Compositions in the form of an injectable aqueous solution comprising at least human insulin a21g and a prandial action glucagon suppressor

Abstract

The invention relates to a composition in the form of an injectable aqueous solution having a pH of 3.5 to 4.4 and comprising at least human insulin A21G and at least one prandial action glucagon suppressor. In one embodiment, the prandial action glucagon suppressor is selected from the group consisting of an amylin analogue, or an amylin receptor agonist, or a GLP-1 analogue, or a GLP-1 receptor agonist (GLP-1 RA). In one embodiment, the prandial action glucagon suppressor is an amylin analogue or an amylin receptor agonist. In one embodiment, the prandial action glucagon suppressor peptide is pramlintide. The invention also relates to a method for obtaining human insulin A21G comprising at least a step of reacting human insulin A21G, B31R, B32R (glargine insulin) with rat carboxypeptidase B, with an insulin / carboxypeptidase ratio of between 500 and 2000, at a pH of 7.5 to 8.5 and a temperature of 20 to 30°C for 10 to 20 hours.

Description

! COMPOSITIONS IN THE FORM OF AN AQUEOUS SOLUTION INJECTABLE COMPRISING AT LEAST HUMAN INSULIN A21G AND A GLUCAGON SUPPRESSANT WITH PRANDIAL ACTION

[0001] The invention relates to injection therapies of a composition comprising at least human insulin A21G, with prandial action, and a glucagon suppressor, in particular with prandial action, for treating diabetes and enabling improved control of postprandial hyperglycemia.

[0002] Type 1 diabetes is an autoimmune disease that leads to the destruction of the beta cells of the pancreas. These cells are known to produce insulin, whose main role is to regulate glucose uptake in peripheral tissues (Gerich 1993 Control of glycaemia). Consequently, patients with type 1 diabetes suffer from chronic hyperglycemia and must administer exogenous insulin to control this hyperglycemia. Insulin therapy has drastically changed the life expectancy of these patients.

[0003] Currently, patients with type 1 diabetes use two types of insulin: short-acting prandial insulins to control their blood glucose levels at mealtimes and long-acting basal insulins to control their blood glucose levels throughout the day and night. Several types of short-acting insulin exist, characterized by their onset of action. For example, regular human insulin has a delayed action compared to rapid-acting insulin analogs, such as insulin lispro (Humalog®, ELI LILLY) or insulin aspart (Novorapid®, NOVO NORDISK). Thus, regular human insulin should be administered on average 30 minutes before meals, while insulin analogs can be administered 15 minutes before or at mealtimes.Furthermore, it is accepted that analog insulins lead to better control of postprandial blood glucose than human insulin, which explains why the vast majority of patients in Europe and the United States now use rapid analog insulins whose action is shorter than that of human insulin.

[0004] However, post-meal blood glucose control achieved with these exogenous prandial insulins is not optimal, even with rapid-acting insulin analogs. This is partly due to the fact that these patients, unlike healthy individuals, produce glucagon after a meal, leading to the release of some of the glucose stored in the liver. This glucose production glucagon-mediated exacerbates the postprandial hyperglycemia problem in these patients and leads to excessive insulin use.

[0005] This problem of regulating postprandial blood glucose is quite similar for patients with type 2 diabetes treated with insulin in cases where their disease has led to a very significant loss of their ability to produce insulin and amylin.

[0006] Glucagon suppressors, particularly peptides and / or hormones, have been shown to inhibit glucagon production after a meal, leading to significantly improved postprandial blood glucose control. For example, amylin, a hormone produced by pancreatic beta cells and whose production is also deficient in patients with type 1 diabetes, plays a key role in regulating postprandial blood glucose. Amylin, also known as islet amyloid polypeptide or IAPP, is a 37-amino-acid peptide that is co-stored and co-secreted with insulin (Schmitz 2004 Amylin Agonists). This peptide is described as blocking glucagon production by pancreatic alpha cells.Thus, insulin and amylin have complementary and synergistic roles, since insulin helps to reduce the concentration of glucose in the blood while amylin helps to reduce the entry of endogenous glucose into the blood by inhibiting the production, or secretion, of endogenous glucagon.

[0007] However, human amylin has properties that are incompatible with pharmaceutical requirements in terms of solubility and stability (Goldsbury CS, Cooper GJ, Goldie KN, Muller SA, Saafi EL, Gruijters WT, Misur MP, Engel A, Aebi U, Kistler J: Polymorphic fibrillar assembly of human amylin. J Struct Biol 119: 17-27, 1997). Amylin is known to form amyloid fibers that lead to the formation of plaques that are insoluble in water. Therefore, it was necessary to develop an analog to overcome these solubility issues.

[0008] Amylin developed an amylin analog, pramlintide, to overcome the lack of physical stability of human amylin. This product, marketed as Symlin®, was approved by the FDA in 2005 for the treatment of type 1 and type 2 diabetes, as an adjunct to insulin therapy. It must be administered subcutaneously three times a day, within an hour before meals, to improve postprandial blood glucose control, given its relatively short half-life of a few hours. This peptide is formulated at H 4.0 and is described to fibrillate when the pH of the solution is greater than 5.5. Variants of analogues are described in US patent 5,686,411.

[0009] Amylin analogs or amylin receptor agonists, also known as "prandial" or "short-acting" amylin receptor agonists, mimic the effects of amylin but with a longer half-life. These amylin derivatives, used to control blood glucose levels at mealtimes, can have a half-life of less than 8 hours. This half-life is the apparent elimination half-life after subcutaneous injection in humans. The half-life of these amylin analogs or amylin receptor agonists can be less than 5 hours, specifically less than 4 hours, or even less than 3 hours.

[0010] However, compositions containing amylin or an amylin analogue, and in particular pramlintide, can cause certain adverse effects in patients. Specifically, these compositions can cause nausea in some patients.

[0011] GLP-1, another physiological peptide, is also described as playing a role similar to that of amylin in suppressing glucagon secretion following a meal. GLP-1 is also known for its role as an insulin secretagogue and is therefore particularly effective as an adjunct to insulin, especially in patients with type 2 diabetes. These actions are glucose-dependent, which minimizes the risk of hypoglycemia. GLP-1 receptor agonists (RAs) are currently approved only for patients with type 2 diabetes.

[0012] Similarly, human GLP-1 cannot be used as a therapeutic treatment due to its extremely short half-life. Various GLP-1 derivatives, known as GLP-1 receptor agonists (GLP-1 RAs) or GLP-1 analogs, replicate the effects of GLP-1 while exhibiting a longer half-life. These GLP-1 derivatives can be divided into three groups according to their respective half-lives: short-acting, or prandial, derivatives to control blood glucose levels at mealtimes (half-life less than 8 hours); daily-acting derivatives to meet daily requirements (half-life greater than 8 hours, or even 10 hours); and weekly-acting derivatives to meet weekly requirements (half-life greater than 48 hours). Regarding GLP-1 RAs with prandial action, the two peptides approved to date are exenatide (Byetta®, ASTRA-ZENECA, two administrations per day) and lixisenatide (Lyxumia®, SANOFI, one administration per day).These two GLP-1 RAs are formulated at a pH close to 4 and are to be administered within one hour before meals, like pramlintide.

[0013] One of the main difficulties for insulin-dependent patients using these various short-acting glucagon-suppressing compounds is related to the number of additional injections, which can range from one to three injections per day beyond the two to four insulin injections. It is therefore essential to be able to combine a prandial insulin with a prandial-acting glucagon suppressant in a solution to allow the use of these complementary compounds alongside insulin without increasing the treatment burden for patients. Furthermore, this would allow for a more precise mimicry of physiological processes, since both hormones are secreted in response to a meal, thus improving postprandial blood glucose control, particularly better control of postprandial hyperglycemia, and therefore better management of diabetes.

[0014] However, prandial insulins and these peptides of interest are not compatible in aqueous solution. Indeed, prandial insulins have optimal chemical stability at a pH close to 7, whereas amylin or GLP-1 derivatives are not physically and chemically stable at a pH close to physiological pH.

[0015] This difficulty led to the design of a pump containing two reservoirs to keep prandial insulin separate from the amylin derivative. US patent application US2016 / 001002 filed by Roche describes such a pump to enable the co-administration of amylin and prandial insulin with a single medical device. However, it would be preferable to be able to mix these hormones in aqueous solution to utilize existing medical devices that are simpler and / or less prone to malfunction.

[0016] Another solution to the problem of mixing these hormones in aqueous solutions involves replacing the water with an organic solvent. XERIS patent application WO2013067022 describes compositions that include amylin and insulin in solution in an organic solvent. However, the use of an organic solvent such as DMSO raises safety concerns for patients treating chronic diseases such as diabetes. Furthermore, organic solvents can be problematic with injection devices, particularly by dissolving some of their components. Therefore, it is preferable to develop these combinations in aqueous solution form.

[0017] The problem of stability in aqueous solution was also circumvented by preparing mixtures in solid form. NOVO NORDISK's patent application EP2060268 describes formulations of insulin and pramlintide in atomized powder form for nasal application. However, the preferred route of administration and the The most commonly used route to date is the subcutaneous route, for which ready-to-use aqueous solutions are required.

[0018] Another approach has been to combine prandial insulin with an amylin derivative at the pH at which this amylin derivative is physically stable. US patent application US20090192075 from Biodel describes a liquid composition comprising human insulin, pramlintide, and a zinc chelator at pH 4.0. This application describes a rapid action of human insulin due to the presence of a zinc chelator. However, since human insulin is known to be chemically unstable at acidic pH, this technique is unlikely to meet the criteria of the EP and / or US Pharmacopoeia.

[0019] An alternative approach involves modifying the structure of prandial insulins to improve their stability at acidic pH levels. Application WO2007104786 exemplifies compositions comprising the rapid-acting analog insulins A21G, B28D desB30 and A21G, B28E desB30, which are soluble at acidic pH.

[0020] Application WO2007104786 also presents rapid insulin compositions, including B28D (insulin aspart) at neutral pH and in the presence of surfactant, and in particular glycerophosphate derivative, more specifically dimyristoyl glycerophosphoglycerol (DMPG), lead to ThT measured stabilities much higher than those of the rapid insulin analogue compositions A21G, B28D desB30 and A21G, B28E desB30 at acidic pH.

[0021] Prior art compositions comprising prandial insulin and pramlintide in combination mostly describe different types of prandial insulins, however their examples are directed towards compositions comprising so-called rapid prandial insulin analogues, the latter being considered more efficient than human insulin.

[0022] Surprisingly, the applicant demonstrated that a composition containing human insulin A21G, known as "regular", i.e. an insulin differing from human insulin only by the replacement of the asparagine residue at position 21 on the A chain with a glycine residue, less rapid than so-called "rapid" insulin analogues, in association with a prandial glucagon suppressant at a pH ranging from 3.5 to 4.4 in aqueous solution, provided better control of postprandial blood glucose than with a so-called rapid prandial insulin analogue.

[0023] Furthermore, the plaintiff has demonstrated, surprisingly, that said composition containing human insulin A21G, in combination with a suppressant of glucagon with prandial action at a pH ranging from 3.5 to 4.4 in aqueous solution exhibited physical and chemical stability compatible with pharmaceutical requirements and superior to solutions proposed in the prior art.

[0024] Obtaining a composition in the form of an injectable aqueous solution exhibiting better control of postprandial hyperglycemia and improved physical and chemical stability properties compared to those described in the prior art, is remarkable because it is well known to those skilled in the art that in the case of combinations the pharmacokinetics of the products in combination and the physical and chemical stability properties are very difficult to predict.

[0025] Furthermore, a composition is also sought that reduces, or even eliminates, all or part of the undesirable effects that can be generated by the active ingredients.

[0026] The invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.4, comprising at least human insulin A21G known as regular and at least one prandial-acting glucagon suppressant.

[0027] According to one embodiment, the prandial-acting glucagon suppressant is an amylin analogue or an amylin receptor agonist, a GLP-1 analogue or a GLP-1 receptor agonist, also called GLP-1 RA.

[0028] The applicant observed that the formulation according to the invention at pH between 3.5 and 4.4 has pharmacokinetic properties compatible with use at mealtimes and allows better control of postprandial blood glucose.

[0029] Furthermore, the applicant demonstrated that these formulations led to a slowing of pramiintide absorption. This is notably characterized by a significantly delayed peak plasma concentration (tmax) of pramiintide and / or by a significantly reduced early plasma exposure to pramiintide (AUCo-3omin) compared to the administration of pramiintide alone.

[0030] This slowing down helps to reduce or even eliminate the side effects of pramiintide, particularly with regard to nausea.

[0031] The invention also relates to the use of a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.4, comprising at least human insulin A21G and a glucagon suppressor, in particular with prandial action, to improve postprandial blood glucose control.

[0032] 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.

[0033] 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 to improve postprandial blood glucose control.

[0034] The invention also relates to a composition according to the invention intended for use in a method of treating diabetes characterized in that it is administered to improve postprandial blood glucose control and to reduce the adverse effects of pramlintide.

[0035] 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 allows for a reduction in insulin-induced food intake.

[0036] According to one embodiment, the reduction in food intake concerns the period from the injection to 4 hours after the injection.

[0037] According to one embodiment, the reduction in food intake concerns the period from the injection to 3 hours after the injection.

[0038] According to one embodiment, the reduction in food intake concerns the period from the injection to 2 hours after the injection.

[0039] According to one embodiment, the reduction in food intake concerns the period from the injection to 1 hour after the injection.

[0040] The invention also relates to stable pharmaceutical formulations comprising such compositions.

[0041] The requirements for obtaining an injectable pharmaceutical formulation for the treatment of diabetes include: a physically and chemically stable aqueous liquid formulation for at least two weeks, or even one month at 30°C (multiple uses) and at least one year, or even two years at 5°C, compatibility with antimicrobial preservatives.

[0042] Similarly, human insulin A21G formulations with a GLP-1 analogue or a GLP-1 receptor agonist, also called a GLP-1 RA, for example exenatide or lixisenatide, at pH levels between 3.5 and 4.4, have physical and chemical stability allowing the development of a liquid formulation that is stable for at least two weeks, or even one month at 30°C, and at least one year, or even two years, at 5°C.

[0043] Regarding stability, the classical method for measuring the stability of proteins or peptides involves measuring fibril formation using Thioflavin T, also known as ThT. This method allows for the measurement, under temperature and agitation conditions that accelerate the phenomenon, of the latency period before fibril formation by measuring the increase in fluorescence. The compositions according to the invention have a latency period before fibril formation significantly longer than those described in the literature. The compositions according to the invention exhibit physical and chemical stability far superior to that described in the prior art using commercial prandial insulins.

[0044] In one embodiment, the formulations according to the invention have a latency time measured by ThT of at least 8 hours.

[0045] In one embodiment, the formulations according to the invention have a latency time measured by ThT of at least 10 hours.

[0046] In one embodiment, the formulations according to the invention have a latency time measured by ThT of at least 15 hours.

[0047] In one embodiment, the formulations according to the invention have a latency time measured by ThT of at least 20 hours.

[0048] In one embodiment, the formulations according to the invention have a latency time measured by ThT of at least 25 hours.

[0049] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.4, comprising at least human insulin A21G and an amylin receptor agonist or an amylin analog. In one embodiment, this is pramlintide.

[0050] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.2, comprising at least human insulin A21G and an amylin receptor agonist or an amylin analog. In one embodiment, this is pramlintide.

[0051] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.8 to 4.2, comprising at least human insulin A21G and an amylin receptor agonist or an amylin analog. In one embodiment, this is pramlintide.

[0052] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 4.0, comprising at least human insulin A21G and an amylin receptor agonist or an amylin analogue. In one embodiment, this is pramlintide.

[0053] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.4, comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0054] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.2, comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0055] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.8 to 4.2, comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0056] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 4.0, comprising at least human insulin A21G and a GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0057] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.4, comprising at least human insulin A21G, an amylin receptor agonist or an amylin analog, and a GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide. In yet another embodiment, said amylin receptor agonist or amylin analog is pramlintide.

[0058] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.2, comprising at least human insulin A21G, at least one amylin receptor agonist or amylin analog, and at least one GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide. In yet another embodiment, said amylin receptor agonist or amylin analog is pramlintide.

[0059] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 3.8 to 4.2, comprising at least human insulin A21G, at least one amylin receptor agonist or an amylin analog, and at least one GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide. In yet another embodiment, said amylin receptor agonist or amylin analog is pramlintide.

[0060] In one embodiment, the invention relates to a composition in the form of an injectable aqueous solution, having a pH of 4.0, comprising at least human insulin A21G, at least one amylin receptor agonist or an amylin analog, and at least one GLP-1 receptor agonist or a GLP-1 analog. In one embodiment, said GLP-1 receptor agonist is exenatide. In another embodiment, said GLP-1 receptor agonist is lixisenatide. In yet another embodiment, said amylin receptor agonist or amylin analog is pramlintide.

[0061] It is particularly interesting to combine human insulin A21G in aqueous solution with an amylin analogue, an amylin receptor agonist or GLP-1 and with a GLP-1 analogue, or a GLP-1 receptor agonist because this so-called "triple" combination makes it possible in particular to potentiate the effects of each hormone and to reduce the doses of each of them.

[0062] The compositions in the form of an injectable aqueous solution according to the invention are clear solutions. "Clear solution" means compositions that meet the criteria described in the American and European pharmacopoeias concerning injectable solutions. In the US Pharmacopoeia, solutions are defined in section <1151> referring to injection (<1>) (referring to <788> according to USP 35 and further specified in <788> according to USP 35 and in <787>). <788> And <790> USP 38 (from 1 er 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.

[0063] In this application, amylin as referred to refers to the compounds described in US patents 5,124,314 and 5,234,906. "Analog," when used by reference to a peptide or protein, is understood to mean a compound in which one or more constituent amino acid residues of the primary sequence have been substituted by other amino acid residues and / or in which one or more constituent amino acid residues have been deleted and / or in which one or more constituent amino acid residues have been added. The percentage of homology permitted for this definition of an analog is 50%. In the case of amylin, an analog may, for example, be derived from the primary amino acid sequence of amylin by substituting one or more natural, non-natural, or peptidomimetic amino acids.

[0064] Exenatide and lixisenatide, respectively described in applications US2004 / 0023871 and WO0104156, are generally considered to be GLP-1 receptor agonists. In one embodiment, the prandial-acting glucagon suppressant is pramlintide (Symlin®) marketed by ASTRAZENECA AB.

[0065] In one embodiment, GLP-1, GLP-1 analogues, or GLP-1 RAs are referred to as "short-acting" or "prandial". "Short-acting" or "prandial" means GLP-1, GLP-1 analogues, or GLP-1 RAs, whose apparent elimination half-life after subcutaneous injection in humans is less than 8 hours, in particular less than 5 hours, preferably less than 4 hours, or even less than 3 hours, such as exenatide and lixisenatide.

[0066] In one embodiment, GLP-1, GLP-1 analogues, or GLP-1 RAs are chosen from the group consisting of exenatide (Byetta®, ASTRAZENECA), lixisenatide (Lyxumia®, SANOFI), their analogues or derivatives and their pharmaceutically acceptable salts.

[0067] 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.

[0068] 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.

[0069] In one embodiment, the concentration of human insulin A21G is between 240 and 3000 μM or 40 and 500 U / mL.

[0070] In one embodiment, the concentration of human insulin A21G is 600 μM or 100 U / mL.

[0071] In one embodiment, the concentration of human insulin A21G is 1200 μM or 200 U / mL.

[0072] In one embodiment, the concentration of human insulin A21G is 1800 μM or 300 U / mL.

[0073] In one embodiment, the concentration of human insulin A21G is 2400 μM or 400 U / mL.

[0074] In one embodiment, the concentration of human insulin A21G is 3000 μM or 500 U / mL.

[0075] In the present application, 100 U / ml of human insulin A21G corresponds to 3.5 mg / ml.

[0076] In one embodiment, the concentration of pramiintide is from 0.32 to 5 mg / m L.

[0077] In one embodiment, the concentration of pramiintide is from 0.4 to 3 mg / mL.

[0078] In one embodiment, the concentration of pramiintide is from 0.5 to 2 mg / mL.

[0079] In one embodiment, the concentration of pramiintide is from 0.5 to 1.5 mg / mL.

[0080] In one embodiment, the concentration of pramiintide is from 0.6 to 1 mg / mL.

[0081] In one embodiment, the concentration of pramiintide is 1.0 mg / ml.

[0082] In one embodiment, the concentration of pramiintide is 0.6 mg / ml.

[0083] In one embodiment, the concentration of exenatide is from 10 to 1000 pg / ml.

[0084] In one embodiment, the concentration of exenatide is from 10 to 500 mg / ml.

[0085] In one embodiment, the concentration of exenatide is between 20 and 400 pg / ml.

[0086] In one embodiment, the concentration of exenatide is between 20 and 300 pg / ml.

[0087] In one embodiment, the concentration of exenatide is from 30 to 300 pg / ml. In another embodiment, the concentration of exenatide is from 30 to 150 pg / ml.

[0088] In one embodiment, the concentration of exenatide is between 40 and 150 pg / ml.

[0089] In one embodiment, the concentration of exenatide is between 40 and 80 μς / πιΙ.

[0090] In one embodiment, the concentration of exenatide is 50 pg / ml.

[0091] In one embodiment, the concentration of lixisenatide is from 20 to 1000 pg / ml.

[0092] In one embodiment, the concentration of lixisenatide is from 20 to 800 pg / ml.

[0093] In one embodiment, the concentration of lixisenatide is between 40 and 600 pg / ml.

[0094] In one embodiment, the concentration of lixisenatide is between 60 and 600 pg / ml.

[0095] In one embodiment, the concentration of lixisenatide is between 60 and 300 pg / ml.

[0096] In one embodiment, the concentration of lixisenatide is between 80 and 300 pg / ml.

[0097] In one embodiment, the concentration of lixisenatide is between 80 and 160 pg / ml.

[0098] In one embodiment, the concentration of lixisenatide is 100 pg / ml.

[0099] In one embodiment, the concentration of exenatide, its analogues or derivatives and their pharmaceutically acceptable salts is in the range of 0.01 to 1.0 mg per 100 U of insulin. [000100] In one embodiment, the concentration of exenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.01 to 0.5 mg per 100 U of insulin. [000101] In one embodiment, the concentration of exenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.02 to 0.4 mg per 100 U of insulin. [000102] In one embodiment, the concentration of exenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.03 to 0.3 mg per 100 U of insulin. [000103] In one embodiment, the concentration of exenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.03 to 0.2 mg per 100 U of insulin. [000104] In one embodiment, the concentration of exenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.03 to 0.15 mg per 100 U of insulin. [000105] In one embodiment, the concentration of exenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.05 mg per 100 U of insulin. [000106] In one embodiment, the concentration of lixisenatide, its analogues or derivatives and their pharmaceutically acceptable salts is in the range of 0.01 to 1 mg per 100 U of insulin. [000107] In one embodiment, the concentration of lixisenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.01 to 0.5 mg per 100 U of insulin. [000108] In one embodiment, the concentration of lixisenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.02 to 0.4 mg per 100 U of insulin. [000109] In one embodiment, the concentration of lixisenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.03 to 0.3 mg per 100 U of insulin. [000110] In one embodiment, the concentration of lixisenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.04 to 0.2 mg per 100 U of insulin. [000111] In one embodiment, the concentration of lixisenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.04 to 0.15 mg per 100 U of insulin. [000112] In one embodiment, the concentration of lixisenatide, its analogues or derivatives and their pharmaceutically acceptable salts is 0.1 mg per 100 U of insulin. [000113] In one embodiment, the compositions according to the invention are made by mixing solutions of amylin analogues or amylin receptor agonists, and solutions of GLP-1, GLP-1 analogue or GLP-1 RA receptor agonist in volume ratios in a range of 10 / 90 to 90 / 10. [000114] In one embodiment, the compositions according to the invention further comprise zinc salts at a concentration of 0 to 800 μM per 100 U of insulin. [000115] In one embodiment, the compositions according to the invention further comprise zinc salts at a concentration of 0 to 500 μM per 100 U of insulin. [000116] In one embodiment, the compositions according to the invention further comprise zinc salts at a concentration of 100 to 500 μM per 100 U of insulin. [000117] In one embodiment, the compositions according to the invention further comprise zinc salts at a concentration of 200 to 400 μM per 100 U of insulin. [000118] In one embodiment, the compositions according to the invention further comprise zinc salts at a concentration of 300 μM per 100 U of insulin. [0001 19] In one embodiment, the compositions according to the invention further comprise buffers. [000120] In one embodiment, the compositions according to the invention comprise a buffer selected from the group consisting of a sodium acetate buffer and Tris. [000121] In one embodiment, the compositions according to the invention further comprise preservatives. [000122] In one embodiment, the preservatives are chosen from the group consisting of m-cresol and phenol, alone or in mixture. [000123] In one embodiment, the concentration of the preservatives is from 10 to 50 mM. [000124] In one embodiment, the concentration of the preservatives is from 10 to 40 mM. [000125] In one embodiment, the compositions according to the invention further comprise a surfactant. [000126] In one embodiment, the surfactant is chosen from the group consisting of Poloxamer 188, T een® 20, also called Polysorbate 20, and Tween® 80, also called Polysorbate 80. [000127] In one embodiment the concentration of Tween® 20 varies from 5 to 50 pg / ml. [000128] In one embodiment the concentration of Teen® 20 varies from 5 to 25 pg / ml. [000129] In one embodiment, the concentration of Tween® 20 is 10 μM. [000130] The compositions according to the invention may further include additives such as tonicity agents. [000131] In one embodiment, the tonic agents are selected from the group consisting of glycerin, sodium chloride, mannitol, and glycine. [000132] In one embodiment, the compositions according to the invention further comprise an antioxidant. [000133] In one embodiment, the antioxidant is methionine. [000134] 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. [000135] 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-l-(l-methylethyl)-2-nitrosohydrazino)-l-propanamine, A / -ethyl-2-(l-ethyl-hydroxy-2-l-nitrosohydrazino)-ethanamine, or S-nitroso- / V-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. [000136] 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. [000137] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator agent. [000138] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator causing hyperpolarization by blocking calcium ion channels. [000139] 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 thereof. [000140] In one embodiment, the pharmaceutical composition further comprises at least one cAMP-mediated vasodilator. [000141] In one embodiment, the pharmaceutical composition further comprises at least one cGMP-mediated vasodilator. [000142] 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. [000143] At least one vasodilating agent is chosen from the group comprising nitric oxide donors, for example, nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, amyl nitrate, erythritol, tetranitrate, and nitroprusside; prostacyclin and its analogues, for example, 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 triphosphate; 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; the; 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. [000144] In one embodiment, the composition according to the invention contains from 3.5 mg / mL to 10.5 mg / mL of human insulin A21G, 0.6 mg / mL to 3 mg / mL of pramiintide, 25 mM of m-cresol, 184 mM of glycerin at a pH of 4.0. This composition may further contain from 300 to 900 μM of zinc.This composition may also include polysorbate 20, in particular 8 to 10 μM, and especially 8 μM. [000145] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 0.6 to 1 mg / mL of pramiintide, 25 to 30 mM of m-cresol, 150 to 200 mM of glycerin, at a pH of 4.0. This composition may further contain 300 μM of zinc. This composition may further comprise polysorbate 20, in particular 8 to 10 μM, and most particularly 8 μM. [000146] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 0.6 mg / mL of pramiintide, 25 mM of m-cresol, 184 mM of glycerin, at a pH of 4.0. This composition may further contain 300 μM of zinc. This composition may further comprise polysorbate 20, in particular 10 μM. [000147] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 0.6 mg / mL of pramiintide, 25 mM of m-cresol, 184 mM of glycerin, at a pH of 4.0. This composition may further contain 300 μM of zinc. This composition may further comprise polysorbate 20, in particular 8 μM. [000148] In one embodiment, the composition according to the invention contains 3.5 mg / mL of human insulin A21G, 1.0 mg / mL of pramiintide, 25 mM of m-cresol, 184 mM of glycerin, at a pH of 4.0. This composition may further contain 300 μM of zinc. This composition may further comprise polysorbate 20, in particular 8 μM. [000149] In one embodiment, the composition according to the invention contains 7.0 mg / mL of human insulin A21G, 1.2 to 2.0 mg / mL of pramiintide, 25 mM of m- cresol, 150 to 200 mM glycerin, at a pH of 4.0. This composition may further contain 600 μM zinc. This composition may further comprise polysorbate 20, in particular 8 to 10 μM, and especially 8 μM. [000150] In one embodiment, the composition according to the invention contains 7.0 mg / mL of human insulin A21G, 1.2 mg / mL of pramlintide, 25 mM of m-cresol, and 184 mM of glycerin, at a pH of 4.0. This composition may further contain 600 μM of zinc. This composition may also include polysorbate 20, in particular 10 μM. [000151] In one embodiment, the composition according to the invention contains 7.0 mg / mL of human insulin A21G, 1.2 mg / mL of pramlintide, 25 mM of m-cresol, and 184 mM of glycerin, at a pH of 4.0. This composition may further contain 600 μM of zinc. This composition may also include polysorbate 20, in particular 8 μM. [000152] In one embodiment, the composition according to the invention contains 7.0 mg / mL of human insulin A21G, 2.0 mg / mL of pramlintide, 25 mM of m-cresol, and 184 mM of glycerin, at a pH of 4.0. This composition may further contain 600 μM of zinc. This composition may also include polysorbate 20, in particular 8 μM. [000153] In one embodiment, the composition according to the invention contains 10.5 mg / mL of human insulin A21G, 1.8 to 3 mg / mL of pramlintide, 25 mM of m-cresol, and 150 to 200 mM of glycerin, at a pH of 4.0. This composition may further contain 900 μM of zinc. This composition may also include polysorbate 20, in particular 8 to 10 μM, and especially 8 μM. [000154] In one embodiment, the composition according to the invention contains 10.5 mg / mL of human insulin A21G, 1.8 mg / mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerin, at a pH of 4.0. This composition may further contain 900 μM of zinc. This composition may further comprise polysorbate 20, in particular 10 μM. [000155] In one embodiment, the composition according to the invention contains 10.5 mg / mL of human insulin A21G, 1.8 mg / mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerin, at a pH of 4.0. This composition may further contain 900 μM of zinc. This composition may further comprise polysorbate 20, in particular 8 μM. [000156] In one embodiment, the composition according to the invention contains 10.5 mg / mL of human insulin A21G, 3 mg / mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerin, at a pH of 4.0. This composition may further contain 900 μM of zinc. This composition may further comprise polysorbate 20, in particular 8 μM. [000157] 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. [000158] According to one embodiment, the composition may be in solid or lyophilized form. This composition can then be used to reconstitute a solution or a formulation. [000159] The envisaged methods of administration are intravenous, subcutaneous, intradermal or intramuscular. [000160] According to a particular embodiment, the method of administration is the subcutaneous route. [000161] Transdermal, oral, nasal, vaginal, ocular, buccal, and pulmonary routes of administration are also considered. [000162] The invention also relates to a pump, implantable or transportable, comprising a composition according to the invention. [000163] The invention further relates to the use of a composition according to the invention intended to be placed in a pump, implantable or transportable. [000164] The invention also relates to unit-dose formulations. [000165] In one embodiment, the formulations are in the form of an injectable solution. [000166] The preparation of a composition according to the invention has the advantage of being able to be carried out by simply mixing an aqueous solution of an amylin analog or an amylin receptor agonist, and human insulin A21G, either in aqueous solution or in lyophilized form. [000167] If necessary, the composition of the mixture is adjusted with excipients such as glycerin, m-cresol, zinc chloride, and polysorbate 20 (Tween® 20). This addition can be carried out by adding concentrated solutions of said excipients. [000168] In one embodiment, the compositions are characterized in that said compositions exhibit solubility at a pH of 4.0 and physical stability measured by ThT superior to that of a reference composition comprising an amylin analogue or an amylin receptor agonist and a commercial prandial insulin. [000169] ThT is measured according to the protocol described in the examples. [000170] In one embodiment, the compositions are characterized in that said compositions exhibit solubility at a pH of 4.0 and physical stability measured by ThT superior to that of a reference composition comprising a GLP-1, a GLP-1 analogue or a GLP-1 receptor agonist and a commercial prandial insulin. [000171] Insulin and insulin analogs can be obtained by recombinant DNA technology methods in bacteria such as Escherichia coli or in yeasts such as Saccharomyces cerevisiae (see, for example, G. Walsh, Appl. Microbiol. Biotechnol. 2005, 67, 151–159). Generally, a proinsulin is produced which is then digested by enzymes such as trypsin and carboxypeptidase B to obtain the desired sequence. [000172] For the manufacture of human insulin A21G, proinsulin is coded to obtain glycine in the A21 region, 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. [000173] The invention also relates to a process for obtaining human insulin A21G comprising at least one step of reacting human insulin A21G, B31R, B32R (insulin glargine) with rat carboxypeptidase B at an insulin / carboxypeptidase ratio of between 500 and 2000, at a pH of between 7.5 and 8.5, and at a temperature of between 20 and 30°C for 10 to 20 hours. The product can then be purified. This purification can be carried out by liquid chromatography. [000174] Human insulin A21G can therefore be obtained by removing the two arginines from glargine insulin by digestion with a carboxypeptidase B. After enzymatic digestion, human insulin A21G is purified by chromatography and then isolated by lyophilization or by crystallization using conventional methods. Description of the figures: Figure 1: Graphical determination of fibrillation latency time. [000175] Figure 1 shows the graphical determination of the fibrillation latency time on a virtual example. The x-axis represents time in minutes, the y-axis represents fluorescence ThT in arbitrary units (u .a ) and LT represents the latency time, or "lag time". Figure 2: Plasma concentrations of pramlintide and insulin after administration of formulation A21-8 (mean ± standard error) [000176] The squares represent the insulin concentration and the triangles the pramlintide concentration. [000177] The abscissa represents the time in minutes after injection, the ordinate on the left represents the insulin concentration in pmol / l and the ordinate represents the pramlintide concentration in pmol / l corrected from the baseline. Figure 3: Blood glucose after administration of formulation A21-8 (mean ± standard error). [000178] On the x-axis, the time in minutes after injection, and on the y-axis, the blood glucose level as a % of the baseline level. Figure 4: Pramlintide concentrations after administration of formulation A21-9 (curve plotted with squares) and PRA (curve plotted with triangles) (mean ± standard error). [000179] On the x-axis is the time in minutes after injection, and on the y-axis are the baseline-corrected pramlintide concentrations (pre-dose concentrations individually subtracted), in pmol / L. Examples Example 1. Preparation of human insulin A21G [000180] Five grams of insulin glargine (Gan & Lee Pharmaceuticals) are mixed with the enzyme carboxypeptidase B (Reference 08039852001; Sigma-Aldrich) at pH 8.0 (pH adjusted by adding Tris buffer) and left at 25°C for 17 hours, resulting in an insulin glargine concentration of approximately 4 mg / mL. The enzyme / glargine ratio is 1 / 500. The mixture is then purified by liquid chromatography, dialyzed against 0.01N hydrochloric acid, and subsequently lyophilized. Human insulin A21G is obtained with a purity of 98% and a yield of approximately 90%. The molar mass of the insulin, measured by mass spectrometry (MALDI-TOF), is 5752 Da. Human insulin A21G can also be obtained from recombinant technology as described by Kohn et al. (Peptides 2007, 28, 935-948). Example 2. Compositions of prandial insulins and pramiintide, exenatide or lixisenatide at acidic pH. Preparation of a solution of human insulin A21G 100 U / mL (3.5 mg / ml) and pramiintide 1 mg / mL containing m-cresol (25 mM), glycerin (184 mM) and zinc chloride (300 μM) at acidic pH of 3.5 or 4.0. [000181] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin A21G solution (300 U / mL at pH 3.5). A concentrated pramiintide (Ambiopharm) solution (10 mg / mL at pH 4) and a concentrated zinc chloride solution are added to this concentrated human insulin A21G and excipient solution to obtain the desired final composition. The final pH, either 3.5 or 4.0, is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of human insulin A21G 100 U / mL and pramiintide 0.6 mg / mL containing m-cresol (25 mM), glycerin (184 mM) and zinc chloride (300 μM) at acidic pH 4.0. [000182] This solution is prepared in the same way as the solution presented above. Preparation of a solution of human insulin A21G 100 U / mL and pramiintide 1 mg / mL containing m-cresol (25 mM) and glycerin (184 mM) at acidic pH of 4.0. [000183] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin A21G solution (800 U / mL at pH 3.5). A concentrated pramiintide solution (10 mg / mL at pH 4) is added to this concentrated human insulin and excipient solution to obtain the desired final composition. The final pH of 4.0 is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through a 0.22 μm filter and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of human insulin A21G 100 U / mL and pramiintide 1 mg / mL containing m-cresol (25 mM), glycerin (184 mM) and Tween 20 (10 pg / ml) at pH 4. [000184] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin A21G solution (300 U / mL at pH 3.5). A concentrated pramiintide solution (10 mg / mL at pH 4) and a concentrated tvveen 20 solution are Added to this concentrated solution of human insulin A21G and excipients to obtain the desired final composition. The final pH is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of human insulin A21G 100 U / mL and pramlintide 1 mg / mL containing m-cresol (25 mM), glycerin (184 mM), zinc chloride (300 μM) and Tween 20 (10 pg / ml) at pH 4. [000185] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin A21G solution (300 U / mL at pH 3.5). A concentrated pramlintide solution (10 mg / mL at pH 4), a concentrated zinc chloride solution, and a concentrated tween 20 solution are added to this concentrated human insulin A21G solution and excipients to obtain the desired final composition. The final pH is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of human insulin A21G 100 U / mL and exenatide 50 pg / mL containing m-cresol (25 mM), glycerin (184 mM) and zinc chloride (300 pM) at acidic pH. [000186] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin A21G solution (300 U / mL at pH 3.5). A concentrated exenatide (Bachem) solution (10.5 mg / mL at pH 4) and a concentrated zinc chloride solution are added to this concentrated human insulin A21G and excipient solution to obtain the desired final composition. The final pH of 4.0 is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of human insulin A21G 100 U / mL and lixisenatide 100 pg / mL containing m-cresol (25 mM), glycerin (184 mM) and zinc chloride (300 pM) at acidic pH. [000187] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin A21G solution (230 U / mL at pH 3.5). A concentrated lixisenatide (Ambiopharm) solution (10.5 mg / mL at pH 4) and a concentrated zinc chloride solution are added to this concentrated human insulin A21G solution and excipients to obtain the desired final composition. The pH The final pH of 4.0 is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through 0.22 μm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of human insulin A21G 100 U / mL, exenatide 50 pg / mL, and pramlintide 0.6 mg / mL containing m-cresol (25 mM), glycerin (184 mM), and Tween 20 (10 pg / mL) at an acidic pH of 4.0. A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin A21G solution (300 U / mL at pH 3.5). A concentrated pramlintide (Ambiopharm) solution (10 mg / mL at pH 4), a concentrated exenatide (Bachem) solution (10.5 mg / mL at pH 4), and a concentrated tween 20 solution are added to this concentrated human insulin A21G solution and excipients to obtain the desired final composition. The final pH of 4.0 is adjusted to the desired value by adding aqueous NaOH or HCl solution. The resulting solution is clear and homogeneous; it is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of human insulin 100 U / mL and pramlintide 1 mg / mL containing m-cresol (25 mM), glycerin (184 mM) and zinc chloride (300 μM) at acidic pH of 3.5 or 4.0. [000188] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated human insulin solution (Amphastar Pharmaceuticals) (800 U / mL at pH 3.5). A concentrated pramlintide solution (10 mg / mL at pH 4) and a concentrated zinc chloride solution are added to this concentrated human insulin and excipient solution to obtain the desired final composition. The final pH, either 3.5 or 4.0, is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of insulin aspart 100 U / mL and pramlintide 1 mg / mL containing m-cresol (25 mM), glycerin (184 mM) and zinc chloride (300 μM) at acidic pH of 3.5 or 4.0. [000189] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated insulin aspart solution (HEC Pharmaceuticals) (500 U / mL at pH 3). A concentrated pramlintide solution (10 mg / mL at pH 4) and a concentrated zinc chloride solution are added to this concentrated insulin aspart and excipient solution to obtain the desired final composition. The final pH, either 3.5 or 4.0, is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The solution adjusted to pH 4.0 is turbid immediately after pH adjustment. The solution adjusted to pH 3.5 is clear. This is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of an insulin Eispro 100 U / mL and pramiintide 1 mg / mL solution containing m-cresol (25 mM), glycerin (184 mM), and zinc chloride (300 μM) at an acidic pH of 3.5 or 4.0. [000190] A concentrated solution of excipients (m-cresol, glycerin) is added to a concentrated insulin lispro solution (Gan and Lee Pharmaceuticals) (650 U / mL at pH 3). A concentrated pramiintide solution (10 mg / mL at pH 4) and a concentrated zinc chloride solution are added to this concentrated insulin lispro and excipient solution to obtain the desired final composition. The final pH, either 3.5 or 4.0, is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution is clear and homogeneous; it is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). Preparation of a solution of insulin glulisine 100 U / mL and pramiintide 1 mg / mL containing the excipients of the commercial product Apidra® (29 mM m-cresol, 50 mM Tris, 86 mM zinc chloride and 8.15 μM Tween 20) at acidic pH of 3.0, 3.5 or 4.0. [000191] The pH of the commercial insulin glulisine solution, Apidra®, is adjusted to pH 2.5 by adding an aqueous solution of HCl. This solution is added to pramiintide in powder form to obtain a solution containing 100 U / mL of insulin and 1 mg / mL of pramiintide. The final pH is adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The solutions adjusted to pH 3.5 and 4.0 are turbid immediately after pH adjustment. The solution adjusted to pH 3.0 is clear. This solution is filtered through 0.22 µm and stored in glass cartridges (1 mL of solution per cartridge). After a few hours of storage, the solution is turbid and heterogeneous. Preparation of a 1 mg / mL pramiintide solution containing m-cresol (20 mM), mannitol (236 mM) and acetic acid / sodium acetate buffer (30 mM) at pH 4.0. [000192] A 10 mg / mL concentrated pramiintide solution is prepared by dissolving pramiintide powder purchased from Ambiopharm. This solution is added to a concentrated solution of excipients (m-cresol, mannitol, acetic acid / sodium acetate buffer) to obtain the desired final composition. The final pH is adjusted to 4.0 ± 0.2 by adding NaOH / HCl. Preparation of a solution of human insulin A21G 100 U / mL and pramlintide 0.6 mg / mL containing m-cresol (25 mM), glycerin (184 mM), acetic acid / sodium acetate buffer (18 mM) and Tween 20 (8 μM) at pH 4. Concentrated solutions of glycerin and m-cresol are added to a concentrated human insulin A21G solution in a pH 4 acetic acid / sodium acetate buffer (300 U / mL at pH 4). A concentrated pramlintide (Ambiopharm) solution (10 mg / mL at pH 4) and a concentrated Tween 20 solution are then added to this concentrated human insulin A21G solution and excipients to obtain the desired final composition. The final pH is adjusted to the desired value by adding aqueous NaOH or HCl solution. The resulting solution is clear and homogeneous; it is then filtered through a 0.22 µm filter. The compositions prepared above are shown in Table 1 below. Table 1: Compositions of insulin and / or glucagon suppressants Example 3. Compatibility study of prandial insulins with pramlintide at acidic pH. Visual appearance of insulin and pramlintide solutions at acidic pH. The observation is carried out at room temperature after 2 to 3 hours of stabilization of the solution stored in cartridges. Table 2 presents the visual appearance of the insulin and pramlintide solutions described previously. Table 2: Visual appearance of insulin and pramlintide solutions. [000193] Among the insulins evaluated, only human insulin, insulin lispro, and human insulin A21G yielded a homogeneous and clear formulation with pramlintide at pH 4, demonstrating the solubility of the species. Aspart and glulisine insulins were not suitable for obtaining a clear formulation with pramlintide at pH 4.0. Example 4. Fibrillation latency study. Principle [000194] Poor peptide stability can lead to the formation of amyloid fibrils, defined as ordered macromolecular structures. These can eventually lead to gel formation within the sample. [000195] The thioflavin T (ThT) fluorescence monitoring assay is used to analyze the physical stability of formulations. Thioflavin T is a small molecule probe having a characteristic fluorescence signature when it binds to amyloid-like fibrils (Naiki et al. (1989) Anal. BioChem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284). [000196] This method allows for monitoring fibril formation at low concentrations of ThT in undiluted formulations. This monitoring is carried out under accelerated stability conditions: with stirring and at 37°C. Experimental conditions [000197] The samples were prepared just before the start of the measurement. The preparation of each composition is described in the accompanying example. Thioflavin T was added to the composition from a concentrated stock solution so as to induce negligible dilution of the composition. The concentration of thioflavin T in the composition is 40 μm. A volume of 150 pL of the composition was introduced into one well of a 96-well plate. Each composition was analyzed in triplicate in the same plate. The plate was sealed with transparent film to prevent evaporation of the composition. [000198] This plate was then placed in the chamber of a plate reader (EnVision 2104 Multilabel, Perkin Elmer). The temperature was set at 37°C, and a lateral agitation of 960 rpm with 1 mm amplitude was imposed. [000199] A fluorescence intensity reading in each well is performed with an excitation wavelength of 442 nm, and an emission wavelength of 482 nm over time. [000200] The fibrillation process is manifested by a strong increase in fluorescence after a delay called the latency time. [000201] For each well, this delay was determined graphically as the intersection between the baseline of the fluorescence signal and the slope of the fluorescence curve as a function of time determined during the strong initial increase in fluorescence, as shown in Figure 1. The reported latency value corresponds to the average of the latency times of the 3 wells. [000202] The clear pramlintide and insulin solutions at pH 3.5 and 4.0 from the previous example are then subjected to the fibrillation test in the presence of ThT. [000203] The latency time given in Table 3 corresponds to the average of the 3 measurements, the uncertainty interval corresponds to the average difference between these 3 results. Table 3: Latency time of insulin and pramlintide solutions. [000204] Unexpectedly, formulations containing human insulin A21G have fibrillation latency times much longer than those of commercial insulins tested at pH 3.5 or pH 4.0, in particular rapid analog insulins, lispro and aspart. Example 5. Study of fibrillation latency time in the presence of Tween 20 Table 4 shows the latency times of human insulin A21G and pramlintide solutions at pH 4 in the presence of Tween 20. Table 4: Latency time of human insulin A21G and pramlintide solutions in the presence of Tween 20. [000205] Physical stability is therefore improved in the presence of Tween 20 at 10 mg / mL Example 6. Physical stability of formulations at 30°C under static conditions [000206] Glass cartridges filled with 1 mL of the composition are placed in an oven maintained at 30°C. These cartridges are visually inspected to detect the appearance of visible particles or turbidity. This inspection is carried out according to the recommendations of the European Pharmacopoeia (EP 2.9.20): the cartridges are subjected to illumination of at least 2000 lux and are observed against a white background and a black background. These results are in accordance with the US Pharmacopoeia (USP < 790>). Table 5: Physical stability of insulin and Pramlintide solutions at 30°C under static conditions. [000207] Insulin aspart formulated with pramlintide at pH 3.5 is less stable than human insulin A21G formulated with pramlintide at pH 3.5 or 4.0. Example 7. Physical stability study of human insulin A21G with exenatide and lixisenatide. Formulations A21-6 and A21-7 are placed in cartridges and then incubated at 30°C for 4 weeks. Fibrillation latency times are measured for freshly prepared formulations and are presented in Table 6. Table 6: Latency time and physical stability at 30°C under static conditions of solutions of human insulin A21G and exenatide or lixisenatide. Example 8. Chemical stability of a formulation of human insulin A21G and pramiintide [000208] All formulations are at pH 4.0 or pH 3.5 and contain 100 U / mL of human insulin A21G, 1 mg / mL of pramiintide, 25 mM of m-cresol, and 184 mM of glycerin. The formulations are stored in glass cartridges and kept at 30°C under static conditions. Human insulin A21G and pramiintide are quantified by reversed-phase high-performance liquid chromatography (HPLC). The measurements are shown in Table 7. Table 7: Evolution of insulin (a) concentrations in U / mL and pramiintide (b) concentrations in mg / mL [000209] Formulations containing human insulin A21G and pramiintide exhibit good chemical stability after 4 weeks at 30°C. Pramiintide formulations with commercial insulins degrade rapidly at pH 4.0, and even more rapidly at pH 3.5. Example 9. Pharmacokinetic and pharmacodynamic studies in dogs [000210] Pharmacokinetic and pharmacodynamic study in dogs of the composition of human insulin A21G (100 U / mL, i.e. 3.5 mg / ml) and pramiintide (0.6 mg / mL). The formulation tested is at pH 4.0 and contains 25 mM of m-cresol and 184 mM of glycerin (Formulation A21-8). [000211] Four animals fasted for approximately 18 hours were injected subcutaneously in the neck with 0.2 U / kg of insulin and 0.12 pg / kg of pramiintide. Within one hour of injection, one or more blood samples were taken to determine baseline glucose, insulin, and pramiintide levels. Blood samples were then taken for 5 hours after administration. of the formulation. Blood glucose is determined using a glucometer. Insulin and pramlintide levels in the plasma are determined by an ELISA test. [000212] The pharmacokinetic parameters of formulation A21-8 are estimated from baseline-corrected plasma concentrations of insulin and pramlintide. A standard non-compartmental analysis is performed using Phoenix WinNonlin software (version 7, Certara). The parameter values ​​(mean ± standard deviation) are reported in Tables 8 and 9 below: Table 8: PK parameters of the total insulin analog Table 9: PK parameters of pramlintide [000213] The mean pharmacokinetic (PK) profiles of total insulin (squares) and pramlintide (triangles) in plasma are shown in Figure 2. Mean blood glucose profiles expressed as percentages of baseline are shown in Figure 3. [000214] It is observed that both pramlintide and human insulin A21G have prandial absorption kinetics resulting in early hypoglycemic activity followed by a return to near-basic blood glucose levels 5 hours post-administration. These pharmacokinetic and pharmacodynamic results clearly indicate that Formulation A21-8 is compatible with use with meals. Example 10: Pharmacokinetic studies of pramlintide in pigs [000215] Pharmacokinetic study in pigs of the composition of human insulin A21G (3.5 mg / mL equivalent to 100 U / mL of insulin) and pramlintide (0.6 mg / mL). [000216] Domestic pigs weighing approximately 50 kg, previously catheterized at the jugular vein, are fasted for 2.5 hours before the start of the experiment. In the hour preceding the insulin injection, 3 blood samples are taken to determine the baseline glucose and insulin levels. [000217] The injection of human insulin formulations A21G combined with pramiintide (A21-9) or pramiintide (PRAM) at a dose of 0.2 U of insulin / kg and 1.2 pg of pramlintide / kg is carried out subcutaneously on the flank of the animal using an insulin pen (Novo, Sanofi or Eli Lilly) equipped with a 31 G needle. [000218] To determine plasma concentrations of pramiintide, blood samples are 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. Pharmacokinetic results of human insulin A21G and pramiintide A21-9 solution and pramiintide PRAM solution in pigs [000219] The results of three studies conducted on the same cohort of pigs were pooled to compare the pharmacokinetics of pramiintide between the A21-9 formulation and the PRAM formulation. The pharmacokinetic parameters of the A21-9 and PRAM formulations were estimated from plasma concentrations of pramiintide corrected for baseline values. A standard non-compartmental analysis was performed using Phoenix WinNonlin software (version 7, Certara). The mean ± deviation values ​​of the parameters are reported in the table below. Table 10: PK parameters of pramiintide from compositions A21-9 and PRAM [000220] With t ma x = time required to observe the maximum plasma concentration; Area under the plasma concentration-time curve between 0 and 30 minutes after injection; AUCo t = area under the plasma concentration-time curve between 0 and the last quantifiable concentration after injection [000221] The pharmacokinetic results of pramiintide obtained with formulations A21-9 and PRAM are shown in Figure 4. Analysis of these profiles and parameters indicates that the combination of human insulin A21G and pramiintide (formulation A21-9, curve plotted with squares) leads to a slowing Significant difference in pramiintide absorption compared to pramiintide alone (PRAM formulation, curve plotted with triangles). The A21-9 formulation leads to a significantly delayed peak plasma concentration (tmax) (approximately 18 min, p<0.05) and a significantly decreased early plasma exposure to pramiintide (AUCi) (approximately 43%, p<0.05) compared to the PRAM formulation. On the other hand, the total plasma exposure to pramiintide (AUCo-t) appears similar between the two formulations, suggesting comparable bioavailabilities. Example 11. Study of food consumption in rats after injection of control compositions and compositions including human insulin A21G and / or pramiintide [000222] This study was carried out on a population of 40 male Sprague Dawley rats of at least 6 weeks. [000223] The rats had free access to food and water, except for a 6-hour fasting period prior to subcutaneous injection of the compositions described in the table below. Table 11: compositions injected into rats and number of rats treated [000224] The control composition is a saline solution, i.e. an aqueous solution comprising 150 mM of NaCl. [000225] Humulin® R is a commercial human insulin solution marketed by ELI LILLY. This product is a 100 U / ml human insulin. The excipients in Humulin® R are glycerol, metacresol, sodium hydroxide, hydrochloric acid for pH adjustment (pH 7.0-7.8), and water. [000226] At t0, immediately after injection, food is distributed (approximately 100 g per rat). Food intake (cumulative average) is measured one, two, and three hours after t0, i.e., t+1h, t+2h, and t+3h. [000227] The results are presented in the following table: Table 12: Food intake 1, 2 and 3 hours after injection [000228] These results show that the A21-9 composition, combining insulin A21G and pramiintide, not only reduces food intake induced by insulin injection, but also limits food intake to a level lower than or equal to that of the control group that underwent an injection of the Control composition (saline solution).

Claims

DEMANDS 1. Composition in the form of an injectable aqueous solution, the pH of which is between 3.5 and 4.4, comprising at least human insulin A21G known as regular and at least one prandial-acting glucagon suppressant.

2. Composition according to claim 1, characterized in that the prandial-acting glucagon suppressor is selected from the group consisting of an amylin analogue or an amylin receptor agonist or a GLP-1 analogue or a GLP-1 receptor agonist (GLP-1 RA).

3. Composition according to any one of the preceding claims, characterized in that the prandial-acting glucagon suppressant is an amylin analogue or an amylin receptor agonist.

4. Composition according to any one of the preceding claims, characterized in that the prandial-acting glucagon-suppressing peptide is pramlintide.

5. Composition according to any one of the preceding claims, characterized in that the prandial-acting glucagon-suppressing peptide is exenatide.

6. Composition according to any one of the preceding claims, characterized in that the prandial-acting glucagon-suppressing peptide is lisixenatide.

7. Composition according to claim 1, characterized in that the concentration of human insulin A21G is from 2 to 20 mg / mL.

8. Composition according to claim 1, characterized in that the concentration of human insulin A21G is 3.5 mg / mL.

9. Composition according to any one of the preceding claims, characterized in that the concentration of prandial-acting glucagon-suppressing peptide is from 0.01 to 10 mg / mL.

10. Composition according to any one of the preceding claims, characterized in that the pH of the solution is from 3.8 to 4.

2.

11. Composition according to any one of the preceding claims, characterized in that the pH of the solution is 4.

0.

12. Composition according to any one of the preceding claims, characterized in that it further comprises a zinc salt.

13. Composition according to any one of the preceding claims, characterized in that it further comprises m-cresol.

14. Composition according to any one of the preceding claims, characterized in that it further comprises a polysorbate excipient (Tween® 20) 15. Composition according to any one of the preceding claims, characterized in that it further comprises an excipient Poloxamer 188.

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

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

18. Composition according to any one of claims 1 to 17, intended for use in a method of treating diabetes characterized in that it is administered to improve postprandial blood glucose control.

19. Composition according to any one of claims 1 to 17, intended for use in a method of treating diabetes characterized in that it is administered to improve postprandial blood glucose control and to reduce the adverse effects of pramlintide.

20. Composition according to any one of claims 1 to 17, intended for use in a method of treating diabetes characterized in that it allows for a reduction in insulin-induced food intake.

21. A process for obtaining human insulin A21G comprising at least one step of reacting human insulin A21G, B31R, B32R (insulin glargine) with rat carboxypeptidase B at an insulin / carboxypeptidase ratio of between 500 and 2000, at a pH of 7.5 to 8.5 and at a temperature of 20 to 30°C for 10 to 20 hours.

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