm, 130 Å, using 0.1% TFA in milliQ water (buffer A) and 0.09% TFA in 90% ACN (buffer B) with 9% acetonitrile as washing buffer, gradient elution over a run time of about 70 min. The column temperature was 50° C., the injection volume was 1 μl, while the flow rate was set at 0.3 ml / min. The chromatography was analyzed by UV detection (215 nm) and the purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0087] result The purity (as a percentage) at time zero and after one week of storage at 45° C. for formulations 16-21 is shown in Table 3. Purity loss is also provided as the difference (in percentage points) between the purity after storage and the purity at time zero. [Table 4]

[0088] In Example 4, the addition of glycerol and mannitol was found to result in increased stability of the insulin formulation, and in Example 3, methionine was found to increase stability.

[0089] In this example, formulation 16, containing 217 mM glycerol and 20 mM mannitol, had a purity loss of 10.5 percentage points at pH 7.2 after one week at 45° C. The addition of 20 mM methionine surprisingly resulted in a large increase in stability, with a purity loss of 3.4 percentage points under the same conditions (formulation 19). The data in Table 3 also show the effect of pH.

[0090] Example 6: Effect of methionine and mannitol on stability In this example, the stabilizing effect of various amounts of methionine and mannitol on the formulation of the insulin derivative of Example 1 was investigated. Chemical stability can be measured in terms of purity, the content of intact insulin derivative determined at time zero and upon storage for a specific time at a specific temperature. The compositions tested are shown in Table 4.

[0091] Preparation process Each formulation was prepared from the following stock solutions: 8 mM insulin derivative of Example 1, 500 mM phenol, 160 mM metacresol, 1 M NaCl, 2.17 M glycerol, 10 mM zinc acetate, 100 mM methionine, 500 mM mannitol, water for injection.

[0092] The formulations were sterilized by filtration through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as needed and adding water to reach the final desired volume. After filtration, the formulations were filled into HPLC vials containing 50 μl for time zero analysis and into 3 ml drug cartridges containing 0.5 ml of formulation for storage stability analysis.

[0093] Methods and assay experiments Cartridges were stored at 37° C. for 4 weeks and analyzed for purity, which was compared to the results of analysis of a time point zero sample.

[0094] The purity (as percentage) of the insulin derivative of Example 1 was determined using a UPLC equipped with a Waters column BEH C18, 2.1×150 mm, 1.7 μm, 130 Å, using 0.1% TFA in milliQ water (buffer A) and 0.09% TFA in 90% ACN (buffer B) with 9% acetonitrile as wash buffer, with gradient elution over a run time of about 64 min. The column temperature was 35° C., the injection volume was 1 μl, while the flow rate was set at 0.3 ml / min. The chromatography was analyzed by UV detection (215 nm) and the purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0095] result The purity (as a percentage) at time zero and after 2 and 4 weeks of storage at 37° C., respectively, for formulations 22-27 are shown in Table 4. Purity loss is also provided as the difference (in percentage points) between the purity after storage and the purity at time zero. [Table 5]

[0096] The data in Table 4 show that formulation 22, with 21 mM methionine and no mannitol, had a purity loss of 5.9 percentage points after 4 weeks at 37° C. Formulation 23, with 29 mM mannitol and no methionine, had a purity loss of 11.0 percentage points. Formulations 24 and 25, which combine similar amounts of methionine and mannitol, surprisingly show that the combination of methionine and mannitol significantly increases stability.

[0097] Example 7: Effect of the Presence of Zinc on Stability In this example, the effect of the presence of zinc on the insulin derivatives of Examples 1 and 2 is shown. Chemical stability can be measured in terms of purity, where the content of intact insulin derivative is determined at time zero and after storage for a specific time at a specific temperature. The compositions tested are shown in Table 5. Typically, the insulin derivatives are more stable in the presence of zinc acetate and sodium chloride, which are known to favor a hexameric oligomeric state for insulin.

[0098] Preparation process Each formulation was prepared from the following stock solution: 1 mM insulin derivative of Example 1 or Example 2, 500 mM phenol, 160 mM metacresol, 1 M NaCl, 2.17 M glycerol, 10 mM zinc acetate, 168 mM methionine, 500 mM mannitol, water for injection.

[0099] The formulations were sterilized by filtering through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as needed and adding water to reach the final desired volume. After filtration, the formulations were filled into HPLC vials to contain 50 μl. One sample was set aside and flash frozen for time zero analysis, and the other sample was used for storage stability at 37° C. for 1 week before analysis.

[0100] Methods and assay experiments The vials were stored at 37° C. for 1 week and analyzed for purity, which was compared to the results of the analysis of the time point zero sample.

[0101] The purity (as percentage) of both insulin derivatives of Example 1 and Example 2 was determined using a UPLC equipped with a Waters column BEH C18, 2.1×150 mm, 1.7 μm, 300 Å, using 0.1% TFA in milliQ water (buffer A) and 0.08% TFA in 80% ACN (buffer B) with gradient elution over a run time of about 100 min using 9% acetonitrile as washing buffer. The column temperature was 55° C. and about 8 μg was loaded onto the column while the flow rate was set at 0.3 ml / min. The chromatography was analyzed by UV detection (215 nm) and the purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0102] result The purity (as a percentage) at time zero and after one week of storage at 37° C. for formulations 58-61 is shown in Table 5. Purity loss is also provided as the difference (in percentage points) between the purity after storage and the purity at time zero. [Table 6]

[0103] The data in Table 5 show that for the insulin derivative of Example 1, the lowest purity loss is observed in the presence of zinc acetate and sodium chloride, whereas for the insulin derivative of Example 2, surprisingly, the lowest purity loss is observed in the absence of zinc acetate and sodium chloride.

[0104] Example 8: Effect of the Presence of Zinc on Stability at Varying pH and API Concentration In this example, the effect of the presence of zinc and varying pH is shown for two concentrations of the insulin derivative of Example 2. Additionally, the effect of using sorbitol instead of glycerol as a tonicity agent was tested. Chemical stability can be measured in terms of purity, and the content of intact insulin derivative is determined at time zero and at a specific temperature and storage time. The compositions tested are shown in Tables 6a and 6b. Typically, insulin derivatives are more stable in the presence of zinc acetate and sodium chloride, which are known to favor a hexameric oligomeric state for insulin.

[0105] Preparation process Each formulation was prepared from the following stock solutions: 5.7 mM insulin derivative of Example 2, 500 mM phenol, 160 mM metacresol, 1 M NaCl, 2.17 M glycerol, 10 mM zinc acetate, 168 mM methionine, 500 mM mannitol, 1 M sorbitol, water for injection.

[0106] The formulations were sterilized by filtering through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as needed and adding water to reach the final desired volume. After filtration, the formulations were filled into HPLC glass vials. One sample of 50 μl size was flash frozen for time zero analysis and another sample of 100 μl size was used for storage stability at 37° C. for 4 weeks before analysis.

[0107] Methods and assay experiments The vials were stored at 37° C. for 1 week and analyzed for purity, which was compared to the results of the analysis of the time point zero sample.

[0108] The purity (as percentage) of both insulin derivatives of Example 1 and Example 2 was determined using a UPLC equipped with a Waters column BEH C8, 2.1×150 mm, 1.7 μm, 130 Å, gradient elution using 0.1% TFA in milliQ water (buffer A) and 0.09% TFA in 80% ACN (buffer B), isocratic, over a run time of about 50 min. The column temperature was 47° C. and 0.3 μl (sample of insulin derivative of Example 2 diluted to 0.6 mM) was loaded onto the column while the flow rate was set to 0.3 ml / min. The chromatography was analyzed by UV detection (214 nm) and purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0109] result The purity (as a percentage) at time zero and after 4 weeks of storage at 37° C. for formulations 62-69 is shown in Table 6a. Purity loss is also provided as the difference (in percentage points) between the purity after storage and the purity at time zero. [Table 7]

[0110] The data in Table 6a show that for the insulin derivative of Example 2, surprisingly, the lowest purity loss is observed around pH 6.6 in the absence of zinc acetate and sodium chloride. [Table 8]

[0111] The data in Table 6b show that replacing glycerol with sorbitol results in formulations with similar stability.

[0112] Example 9: Effect of various excipients on stability In this example, formulations with varying amounts of the insulin derivative of Example 2, varying amounts of glycerol and mannitol, varying amounts of the antioxidant methionine, and varying pH were prepared and tested for stability. Chemical stability can be measured in terms of purity, with the content of intact insulin derivative determined at time zero and upon storage for a specific time at a specific temperature. The compositions tested are shown in Tables 7a, 7b, 7c, and 7d.

[0113] Preparation process Each formulation was prepared from the following stock solutions: 5.7 mM insulin derivative of Example 1, 500 mM phenol, 160 mM metacresol, 1 M sodium chloride, 2.17 M glycerol, 10 mM zinc acetate, 167.5 mM methionine, 500 mM mannitol, 1000 mM sorbitol, water for injection.

[0114] The formulations were sterilized by filtration through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as needed and adding water for injection to reach the final desired volume. After filtration, the formulations were filled into flat-bottom glass UPLC vials containing 0.3 ml of formulation for storage stability.

[0115] Methods and assay experiments The vials were stored at 37° C. for 4 weeks and analyzed for purity, which was compared to the results of the analysis of the time point zero sample.

[0116] The purity (as a percentage) of the insulin derivatives was determined using a UPLC equipped with a Waters column BEH C8, 2.1×150 mm, 1.7 μm, 130 Å, with gradient elution over a run time of about 50 min using 0.1% TFA in milliQ water (buffer A) and 0.09% TFA in 90% ACN (buffer B) and 9% acetonitrile as wash buffer. The column temperature was 47° C., the injection volume was 0.3 μl for samples containing 0.6 mM insulin derivatives, while the flow rate was set at 0.3 ml / min. Samples containing 1.2 mM insulin derivatives were diluted to 0.6 mM with water immediately before loading. The chromatography was analyzed by UV detection (214 nm) and the purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0117] result The purity (as a percentage) at time zero and after 4 weeks of storage at 37° C. for formulations 28-38 is shown in Tables 7a, 7b, 7c, and 7d. Purity loss is also provided as the difference (in percentage points) between the purity after storage and the purity at time zero.

[0118] Comparing formulations 28-30 (Table 7a) shows that there is a decrease in stability when the pH is reduced to 5.8 compared to when the pH is 6.2 and 6.6, respectively.

[0119] Formulations 32, 34, 35, and 36 (Table 7b) show that stable formulations can be achieved with various concentrations of methionine, mannitol, and glycerol, with the most stable formulations being achieved with a methionine concentration of at least 30 mM and a mannitol concentration of at least 40 mM.

[0120] Formulations 32 and 33 (Table 7c) show that stable formulations can be achieved using the preservatives phenol and meta-cresol alone and in combination. [Table 9] [Table 10] [Table 11] [Table 12]

[0121] In formulations 30, 31, 32, 37, and 38 (Table 7d), the concentration of insulin derivative varies from 0.6 mM to 4.8 mM, showing that stable formulations can be achieved throughout this concentration range.

[0122] Example 10: Effect of various excipients on stability In this example, formulations containing the insulin derivative of Example 2 were prepared containing various amounts of preservatives, methionine, mannitol, and glycerol, and tested for stability. Chemical stability can be measured in terms of the formation of high molecular weight products (HMWP), and the content of HMWP is determined at time zero and after storage for a specific time at a specific temperature. The compositions tested are shown in Tables 8a, 8b, and 8c.

[0123] Preparation process Each formulation was prepared from the stock solution of insulin derivative of Example 2 and excipients as shown in Tables 8a, 8b, and 8c. The formulations were sterilized by filtration through a 0.22 μm sterile filter after adjusting the pH using either HCl and NaOH or acetic acid as necessary and adding water for injection to reach the final desired volume. After filtration, the formulations were filled into HPLC vials for storage stability.

[0124] Methods and assay experiments The vials were stored at 37° C. for 6 weeks and analyzed for the presence of high molecular weight proteins (HMWPs) and compared with the results of analysis of the time point zero samples.

[0125] The HMWP (as a percentage) of insulin derivatives was determined using SE-UPLC equipped with a Waters Acquity BEH SEC Insulin column, 4.6×150 mm, 1.7 μm, 125 Å, with an isocratic elution using 300 mM sodium chloride, 10 mM sodium phosphate, 5 mM phosphoric acid, 15% isopropanol, and 35% ACN over a run time of approximately 10 minutes. The column temperature was 50° C., the injection volume was 0.6 μl for a sample diluted (10-fold) to contain 0.24 mM insulin derivative from Example 2, while the flow rate was set at 0.3 ml / min. The chromatography was analyzed by UV detection (214 nm) and the percentage of HMWP was evaluated as the area of ​​the HMWP peak relative to the total peak area (100%).

[0126] result The tested compositions of formulations 39-57 are shown in Tables 8a, 8b, and 8c along with the results of HMWP (as a percentage) at time zero and after 6 weeks of storage at 37°C. [Table 13]

[0127] From Table 8a, it can be seen that formulations containing either methionine or mannitol were found to have lower HMWP formation during 6 weeks of storage compared to formulation 39 which did not. Formulations containing both methionine and mannitol further reduced HMWP formation.

[0128] In formulations 42, 49, 50, 51, and 52 (Table 8b), the concentration of methionine varies from 15 mM to 45 mM and the concentration of mannitol varies from 20 mM to 80 mM, showing that stable formulations can be achieved across the entire concentration range. [Table 14]

[0129] Formulations 42, 43, 44, 45, 46, 47, and 48 (Table 8c) show that the preservatives phenol and meta-cresol can be used alone and in combination at various concentrations to achieve stable formulations. [Table 15]

[0130] Example 11: Stability data of formulations from the insulin derivative of Example 2 This example describes long-term stability data for formulations of the insulin derivative of Example 2. Chemical stability can be measured in terms of purity, with the content of intact insulin derivative determined at time zero and upon storage for specific times at specific temperatures. The compositions tested are shown in Table 9.

[0131] Preparation process The formulations were prepared from crude pharmacopoeia grade excipients, phenol, metacresol, mannitol, methionine, glycerol, and water for injection. The excipient formulations were prepared at 80% of the final volume in which the insulin derivative of Example 2 was dissolved. The formulations were sterilized by filtering through a 0.22 μm sterile filter after adjusting the pH using HCl and NaOH as necessary and adding water for injection to reach the final desired volume. After filtration, the formulations were filled into glass penfills containing 50 μl for time zero analysis and into 3 ml size drug cartridges containing 1 ml of formulation for storage stability analysis.

[0132] Methods and assay experiments The penfills were analyzed at time zero, while the remaining penfills were stored at 5° C. and 25° C. for up to 6 months, and at 30° C. and 37° C. for up to 3 months, and then analyzed for purity. The results were compared to those of the analysis of the time zero samples.

[0133] The purity (as percentage) of the insulin derivative of Example 2 was determined using a UPLC equipped with a Waters column BEH C8, 2.1×150 mm, 1.7 μm, 130 Å, gradient elution with 0.1% TFA in Milli-Q water (buffer A) and 0.09% TFA in 90% ACN (buffer B) over a run time of about 40 min. The column temperature was 41° C., about 8 μg was loaded onto the column, while the flow rate was set at 0.3 ml / min. The chromatography was analyzed by UV detection (214 nm) and purity was evaluated as the area of ​​the main peak divided by the area of ​​all peaks × 100%.

[0134] result For Formulation 72, purity (as a percentage) at time zero and after storage for up to 6 months at 5° C. and 25° C., and after storage for up to 3 months at 30° C. and 37° C. is shown in Table 9. Purity loss is also provided as the difference (in percentage points) between the purity after storage and the purity at time zero.

[0135] The data in Table 9 show the stability of the formulations of the invention after 3 and 6 months at 5°C, 25°C, 30°C, and 37°C, respectively. [Table 16]

[0136] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. 1. A liquid pharmaceutical composition comprising: an insulin derivative containing at least one aryl boron moiety, such as an aryl boronic acid or aryl boroxol; methionine.

2. 10. The liquid pharmaceutical composition of claim 1, further comprising glycerol, or a sugar alcohol, such as mannitol or sorbitol, either alone or in combination.

3. 3. The liquid pharmaceutical composition of claim 2, comprising mannitol.

4. 3. The liquid pharmaceutical composition of claim 2, comprising mannitol and glycerol.

5. The liquid pharmaceutical composition according to any one of claims 1 to 4, wherein the pH is within the range of 5.0 to 8.

0.

6. The liquid pharmaceutical composition according to any one of claims 1 to 5, further comprising a phenolic preservative or a mixture of phenolic preservatives.

7. 7. The liquid pharmaceutical composition according to any one of claims 1 to 6, wherein the insulin derivative is the insulin derivative of Example 1, or a pharma- ceutically acceptable salt thereof, or the insulin derivative of Example 2, or a pharma- ceutically acceptable salt thereof.

8. The composition, The insulin derivative of Example 2, or a pharma- ceutically acceptable salt thereof; Glycerol, Mannitol, 8. The liquid pharmaceutical composition according to any one of claims 1 to 7, comprising:

9. 9. The liquid pharmaceutical composition of claim 8, wherein the composition is substantially free of zinc.

10. The composition, 0.6-4.8 mM of the insulin derivative of Example 2, or a pharma- ceutically acceptable salt thereof; 30 mM methionine; 152 mM glycerol; 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; The liquid pharmaceutical composition according to any one of claims 1 to 9, having a pH of 6.

6.

11. The composition, 1.2 mM of the insulin derivative of Example 2, or a pharma- ceutically acceptable salt thereof; 30 mM methionine; 152 mM glycerol; 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; 11. The liquid pharmaceutical composition of claim 10, having a pH of 6.

6.

12. 1. A liquid pharmaceutical composition comprising: 1.2 mM of the insulin derivative of Example 2, or a pharma- ceutically acceptable salt thereof; 30 mM methionine; 152 mM glycerol; 40 mM mannitol, 25 mM phenol, 25 mM meta-cresol; A liquid pharmaceutical composition having a pH of 6.

6.

13. A liquid pharmaceutical composition according to any one of claims 1 to 12 for use as a medicament.

14. 13. A liquid pharmaceutical composition according to any one of claims 1 to 12 for use in the prevention or treatment of diabetes, including type 1 diabetes and type 2 diabetes.

15. A method for treating diabetes, such as type 1 diabetes and type 2 diabetes, in a subject, comprising administering to the subject a liquid pharmaceutical composition according to any one of claims 1 to 12.

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