THERMOSTATIC FORMULATION OF HUMAN INSULIN A21G

Description

[0001] The present invention, as defined in the claims, relates to a composition in the form of an injectable aqueous solution, having a pH between 7.2 and 8.0, comprising at least human insulin A21G. This composition exhibits improved physical and / or chemical stability, particularly at 40 and 50°C. Human insulin and its analogs, produced by genetic engineering since the early 1980s, have revolutionized the treatment of diabetic patients requiring insulin therapy.

[0002] Observance (also called " compliance " and treatment can be further improved by the choice of insulins, rapid or basal, as well as by the choice of injection devices, such as pre-filled pens or pumps.

[0003] In most developed countries, access to insulin is easy and a person with diabetes can, in general, lead a "normal" life.

[0004] All currently marketed insulins must be stored at a temperature between 2 and 8°C. After first use, most insulins can be stored for approximately 1 month at a maximum temperature of 30°C (Grajower et al. Diabetes Care 2003, 26,2665-2669).

[0005] When using a pump-type injection device, the insulin reservoir may come into contact with a surface exceeding 30°C. This is particularly true when using an implantable pump with a reservoir placed under the skin. One of the problems that the present invention aims to solve is the development of a thermally stable insulin formulation, especially at temperatures above 30°C, and in particular at approximately 37°C.

[0006] Furthermore, in some regions of the world, the average temperature is around 25°C and can very regularly exceed 30°C, even locally reaching 40°C to 45°C during the day. The use of terracotta pots, common in some hot countries, can reduce the average temperature by 3 to 8°C, but this is often insufficient for storing insulin-based preparations for more than a month (Ogle et al. Diabet. Med. 2016, 33, 1544-1553).

[0007] Thus, in some cases, insulin is difficult for a portion of the population to access due to its price and problems maintaining the cold chain. This is particularly acute in regions with low living standards, hot or even tropical climates, and / or those far from centers with infrastructure that allows for proper insulin storage.

[0008] One of the challenges to be addressed is therefore improving access to insulin for a greater number of patients through an insulin formulation with enhanced high-temperature stability, particularly compared to currently marketed insulins. It is also advantageous if this formulation is inexpensive. A thermally stable insulin formulation over a relatively long period can also be useful in areas without electricity, in war zones, or in the event of a natural disaster.

[0009] It is known that the chemical and physical stability of insulins can be partly dependent on their primary structure or sequence, pH and the nature of the excipients.

[0010] As examples chosen from commercial products, the following products can be cited.

[0011] Human insulin, as sold in vials (also called "vials") and formulated at pH 7.4, cannot maintain a stable formulation at 40°C for more than three weeks due to its physical instability. Beyond this period, it no longer meets the European Pharmacopoeia specifications for insulin. In particular, the chemical aggregate content rapidly exceeds 2%, and recovery falls below 90%. At 50°C, human insulin is not stable for more than a few days based on chemical stability. Physical stability at 50°C is less than two weeks. The manufacturer recommends storing this product for a maximum of 31 days at a temperature not exceeding 30°C.

[0012] Insulin lispro, an analog of human insulin formulated at pH 7.4, with a lysine residue at position B28 and a proline residue at position B29, is a prandial insulin marketed under the name Humalog®, and is physically stable for less than one week at 50°C. The manufacturer recommends storing this product for a maximum of 28 days at a temperature not exceeding 30°C.

[0013] Insulin glulisine, an analog of human insulin formulated at pH 7.3, with a lysine residue at position B3 and a glutamic acid residue at position B29, is a prandial insulin marketed under the name Apidra®. The manufacturer recommends storing this product for a maximum of 28 days at a temperature not exceeding 25°C.

[0014] Insulin glargine, an analog of human insulin formulated at pH 4.0, which has a glycine residue at position A21 and two arginine residues at positions B30 and B31, is a basal insulin marketed under the name Lantus®, and is physically stable for less than one week at 50°C. The manufacturer recommends storing this product for a maximum of 28 days at a temperature not exceeding 30°C.

[0015] Numerous studies have investigated the role of excipients in improving the stability of insulin formulations. They specifically describe the significant role that zinc and preservatives can play in this regard. For a review on insulin stability, see the following publications by Brange J. et al.: Insulin Structure and Stability. In: Wang YJ, Pearlman R. (eds) Stability and Characterization of Protein and Peptide Drugs. Pharmaceutical Biotechnology, vol 5. Springer, Boston, MA) 1993, Chemical Stability of Insulin. 1. Pharmaceutical Research 1992, 9, 715-726, Chemical Stability of Insulin. 2. Pharmaceutical Research 1992, 9, 727-734, Chemical Stability of Insulin. 3. Acta. Pharm. North. 1992, 4, 149-158, Chemical Stability of Insulin. 4. Acta. Pharm. North. 1992, 4, 209-222.

[0016] Known pathways of chemical degradation of human insulin include the deamidation of asparagines at B3 and A21, the formation of covalent aggregates, and the cleavage and rearrangement of disulfide bonds. These degradation mechanisms are dependent, at least in part, on the pH of the formulation and the presence of excipients. Deamidation at A21 is predominant at acidic pH, while deamidation at B3 is more significant at neutral pH.

[0017] Regarding physical stability, it is known that insulins can form fibrils or aggregates, particularly when heated or shaken. These fibrils or aggregates can be immunogenic and should therefore be avoided.

[0018] Obtaining physically and chemically stable insulin over extended periods at temperatures above 30°C, particularly at 40 and / or 50°C, is challenging because chemical degradation and the formation of fibrils or aggregates result from distinct phenomena whose mechanisms, kinetics, and triggering factors are not fully understood. Indeed, even though the main impurities resulting from insulin degradation are known, many impurities remain unidentified and, consequently, originate from unknown mechanisms.

[0019] Understanding some of the degradation mechanisms allows us to find solutions to improve physical and / or chemical stability. However, significant improvements with practical applications, particularly those leading to physical stability exceeding one month at 40°C and enabling at least postprandial insulin therapy, have not yet been described.

[0020] In this context, a prandial insulin formulation exhibiting good physical and / or chemical stability at temperatures above 30°C, such as 40 and 50°C, would be highly desirable. In particular, a "thermostatable" prandial insulin formulation with the following characteristics would be a significant advantage over all currently marketed products: physical and chemical stability of at least one month at 40°C and at least two weeks at 50°C, good compatibility with antimicrobial preservatives, possible use in both vials and / or cartridges, pharmacokinetic profile similar to that of human insulin at the same concentration, and ease of manufacture.

[0021] Surprisingly, the applicant has developed a composition, in the form of an injectable aqueous solution, comprising human insulin A21G at a pH between 7.2 and 8.0 exhibiting physical and / or chemical stability at 40°C for at least 3 weeks and / or at 50°C for at least one week.

[0022] Note that the accuracy of the pH value measurement is ± 0.1.

[0023] According to one embodiment, the pH of the compositions is between 7.2 and 8.0.

[0024] According to another embodiment, the pH of the compositions is between 7.2 and 7.8.

[0025] According to yet another embodiment, the pH of the compositions is between 7.2 and 7.6.

[0026] According to one embodiment, the composition is physically and / or chemically stable for at least one month (i.e., 4 weeks) at 40°C and at least two weeks at 50°C.

[0027] According to one embodiment, the composition is physically stable for at least one month at 40°C and at least two weeks at 50°C.

[0028] According to one embodiment, the composition is chemically stable for at least one month (i.e., 4 weeks) at 40°C and at least two weeks at 50°C.

[0029] According to one embodiment, the composition is chemically stable for at least 4 weeks at 40°C.

[0030] According to one embodiment, the composition is chemically stable for at least 8 weeks at 40°C.

[0031] According to one embodiment, the composition is chemically stable for at least 12 weeks at 40°C.

[0032] According to one embodiment, the composition is chemically stable for at least two weeks at 50°C.

[0033] According to one embodiment, the composition is physically stable for at least 8 weeks at 40°C.

[0034] According to one embodiment, the composition is physically stable for at least 12 weeks at 40°C.

[0035] According to one embodiment, the composition is physically stable for at least 16 weeks at 40°C.

[0036] According to one embodiment, the composition is physically stable for at least 4 weeks at 50°C.

[0037] According to one embodiment, the composition is physically stable for at least 6 weeks at 50°C.

[0038] According to one embodiment, the composition is physically stable for at least 8 weeks at 50°C.

[0039] According to one embodiment, the composition is physically stable for at least 12 weeks at 50°C.

[0040] The term "thermostat formulation" means a formulation exhibiting physical and chemical stability of at least 4 weeks at 40°C and at least 2 weeks at 50°C.

[0041] A "chemically stable composition" is defined as a composition with an insulin coverage of at least 90% and an HMWP ("aggregates") level of less than 2%.

[0042] Physically stable composition means compositions that meet the visual inspection criteria described in the European, American and international pharmacopoeias, i.e. compositions that are clear and do not contain visible particles, but are also colorless.

[0043] The term "injectable aqueous solution" refers to water-based solutions that meet the conditions of the EP and US pharmacopoeias.

[0044] The compositions in the form of an injectable aqueous solution according to the invention are clear solutions. A "clear solution" is defined as a composition that meets the criteria described in the American and European Pharmacopoeias concerning injectable solutions. In the US Pharmacopoeia, solutions are defined in Part <1151> referring to the injection ( <1> (referring to <788> according to USP 35 and specified in <788> according to USP 35 and in <787> , <788> And <790> USP 38 (as of 1 August 2014), according to USP 38). In the European Pharmacopoeia, injectable solutions must meet the criteria given in sections 2.9.19 and 2.9.20.

[0045] The term "monthly" or "one month" refers to a period of 4 weeks.

[0046] The term "bi-monthly" refers to a period of 2 weeks.

[0047] In the examples, "dB3 impurity" refers to an insulin in which the asparagine located in the third position of the B chain is deamidated and converted into aspartic acid.

[0048] In the examples, "disoB3 impurity" refers to insulin in which the asparagine located in the third position of the B chain is deamidated and converted into isoaspartic acid.

[0049] Human insulin A21G is an insulin with the same sequence as human insulin, but in which the asparagine at position A21 is mutated to glycine. In other words, this human insulin A21G has an identical sequence to that of human insulin, except at position A21 where a glycine residue replaces the asparagine residue present in human insulin.

[0050] The sequence of chain A is SEQ ID NO: 1 GIVEQCCTSICSLYQLENYCG and the sequence of chain B is SEQ ID NO: 2 FVNQHLCGSHLVEALYLVCGERGFFYTPKT.

[0051] This human insulin A21G is the main metabolite of insulin glargine, marketed under the name Lantus® (SANOFI) (Bolli et al. Diabetes Care 2012, 35, 2626-2630). This insulin has a similar duration of action to human insulin and can therefore be used with meals; it is thus a so-called "prandial" insulin.

[0052] Obtaining a composition in the form of an injectable aqueous solution exhibiting improved physical and chemical stability properties compared to those of prandial insulins described in the prior art at temperatures above 30°C and at a pH close to 7, is remarkable because it is well known to those skilled in the art that physical and chemical stability properties are very difficult to predict, especially in the case of combinations.

[0053] The invention further relates to containers comprising the composition according to the invention. These containers may be vials, cartridges, pre-filled syringes, and reservoirs. These containers may be included or contained within injection pens or insulin pumps.

[0054] The invention also relates to compositions for their use as a medicinal product.

[0055] The invention further relates to compositions for their use in the treatment of diabetes.

[0056] According to one embodiment, this is type I diabetes.

[0057] According to one embodiment, this is type II diabetes.

[0058] The invention also relates to pharmaceutical formulations comprising such compositions.

[0059] The invention also relates to the use of a composition according to the invention.

[0060] The disclosure further relates to a method of treating diabetes comprising the administration of a composition according to the invention. More particularly, it relates to a method comprising the administration of a composition according to the invention as prandial insulin.

[0061] In one respect, it is a treatment method intended to treat human beings.

[0062] The invention also relates to a composition according to the invention intended to be used in a method of treating diabetes characterized in that it is administered as a bolus before meals.

[0063] It relates more specifically to a composition intended for use in a method of treating diabetes characterized in that it is administered as prandial insulin.

[0064] According to one embodiment, human insulin A21G is in hexameric form.

[0065] In this application, 100 U of human insulin A21G corresponds to 3.5 mg.

[0066] In one embodiment, the concentration of human insulin A21G is between 240 and 6000 µM or between 40 and 1000 U / mL.

[0067] In one embodiment, the concentration of human insulin A21G is between 240 and 3000 µM or between 40 and 500 U / mL.

[0068] In one embodiment, the concentration of human insulin A21G is 600 µM or 100 U / mL.

[0069] In one embodiment, the concentration of human insulin A21G is 1200 µM or 200 U / mL.

[0070] In one embodiment, the concentration of human insulin A21G is 1800 µM or 300 U / mL.

[0071] In one embodiment, the concentration of human insulin A21G is 2400 µM or 400 U / mL.

[0072] In one embodiment, the concentration of human insulin A21G is 3000 µM or 500 U / mL.

[0073] In one embodiment, the concentration of human insulin A21G is 3600 µM or 600 U / mL.

[0074] In one embodiment, the concentration of human insulin A21G is 4200 µM or 700 U / mL.

[0075] In one embodiment, the concentration of human insulin A21G is 4800 µM or 800 U / mL.

[0076] In one embodiment, the concentration of human insulin A21G is 5400 µM or 900 U / mL.

[0077] In one embodiment, the concentration of human insulin A21G is 6000 µM or 1000 U / mL.

[0078] In one embodiment, the compositions according to the invention further comprise zinc salts.

[0079] In one embodiment, the zinc is derived from ZnCl2 or ZnO.

[0080] In one embodiment, the compositions according to the invention comprise zinc salts at a concentration of between 50 and 600 µM per 100 U of human insulin A21G.

[0081] In one embodiment, the compositions according to the invention comprise zinc salts at a concentration of between 100 and 500 µM per 100 U of human insulin A21G.

[0082] In one embodiment, the compositions according to the invention comprise zinc salts at a concentration of between 150 and 400 µM per 100 U of human insulin A21G.

[0083] In one embodiment, the compositions according to the invention comprise zinc salts at a concentration of between 200 and 350 µM per 100 U of human insulin A21G.

[0084] In one embodiment, the compositions according to the invention comprise zinc salts at a concentration of between 200 and 300 µM per 100 U of human insulin A21G.

[0085] In one embodiment, the compositions according to the invention comprise zinc salts at a concentration of 230 µM per 100 U of human insulin A21G.

[0086] In one embodiment, the compositions according to the invention further comprise at least one buffer.

[0087] In one embodiment, the compositions according to the invention comprise a buffer selected from the group consisting of a sodium acetate buffer and a Tris buffer, short for trishydroxymethylaminomethane.

[0088] In one embodiment, the compositions according to the invention include a Tris buffer.

[0089] In one embodiment, trihydroxymethylaminomethane is present at a concentration between 2 and 100 mM.

[0090] In one embodiment, trihydroxymethylaminomethane is present at a concentration between 4 and 80 mM.

[0091] In one embodiment, trihydroxymethylaminomethane is present at a concentration between 5 and 50 mM.

[0092] In one embodiment, trihydroxymethylaminomethane is present at a concentration between 6 and 40 mM.

[0093] In one embodiment, trihydroxymethylaminomethane is present at a concentration between 6 and 20 mM.

[0094] In one embodiment, the compositions according to the invention comprise arginine.

[0095] In one embodiment, arginine is present at a concentration between 5 and 100 mM.

[0096] In one embodiment, arginine is present at a concentration between 10 and 90 mM.

[0097] In one embodiment, arginine is present at a concentration between 20 and 80 mM.

[0098] In one embodiment, arginine is present at a concentration between 30 and 70 mM.

[0099] In one embodiment, arginine is present at a concentration between 40 and 60 mM.

[0100] In one embodiment, the compositions according to the invention comprise arginine and a tris buffer.

[0101] In one embodiment, the compositions according to the invention further comprise preservatives.

[0102] In one embodiment, the preservatives are phenolic preservatives.

[0103] In one embodiment, the phenolic preservatives are chosen from the group consisting of m-cresol and phenol, alone or in mixture.

[0104] In one embodiment, the concentration of phenolic preservative(s) is between 10 and 100 mM.

[0105] In one embodiment, the concentration of phenolic preservative(s) is between 15 and 100 mM.

[0106] In one embodiment, the concentration of phenolic preservative(s) is between 20 and 75 mM.

[0107] In one embodiment, the concentration of phenolic preservative(s) is between 20 and 60 mM.

[0108] In one embodiment, the preservative is phenol.

[0109] In one embodiment, the phenol concentration is between 30 and 75 mM.

[0110] In one embodiment, the phenol concentration is between 40 and 60 mM.

[0111] In one embodiment, the phenol concentration is 50 mM.

[0112] In one embodiment, the preservative is m-cresol.

[0113] In one embodiment, the concentration of m-cresol is between 15 and 50 mM.

[0114] In one embodiment, the concentration of m-cresol is between 20 and 30 mM.

[0115] In one embodiment, the concentration of m-cresol is 25 mM.

[0116] In one embodiment, the composition comprises a mixture of m-cresol and phenol.

[0117] In one embodiment, the total concentration of m-cresol and phenol is between 20 and 75 mM.

[0118] According to one embodiment, the composition is devoid of a zinc chelating agent.

[0119] A "zinc chelating agent" is defined as an agent capable of complexing zinc. This agent must have a metal binding stability constant (logK) for zinc of at least 4.5, determined at 25°C.

[0120] The metal bond stability constants listed in the National Institute of Standards and Technology Reference Database 46 (Critically Selected Stability Constants of Metal Complexes) can be used. This database lists the logK constants determined at 25°C. Therefore, the conformity of a chelating agent according to the present invention can be determined based on its bond stability constant for zinc, measured at 25°C, and as described in this database.

[0121] Among the zinc chelating agents, we can mention polydentate organic anions.

[0122] According to one embodiment, the chelating agent is chosen from the following compounds: EDTA (logK = 14.5), citrate (logK = 4.93), EGTA (logK = 12.6), pyrophosphate (logK = 8.71) and alginate (logK = 6.91).

[0123] In another embodiment, the chelating agent is a compound comprising a lone pair of electrons or an electron density that allows interaction with ionic zinc. Such compounds may be polydentate amines. These may be chosen from the following compounds: ethylenediamine, aromatic or heteroaromatic substances, in particular those comprising an imidazole unit, such as histidine (logK 6.91).

[0124] In one embodiment, the compositions according to the invention further comprise a surfactant.

[0125] In one embodiment, the surfactant is chosen from the group consisting of Poloxamer 188, and polysorbates, in particular Tween ®< 20, also called Polysorbate 20, and Tween ®< 80, also called Polysorbate 80.

[0126] In one embodiment, the surfactant is chosen from among the polysorbates.

[0127] In one embodiment the polysorbate concentration is between 4 and 20 µM (4 µM ≤ polysorbate concentration ≤ 20 µM).

[0128] In one embodiment, the surfactant is chosen from Tween ®< 20, also called Polysorbate 20, and Tween ®< 80, also called Polysorbate 80.

[0129] In one embodiment, the surfactant is Tween ®< 20, also known as Polysorbate 20.

[0130] In one embodiment the concentration of Tween ®< 20 is between 4 and 20 µM.

[0131] In one embodiment the concentration of Tween ®< 20 is between 5 and 10 µM.

[0132] In one embodiment the concentration of Tween ®< 20 is 8 µM.

[0133] In one embodiment the concentration Poloxamer 188 is between 0.5 and 2.5 mg / ml.

[0134] In one embodiment, the Poloxamer 188 concentration is between 0.8 and 1.6 mg / ml.

[0135] In one embodiment the concentration Poloxamer 188 is 1.2 mg / ml.

[0136] According to one embodiment, the composition is free of polysorbate 80.

[0137] According to one embodiment, the composition is devoid of at least one zinc chelating agent and polysorbate 80.

[0138] In one embodiment, the composition includes Tween ®< 20 and Tris buffer.

[0139] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 7.2 to 7.6, comprising at least human insulin A21G, a surfactant, and a buffer. In one embodiment, this buffer is Tween® < 20 and Tris buffer.

[0140] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 7.4, comprising at least human insulin A21G, a surfactant, and a buffer. In one embodiment, this buffer is Tween® < 20 and Tris buffer.

[0141] The compositions according to the invention may further include additives such as tonicity agents, also called osmotic agents.

[0142] In one embodiment, the tonic agents are chosen from the group consisting of glycerin, sodium chloride, mannitol, trehalose and glycine.

[0143] In one embodiment, the tonic agent is glycerin.

[0144] In one embodiment, the composition comprises an osmotic agent concentration of between 20 and 500 mM.

[0145] In one embodiment, the concentration of the tonic agent is between 30 and 400 mM.

[0146] In one embodiment, the concentration of the tonic agent is between 50 and 250 mM.

[0147] In one embodiment, the composition according to the invention contains from 3.5 mg / mL to 10.5 mg / mL of human insulin A21G, 50 mM of phenol, 50 to 250 mM of glycerin at a pH of 7.4. This composition may further contain from 200 to 900 µM of zinc.

[0148] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 50 mM of phenol, 150 to 250 mM of glycerin, and 10 mM of TRIS buffer at pH 7.4. This composition may further contain 200 to 250 µM of zinc. This composition may also include polysorbate 20, particularly at 8 µM, or poloxamer 188, particularly at a concentration of 1.2 mg / mL.

[0149] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 50 mM of phenol, 50 to 100 mM of glycerin, and 50 mM of TRIS buffer at pH 7.4. This composition may further contain 200 to 250 µM of zinc. This composition may also include polysorbate 20, particularly at 8 µM, or poloxamer 188, particularly at a concentration of 1.2 mg / mL.

[0150] In one embodiment, the composition according to the invention contains 7.0 mg / mL of human insulin A21G, 50 mM of phenol, and 150 to 250 mM of glycerin, at a pH of 7.4. This composition may further contain 400 to 500 µM of zinc. This composition may also include polysorbate 20, in particular at 8 µM, or poloxamer 188, in particular at a concentration of 1.2 mg / mL.

[0151] In one embodiment, the composition according to the invention contains 10.5 mg / mL of human insulin A21G, 50 mM of phenol, and 150 to 250 mM of glycerin, at a pH of 7.4. This composition may further contain 600 to 750 µM of zinc. This composition may also include polysorbate 20, in particular at 8 µM, or poloxamer 188, in particular at a concentration of 1.2 mg / mL.

[0152] In one embodiment, the composition according to the invention contains from 3.5 mg / mL to 10.5 mg / mL of human insulin A21G, 25 mM of m-cresol, 150 to 250 mM of glycerin at a pH of 7.4. This composition may further contain from 200 to 1000 µM of zinc.

[0153] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 50 mM of phenol, 150 to 250 mM of glycerin, and 10 mM of TRIS buffer at pH 7.4. This composition may further contain 200 to 250 µM of zinc. This composition may also include polysorbate 20, particularly at 8 µM, or poloxamer 188, particularly at a concentration of 1.2 mg / mL.

[0154] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 25 mM of m-cresol, 150 to 250 mM of glycerin, and 10 mM of TRIS buffer at pH 7.4. This composition may further contain 200 to 250 µM of zinc. This composition may also include polysorbate 20, particularly at 8 µM, or poloxamer 188, particularly at a concentration of 1.2 mg / mL.

[0155] In one embodiment, the composition according to the invention contains 7.0 mg / mL of human insulin A21G, 25 mM of m-cresol, and 150 to 250 mM of glycerin, at a pH of 7.4. This composition may further contain 400 to 500 µM of zinc. This composition may also include polysorbate 20, in particular at 8 µM, or poloxamer 188, in particular at a concentration of 1.2 mg / mL.

[0156] In one embodiment, the composition according to the invention contains 10.5 mg / mL of human insulin A21G, 25 mM of m-cresol, and 150 to 250 mM of glycerin, at a pH of 7.4. This composition may further contain 600 to 750 µM of zinc. This composition may also include polysorbate 20, in particular at 8 µM, or poloxamer 188, in particular at a concentration of 1.2 mg / mL.

[0157] According to one embodiment, the compositions described above comprise polysorbate 20, in particular at 8 µM.

[0158] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analogue.

[0159] In one embodiment, GLP-1, GLP-1 analogues, or GLP-1 RA are chosen from the group consisting of exenatide (Byetta ®< , ASTRA-ZENECA), lixisenatide (Lyxumia ®< , SANOFI), their analogues or derivatives and their pharmaceutically acceptable salts.

[0160] Exenatide and lixisenatide, respectively described in applications US2004 / 0023871 and WO0104156, are generally considered to be GLP-1 receptor agonists.

[0161] In one embodiment, GLP-1, GLP-1 analogue, or GLP-1 RA is exenatide or Byetta ®<, its analogues or derivatives and their pharmaceutically acceptable salts.

[0162] In one embodiment, GLP-1, GLP-1 analogue, or GLP-1 RA is lixisenatide or Lyxumia ®<, its analogues or derivatives and their pharmaceutically acceptable salts.

[0163] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH between 7.2 and 8.0, comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analogue.

[0164] According to one embodiment, said GLP-1 receptor agonist is exenatide.

[0165] According to another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0166] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 7.2 to 7.6, comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analogue.

[0167] According to one embodiment, said GLP-1 receptor agonist is exenatide.

[0168] According to another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0169] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 7.4, comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analogue.

[0170] According to one embodiment, said GLP-1 receptor agonist is exenatide.

[0171] According to another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0172] In one embodiment, the composition further comprises a nicotine compound or one of its derivatives.

[0173] In one embodiment, the composition includes nicotinamide.

[0174] In one embodiment, the concentration of nicotinamide is between 10 and 160 mM.

[0175] In one embodiment, the concentration of nicotinamide is between 20 and 150 mM.

[0176] In one embodiment, the concentration of nicotinamide is between 40 and 120 mM.

[0177] In one embodiment, the concentration of nicotinamide is between 60 and 100 mM.

[0178] In one embodiment, the pharmaceutical composition further comprises at least one absorption promoter selected from absorption promoters, diffusion promoters or vasodilating agents, alone or in mixture.

[0179] Absorption promoters include, but are not limited to, surfactants, for example, bile salts, fatty acid salts, or phospholipids; nicotinic agents, such as nicotinamides, nicotinic acids, niacin, niacinamide, vitamin B3 and their salts; pancreatic trypsin inhibitors; magnesium salts; polyunsaturated fatty acids; didecanoyl phosphatidylcholine; aminopolycarboxylates; tolmetin; sodium caprate; salicylic acid; oleic acid; linoleic acid; eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); benzyl acid; nitric oxide donors, for example, 3-(2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine, N-ethyl-2-(1-ethyl-hydroxy-2-1-nitrosohydrazino)-ethanamine, or S-nitroso-N-acetylpenicillamine; bile acids, glycine in its conjugated form with a bile acid; sodium ascorbate, potassium ascorbate;sodium salicylate, potassium salicylate, acetylsalicylic acid, salicylosalicylic acid, aluminum acetylsalicylate, choline salicylate, salicylamide, lysine acetylsalicylate; exalamide; diflunisal; ethenzamide; EDTA; alone or in mixture.

[0180] In one embodiment, the pharmaceutical composition further comprises at least one diffusion promoter. Examples of diffusion promoters include, but are not limited to, glycosaminoglycanases, for example hyaluronidase.

[0181] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator.

[0182] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator causing hyperpolarization by blocking calcium ion channels.

[0183] In one embodiment, the vasodilator causing hyperpolarization by blocking calcium ion channels is adenosine, an endothelium-derived hyperpolarizing agent, a phosphodiesterase type 5 (PDE5) inhibitor, a potassium channel-opening agent, or any combination of these agents.

[0184] In one embodiment, the pharmaceutical composition further comprises at least one cAMP-mediated vasodilator.

[0185] In one embodiment, the pharmaceutical composition further comprises at least one cGMP-mediated vasodilator.

[0186] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator selected from the group comprising vasodilators that act by causing hyperpolarization by blocking calcium ion channels, cAMP-mediated vasodilators, and cGMP-mediated vasodilators.

[0187] At least one vasodilator is chosen from the group including nitric oxide donors (e.g., nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, amyl nitrate, erythrityl tetranitrate, and nitroprusside); prostacyclin and its analogues (e.g., epoprostenol sodium, iloprost, epoprostenol, treprostinil, or selexipag); histamine, 2-methylhistamine, 4-methylhistamine; 2-(2-pyridyl)ethylamine, 2-(2-thiazolyl)ethylamine; papaverine, papaverine hydrochloride; minoxidil; dipyridamole; hydralazine; adenosine, adenosine triphosphate; uridine trisphosphate; GPLC; L-carnitine; arginine; prostaglandin D2; potassium salts; and in some cases, α1 and α2 receptor antagonists, for example, prazosin, phenoxybenzamine, phentolamine, dibenamine, moxisylyte hydrochloride and tolazoline), betazole, dimaprit;β2 receptor agonists, for example, isoproterenol, dobutamine, albuterol, terbutaline, aminophylline, theophylline, caffeine; alprostadil, ambrisentan; cabergoline; diazoxide; dihydralazine mesylate; diltiazem hydrochloride; enoximone; flunarizine hydrochloride; Ginkgo biloba extract; levosimendan; molsidomine; naftidrofuryl acid oxalate; nicorandil; pentoxifylline; phenoxybenzamine chloride; piribedil base; piribedil mesylate; regadenoson monohydrate; riociguat; sildenafil citrate, tadalafil, vardenafil hydrochloride trihydrate; trimetazidine hydrochloride; nitroglycerin; verapamil hydrochloride; endothelin receptor antagonists, for example avanafil and bosentran monohydrate;and calcium channel blockers, for example, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, isradipine, efonidipine, felodipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, prandipine; alone or in combination.

[0188] The compositions according to the invention may further include all excipients conforming to the Pharmacopoeias, in particular EP and / or US, and compatible with the insulins used at the concentrations of use.

[0189] In one embodiment, the composition can be in solid or lyophilized form. This composition can then be used to reconstitute a solution or formulation.

[0190] The proposed methods of administration are intravenous, subcutaneous, intradermal or intramuscular.

[0191] According to a particular embodiment, the method of administration is the subcutaneous route.

[0192] Transdermal, oral, nasal, vaginal, ocular, buccal, and pulmonary routes of administration are also being considered.

[0193] The invention also relates to a pump, implantable or transportable, comprising a composition according to the invention.

[0194] The invention further relates to the use of a composition according to the invention intended to be placed in a pump, implantable or transportable.

[0195] In one embodiment, the pump is an implantable pump placed under the skin. Examples of such pumps include those that allow for intraperitoneal insulin administration.

[0196] In one embodiment, the pump delivering the composition according to the invention via the intraperitoneal route is the Medtronic MiniMed MIP2007 pump.

[0197] The invention also relates to single-dose formulations.

[0198] In one embodiment, the formulations are in the form of an injectable solution.

[0199] The preparation of a composition according to the invention has the advantage of being able to be carried out by simple mixing of an aqueous solution of human insulin A21G or in lyophilized form.

[0200] The composition of the mixture is adjusted with excipients such as glycerin, m-cresol and / or phenol, zinc chloride, and polysorbate 20 (Tween® < 20). This addition can be carried out by adding concentrated solutions of said excipients.

[0201] In one embodiment, the compositions are characterized in that said compositions are clear at a pH of 7.4 and exhibit physical and chemical stability of at least one month at 40°C.

[0202] In one embodiment, the compositions are characterized in that said compositions exhibit a physical and chemical stability superior to that of a commercial prandial insulin.

[0203] Insulin and insulin analogs can be obtained by recombinant DNA technology methods in bacteria such as Escherichia coli or yeasts such as Saccharomyces cerevisiae (see, for example, G. Walsh, Appl. Microbiol. Biotechnol. 2005, 67, 151–159). Typically, a proinsulin is produced and then digested by enzymes such as trypsin and carboxypeptidase B to obtain the desired sequence.

[0204] For the production of human insulin A21G, proinsulin is coded to glycine A21, and after digestion by trypsin and carboxypeptidase B, the desired insulin is obtained. A procedure is described by Kohn et al., in Peptides 2007, 28, 935-948.

[0205] In one embodiment, the composition according to the invention is characterized in that insulin A21G is obtained from human insulin A21G, B31R, B32R (insulin glargine).

[0206] In one embodiment, the composition according to the invention is characterized in that insulin A21G is obtained from human insulin A21G, B31R, B32R (insulin glargine) reacted with rat carboxypeptidase B at an insulin / carboxypeptidase ratio of 500 to 2000, at a pH of 7.5 to 8.5, and at a temperature of 20 to 30 °C for 10 to 20 hours. The product can then be purified. This purification can be carried out by liquid chromatography.

[0207] A route for preparing human insulin A21G can therefore be obtained by removing the two arginines from insulin glargine by digestion with a carboxypeptidase B. After enzymatic digestion, human insulin A21G can be purified by chromatography and then isolated by lyophilization or by crystallization using conventional methods. Description of the figures Figure 1:

[0208] There figure 1 represents the pharmacodynamics of composition B5-1 at t0 (i.e. stored at 4°C) and at t12 weeks (composition B5-1 "aged" at 40°C over 12 weeks) and of composition B4, commercial formulation of human insulin Humulin ®< .

[0209] In this figure the x-axis represents the time after injection (in minutes) and the y-axis represents the blood glucose level (in mg / dL). Figure 1:

[0210] Represented at the Figure 1the pharmacodynamic results obtained with composition B5-1 administered at t0 (curve drawn with squares) and at t12 weeks at 40 °C (curve drawn with triangles) compared with the results obtained with composition B4 (reference human insulin) (curve drawn with dotted lines and circles).

[0211] The following examples illustrate, in a non-limiting manner, the invention. Examples Chemistry Example A1: Preparation of human insulin A21G (insulin A21G)

[0212] Insulin glargine (5 g; Gan & Lee Pharmaceuticals) was mixed with carboxypeptidase B enzyme (Reference 08039852001; Sigma-Aldrich) at a ratio of 1 / 500 (w / w) at pH 8.0 (pH adjusted by adding trihydroxymethylaminomethane buffer), the final insulin glargine concentration being approximately 4 mg / ml.

[0213] The solution was left under gentle stirring at 25 °C for 17 h. The mixture was then purified by liquid chromatography, dialyzed against 0.01 N hydrochloric acid and then lyophilized.

[0214] Human insulin A21G was obtained with a purity of 98% and a yield of approximately 90%.

[0215] The molar mass of insulin A21G measured by mass spectrometry (Maldi-Tof) is 5752 Da. B Compositions Example B2 : Rapid-acting insulin analogue composition (Humalog®) at 100 U / mL

[0216] This composition is a commercial lispro insulin solution marketed by ELI LILLY under the name Humalog®. This product is a rapid-acting insulin analog. The excipients in Humalog® are metacresol (3.15 mg / mL), glycerol (16 mg / mL), disodium phosphate (1.88 mg / mL), zinc oxide (to provide 0.0197 mg of zinc ion / mL), sodium hydroxide, and hydrochloric acid for pH adjustment (pH 7-7.8) and water. Example B3 : Composition of long-acting insulin analogue (Lantus®) at 100 U / mL

[0217] This composition is a commercial solution of insulin glargine marketed by SANOFI under the name Lantus®. This product is a long-acting insulin analog. The excipients in Lantus® are zinc chloride (30 µg / mL), m-cresol (2.7 mg / mL), glycerol (20 mg / mL), polysorbate 20 (16 µM), sodium hydroxide, hydrochloric acid for pH adjustment (pH 4), and water. Example B4 : Composition of human insulin (Humulin®< R) at 100 IU / mL

[0218] This composition is a commercial solution of human insulin from ELI LILLY sold under the name Humulin®< R. This product is human insulin. The excipients in Humulin®< R are glycerol, metacresol, sodium hydroxide, hydrochloric acid for pH adjustment (pH 7.0-7.8), and water. Example B5: Preparation of insulin A21G compositions at concentrations between 100 and 300 U / mL and excipients

[0219] Compositions of human insulin A21G were prepared according to the following protocol.

[0220] An aqueous solution of human insulin A21G at pH 7.3 ± 0.2 is prepared from the lyophilized powder obtained in Example A1 and an amount of sodium hydroxide or hydrochloric acid necessary for pH adjustment. This solution is added to a mixture of concentrated solutions of the excipients (zinc chloride, preservative, osmotic agent, trihydroxymethylaminomethane, arginine). A surfactant solution is then added to the mixture. The pH is measured and adjusted, if necessary, between 7.1 ± 0.1 and 8.0 ± 0.1 by adding concentrated solutions of sodium hydroxide or hydrochloric acid. A clear solution free from visible particles is obtained. This inspection is carried out according to the recommendations of the European Pharmacopoeia (EP 2.9.20) and the United States Pharmacopoeia (USP). <790> The preservative is either phenol (25 to 50 mM) or meta-cresol (25 mM).The concentrations of the stock solutions are chosen to obtain a trihydroxymethylaminomethane concentration of 0 to 50 mM and / or an arginine concentration of 0 to 50 mM in the final composition. The concentration of the osmotic agent stock solution, in this case glycerol, is chosen to obtain a glycerol concentration between 50 and 210 mM in the final composition. The surfactant used may be Tween® < 20, at a concentration of at least 8 µM, or Poloxamer® < 188, at a concentration of 1.2 mg / mL, in the final composition. The concentration of human insulin A21G, C IHA21G, is between 100 and 300 U / mL. The concentration of zinc chloride (µM) corresponds to 2 to 3 times the concentration of human insulin A21G (U / ml), i.e. C ZnCl2 (µM) = 2-3 C IHA21G (U / ml).

[0221] These compositions are presented in Table 1 below. Example B6: Preparation of human insulin A21G compositions at concentrations between 100 and 300 U / mL (corresponding to 3.5 and 10.5 mg / ml) and excipients at pH 4

[0222] These compositions were prepared according to the protocol described in Example B5, using an aqueous solution of human insulin A21G at pH 4.0 ± 0.2, prepared from the lyophilized powder obtained in Example A1 and an amount of sodium hydroxide or hydrochloric acid sufficient to adjust the pH. The concentration of trihydroxymethylaminomethane is between 0 and 50 mM in the final composition. The concentrations of the other excipients are similar to those mentioned in Example B5.

[0223] These compositions are presented in Table 1 below. Table 1: Insulin A21G compositions prepared according to protocols B5 (B5-1 to B5-13) and B6 (B6-1 to B6-3) Composition Insulin (mq / mL) Tris (mM) Additive Surfactant [Zn] (µM) Glycerol (mM) pH (±0.1) B5-1 3,5 10 Phenol (50 mM) Tween ® < 20 (8 µM) 230 200 7,4 B5-2 3,5 10 Phenol (50 mM) Poloxamer ® < 188 (1.2 mg / mL) 230 184 7,4 B5-3 3,5 - m-cresol (25 mM) Poloxamer ® < 188 (1.2 mg / mL) 300 184 7,4 B5-4 3,5 10 Phenol (50 mM) Tween ® < 20 (8 µM) 230 200 7,0 B5-5 3,5 10 Phenol (50 mM) Tween ® < 20 (8 µM) 230 200 7,2 B5-6 3,5 10 Phenol (50 mM) Tween ® < 20 (8 µM) 230 200 7,5 B5-7 3,5 10 Phenol (50 mM) Tween ® < 20 (8 µM) 230 200 8,0 B5-8 10,5 - m-cresol (25 mM) Poloxamer ® < 188 (1.2 mg / mL) 900 184 7,4 B5-9 3,5 - Phenol (50 mM) Arginine (50 mM) Tween ® < 20 (8 µM) 230 184 7,4 B5-10 7 10 Phenol (50 mM) Tween ® < 20 (8 µM) 460 184 7,4 B5-11 10,5 10 Phenol (50 mM) Tween ® < 20 (8 µM) 690 184 7,4 B5-12 3,5 50 Phenol (50 mM) Arginine (40 mM) Tween ® < 20 (8 µM) 230 60 7,4 B6-1 3,5 - m-cresol (25 mM) Poloxamer ® < 188 (1.2 mq / mL) 300 184 4,0 B6-2 3,5 - m-cresol (25 mM) Poloxamer ® < 188 (1.2 mg / mL) - 184 4,0 B6-3 10,5 - m-cresol (25 mM) Tween ® < 20 (8 µM) - 184 4,0 B5-13 10,5 10 Phenol (50 mM) Tween ® < 20 (8 µM) 690 200 7,4 C Stability Example C1 : Physical stability study at 50 °C of the prepared compositions in comparison with commercial insulins at a concentration of 100 U / mL in vials (vials).

[0224] At least five 3 mL vials, each filled with 1 mL of the composition, are placed vertically in an oven maintained at 50 °C. The vials are visually inspected at least weekly to detect the appearance of visible particles or turbidity. This inspection is performed according to the recommendations of the European Pharmacopoeia (EP 2.9.20) and the United States Pharmacopoeia (USP). <790> The vials are subjected to illumination of at least 2000 lux and are observed against a white background and a black background. The number of weeks of stability corresponds to the duration during which at least half of the vials remain clear and do not contain particles visible upon inspection.

[0225] The physical stability results obtained with different compositions are presented in the table below. Table 2: Results of physical stability studies at 50 °C of compositions B5-1 to B5-3, B5-9, B5-12, B6-1 to B6-3 and B2 to B4. Composition Insulin Insulin concentration (mg / mL) pH 50 °C B5-1 A21G 3,5 7,4 No particles at 15 weeks B5-2 A21G 3,5 7,4 No particles at 15 weeks B5-3 A21G 3,5 7,4 No particles at 8 weeks B5-9 A21G 3,5 7,4 No particles at 9 weeks B5-12 A21G 3,5 7,4 No particles at 6 weeks B6-1 A21G 3,5 4,0 Particles at 1 week B6-2 A21G 3,5 4,0 Particles at 1 week B6-3 A21G 3,5 4,0 Particles at 4 weeks B2 (Humalog ®< ) Insulin Lispro 3,5 7,4 Particles at 1 week B3 (Lantus ®< ) Insulin glargine 3,6 4,0 Particles at 1 week B4 (Humulin ®< R) Human insulin 3,5 7,4 Particles at 2 weeks

[0226] Human insulin A21G compositions at pH 4.0 (B6-1 to B6-3) and commercial formulations (B2 to B4) are less stable at 50°C than compositions containing human insulin A21G at pH 7.4 (B5-1 to B5-3, B5-9, and B5-12). These latter compositions (B5-1 to B5-3, B5-9, and B5-12) provide physical stability for at least 6 weeks at 50°C. Example C2 : Chemical stability study at 40 °C and 50 °C.

[0227] The compositions, presented in the following table, are placed in an oven maintained at 40 °C or 50 °C.

[0228] The compositions are analyzed at 4, 8, and 12 weeks at 40 °C and at 2 weeks at 50 °C. At each time point, a sample is analyzed by RP-HLPC-UV (214 nm) with a C18 column and a phosphate / acetonitrile buffer elution gradient in the presence of sodium heptanesulfonate to determine, by surface percentage, the proportion of deamidated dB3 / disoB3 products as well as other impurities (denoted as total impurities). At each time point, a sample is also analyzed by SE-HPLC-UV (276 nm) with a silica column coated with 80 Å pores and a water / acetonitrile / trifluoroacetic acid mobile phase to determine, by surface percentage, the proportion of high molecular weight products (HMWP).

[0229] The results are given as percentages in the table below. Table 3: Chemical stability results of solutions B5-1 to B5-3, B5-9, and B5-12 and commercial compositions of Humulin® human insulin. Composition Insulin (mg / mL) Decontamination products TO 40 °C 4S 40 °C 8S 40 °C 12S 50 °C 2S B5-1 A21G dB3 / disoB3 0,4 1,4 2,8 4,2 1,8 (3,5) HMWP 0,1 0,3 0,7 0,9 0,3 Total Imp. ∗< 0,3 1,9 3,7 5,0 2,2 B5-2 A21G dB3 / disoB3 0,1 - - - 2,0 (3,5) HMWP 0,2 0,5 Total Imp. ∗< 0,3 2,5 B5-3 A21G dB3 / disoB3 0,2 3,0 5,7 8,7 4,2 (3,5) HMWP 0,1 0,4 1,0 1,9 0,5 Total Imp. ∗< 0,6 1,4 4,1 5,2 2,3 B5-9 A21G dB3 / disoB3 0,1 - - - 1,8 (3,5) HMWP 0,1 0,1 Total Imp. ∗< 0,3 2,5 B5-12 A21G dB3 / disoB3 0,1 1,4 2,8 4,1 1,6 (3,5) HMWP 0,1 0,2 0,2 0,3 0,1 Total Imp. ∗< 0,4 2,3 4,3 5,6 2,5 B4 Human insulin (3.5) dB3 / disoB3 2,5 11,7 - - 6,4 HMWP 0,3 2,1 - - 3,4 Total Imp. ∗< 1,4 12,8 - - 6,5 "*<" by "total imp." means all impurities except dB3 / disoB3 and HMWP. "-" means not measured.

[0230] The commercial formulation Humulin®< R (B4) was analyzed after 4, 8, and 12 weeks at 40°C and after 2 weeks at 50°C. At these intervals, this formulation no longer met the specifications of the European Pharmacopoeia in terms of physical stability (see above), nor in terms of impurity percentage. Formulations B5-1 to B5-3, B5-9, and B5-12 are significantly more stable. Example C3 : Effect of pH on the physical and chemical stability of compositions including human insulin A21G

[0231] Formulations B5-4 to B5-7 have an identical composition to formulation B5-1, except for the pH. Table 4: Results of the effect of pH on the physical and chemical stability of solutions B5-4 to B5-7 Composition pH Vial 40 °C Degradation products after 4 weeks at 40 °C (relative to t0) B5-4 7,0 ± 0,1 Particles observed immediately at t0 - B5-5 7,2 ± 0,1 No particles at 11 weeks dB3 / disoB3 1,3 % HMWP 0,1 % Total Imp. ∗< 1,8 % B5-6 7,5 ± 0,1 No particles at 11 weeks dB3 / disoB3 1,4 % HMWP 0,2 % Total Imp. ∗< 1,9 % B5-7 8,0 ± 0,1 No particles at 11 weeks dB3 / disoB3 1,6 % HMWP 0,3 % Total Imp. ∗< 2,4 % * By "total impurities" we mean all impurities excluding dB3 / disoB3 and HMWP. "-" means not measured.

[0232] The compositions exhibit good stability between 7.2 and 8.0. It should be noted that even better stability at 40 °C is obtained in a pH range between 7.2 and 7.5. Example C4: Physical and chemical stability of human insulin A21G at 100 and 300 U / mL (i.e. 3.5 and 10.5 mg / ml), at pH 7.4.

[0233] Table 5: Results of the physical stability at 50 °C of the compositions at 100 and 300 U / mL of insulin A21G (B5-3 and B5-8). Formulation Insulin A21G (mg / mL) 50 °C B5-3 3,5 No particles at 8 weeks B5-8 10,5 No particles at 4 weeks. Particles at 5 weeks. Table 6: Results of chemical stabilities at 40 and 50 °C of the compositions at 100 and 300 U / mL of insulin A21G (B5-3 and B5-8). Composition Insulin A21G (mg / mL) Degradation products TO 40°C 4S 40°C 8S 40°C 12S 50°C 2S B5-3 3,5 dB3 / disoB3 0,2 3,0 5,7 8,9 4,2 HMWP 0,1 0,4 1,0 1,9 2,3 Total Imp. 0,6 1,4 4,1 5,2 0,5 B5-8 10,5 dB3 / disoB3 0,4 1,9 3,8 5,9 2,7 HMWP 0,1 0,2 0,5 0,9 2,0 Total Imp. 1,2 1,3 3,1 3,9 0,4 * By "total impurities" we mean all impurities excluding dB3 / disoB3 and HMWP

[0234] The results are expressed as percentages in the table below.

[0235] The results presented above show that composition B5-8 (U300) exhibits good physical stability and excellent chemical stability. Example C5: Physical and chemical stability of human insulin A21G at 100 and 300 U / mL (i.e. 3.5 and 10.5 mg / ml) in cartridges (3 mL), at 40°C.

[0236] Compositions B5-1 and B5-13 of human insulin A21G are inserted into 3 mL cartridges and placed in an oven at 40°C under static conditions. The cartridges are visually inspected every two weeks for the appearance of visible particles or turbidity. This inspection is performed according to the recommendations of the European (EP 2.9.20) and American (USP) pharmacopoeias. <790> ) described for vial solutions. Thus, the cartridges are subjected to illumination of at least 2000 Lux and are observed against a white background and a black background. The number of weeks of stability corresponds to the duration during which at least half of the cartridges remain clear and do not contain particles visible upon inspection.

[0237] The cartridges are analyzed at 4, 8, and 12 weeks at 40 °C. At each time point, one cartridge is sampled and an aliquot is analyzed by RP-HLPC-UV (214 nm) with a C18 column and a phosphate / acetonitrile buffer gradient elution in the presence of sodium heptanesulfonate to determine, by surface percentage, the proportion of deamidated dB3 / disoB3 products as well as other impurities (denoted as total impurities). At each time point, an aliquot is also analyzed by SE-HPLC-UV (276 nm) with a silica-coated column with 80 Å pores and a water / acetonitrile / trifluoroacetic acid mobile phase to determine, by surface percentage, the proportion of high molecular weight products (HMWP).

[0238] The results are given as percentages in the table below. Table 6a: Results of physical stability at 40 °C of the compositions at 100 and 300 U / mL of insulin A21G (B5-1 and B5-13). Composition Insulin A21G (mg / mL) 40 °C B5-1 3,5 No particles at 12 weeks B5-13 10,5 No particles at 10 weeks Table 6b: Results of the chemical stabilities of solutions B5-1 and B5-13. Composition Insulin (mg / mL) Degradation products T0 40 °C 4S 40 °C 8S 40 °C 11S 40 °C 12S B5-1 A21G dB3 / disoB3 0,1 1,4 2,6 - 3,8 (3,5) HMWP 0,2 0,3 0,4 - 0,7 Total Imp. ∗< 0,6 1,9 3,1 - 4,5 B5-13 A21G 10.5) dB3 / disoB3 0,3 - - 3,2 - HMWP 0,2 - - 0,5 - Total Imp. ∗< 0,4 - - 4,0 - "*<" by "total impurities" means all impurities excluding dB3 / disoB3 and HMWP

[0239] The results are expressed as percentages in the table below.

[0240] The results presented above show that compositions B5-1 (U100) and B5-13 (U300) exhibit excellent physical and chemical stability. D Pharmacodynamics Example D1 Protocol for measuring the pharmacodynamics of insulin solutions

[0241] Domestic pigs weighing approximately 50 kg, previously catheterized via the jugular vein, are fasted for 2.5 hours before the start of the experiment. Within the hour preceding insulin injection, three blood samples are taken to determine the baseline glucose level.

[0242] The injection of M1 insulin formulations or human insulin at a dose of 0.2 IU / kg is carried out subcutaneously on the flank of the animal using an insulin pen (Novo, Sanofi or Lilly) equipped with a 31 G needle.

[0243] Blood samples are then taken at the following times: 4, 8, 12, 16, 20, 30, 40, 50, 60, 70, 80, 100, 120, 150, and 180 minutes. After each sample is taken, the catheter is flushed with a diluted heparin solution. A drop of blood is collected to determine blood glucose levels using a glucometer. Average blood glucose curves, expressed in mg / dL, are then plotted.

[0244] Example D2 : Pharmacodynamic results of the A21G B5-1 insulin solution at time zero (t0, i.e. stored at 4°C) and after 12 weeks at 40°C and of a human insulin solution. Table 7: Compositions used for pharmacodynamics Composition Human insulin A21G (U / mL) Number of pigs B5-1 100 - 12 B4 - 100 11

[0245] The pharmacodynamic results obtained with composition B5-1 are illustrated in the FIG. 1Analysis of these profiles indicates that similar hypoglycemic effects are obtained with the B5-1 composition administered at t0 (curve plotted with squares) and at t12 weeks at 40°C (curve plotted with triangles). Furthermore, the B5-1 composition administered at t0 or at t12 weeks at 40°C exhibits a hypoglycemic effect similar to that obtained with the B4 composition (reference human insulin) (dotted curve plotted with circles). SEQUENCE LISTING

[0246] <110> ADOCIA <120> Thermostable formulation of insulin A21G <130> B1458PC00 <150> FR1856479 <151> 2018-07-13 <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 21 <212> PRT <213> Homo sapiens <400> 1 <210> 2 <211> 30 <212> PRT <213> Homo sapiens <400> 2

Claims

1. Composition in the form of an injectable aqueous solution, the pH of which is between 7.2 and 8.0 (7.2 ≤ pH ≤ 8.0) comprising at least A21G human insulin.

2. Composition according to claim 1, characterized in that the concentration of A21G human insulin is between 40 and 1000 U / mL (40 U / mL ≤ concentration of A21G human insulin ≤ 1000 U / mL).

3. Composition according to claim 1 or 2, characterized in that the concentration of A21G human insulin is 100 U / mL.

4. Composition according to any one of claims 1 to 3, characterized in that the concentration of A21G human insulin is 300 U / mL.

5. Composition according to any one of claims 1 to 4, characterized in that the zinc salt concentration is between 50 and 600 µM per 100 U / mL of A21G insulin.

6. Composition according to any one of the preceding claims, characterized in that the composition further comprises a phenolic preservative, in particular chosen from phenol and m-cresol.

7. Composition according to claim 6, characterized in that the concentration of phenolic preservative is between 15 and 100 mM.

8. Composition according to any one of the preceding claims, characterized in that it further comprises a surfactant.

9. Composition according to claim 8, characterized in that the surfactant is chosen from polysorbates.

10. Composition according to claim 9, characterized in that the surfactant is chosen from polysorbate 20 or "Tween® 20".

11. Composition according to any one of the preceding claims, characterized in that it further comprises arginine.

12. Composition according to any one of the preceding claims, characterized in that it comprises trishydroxymethylaminomethane.

13. Composition according to the preceding claim 12, characterized in that it comprises trishydroxymethylaminomethane at a concentration between 2 and 100 mM (2 mM ≤ trishydroxymethylaminomethane concentration ≤ 100 mM).

14. Composition according to any one of the preceding claims, intended for use in a method for treating diabetes, characterized in that it is administered as a bolus before meals.

15. Composition according to any one of the preceding claims, intended for use in a method for treating diabetes, characterized in that it is administered as prandial insulin.