A composition in the form of an injectable aqueous solution comprising at least human insulin A21G and a prandial action glucagon inhibitor

Abstract

The present 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 acting glucagon inhibitor. In one embodiment, the prandial acting glucagon inhibitor is selected from 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 acting glucagon inhibitor is an amylin analogue or an amylin receptor agonist. In one embodiment, the prandial acting glucagon inhibitor peptide is pramlintide. The present invention also relates to a process for obtaining human insulin A21G comprising at least the step of reacting human insulin A21G, B31R, B32R (insulin glargine) with rat carboxypeptidase B in a ratio of insulin / carboxypeptidase of 500 to 2000 at a pH of 7.5 to 8.5 and a temperature of 20 to 30 °C for 10 to 20 hours.

Description

[0001] The present invention relates to a treatment for diabetes by injecting a composition comprising at least human insulin A21G with a mealtime effect and, in particular, a glucagon inhibitor with a mealtime effect, a combination that allows for improved control of postprandial hyperglycemia.

[0002] Type 1 diabetes is an autoimmune disease that leads to the destruction of pancreatic beta cells. These cells are known to produce insulin, whose primary function is to regulate glucose use in peripheral tissues (Gerich 1993 Control of glycaemia). Therefore, people with type 1 diabetes suffer from chronic hyperglycemia and must self-administer exogenous insulin to control this hyperglycemia. Insulin therapy has dramatically altered the life expectancy of these patients.

[0003] To date, people with type 1 diabetes use two types of insulin: short-acting prandial insulin to control blood sugar during meals, and long-acting basal insulin to control blood sugar throughout the day and night. Several types of short-acting insulin exist, characterized by their onset of action. For example, there are so-called rapid-acting insulin analogs (such as lispro insulin). ELI LILLY) or aspart insulin ( Compared to NOVO NORDISK, human insulin, also known as regular insulin, has a delayed effect. Therefore, human insulin must be administered an average of 30 minutes before meals, while insulin analogs can be administered 15 minutes before or during meals. Furthermore, insulin analogs are considered to control postprandial blood glucose better than human insulin, which explains why the vast majority of patients in Europe and the United States today use rapid-acting insulin analogs, which have a shorter duration of action than human insulin.

[0004] However, even with rapid-acting insulin analogs, ensuring optimal glycemic control through these exogenous mealtime insulins after meals is not optimal. This is partly due to the fact that, compared to healthy individuals, these patients produce glucagon after meals, which leads to the removal of some glucose stored in the liver. This glucagon-mediated glucose production exacerbates postprandial hyperglycemia in these patients and results in excessive insulin use.

[0005] This problem of regulating postprandial blood sugar is quite similar to that of patients with type 2 diabetes treated with insulin, in which the disease has led to a very significant loss of their ability to produce insulin and amylin.

[0006] It has been shown that glucagon inhibitors (particularly peptides and / or hormones) can inhibit postprandial glucagon production, leading to significant improvements in postprandial glycemic control. For example, amylin, a hormone produced by pancreatic β-cells and lacking in patients with type 1 diabetes, plays a crucial role in regulating postprandial blood glucose. Also known as "islet amyloid polypeptide" or IAPP, a 37-amino acid peptide co-stored and co-secreted with insulin (Schmitz 2004 Amylin Agonists). This peptide is described as an inhibitor of glucagon production by pancreatic α-cells. Therefore, insulin and amylin have complementary and synergistic effects, as insulin lowers blood glucose levels, while amylin reduces the entry of endogenous glucose into the bloodstream by inhibiting the production or secretion of endogenous glucagon.

[0007] However, human amylin possesses properties 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 Truct Biol 119:17-27, 1997). Amylin is known to form amyloid fibrils, leading to the formation of water-insoluble plaques. Therefore, it is necessary to develop analogs to address these solubility issues.

[0008] Amylin has developed pramlintide, an analogue of amylin, to compensate for the lack of physical stability in human amylin. The product is marketed under the name... This product is marketed and approved by the FDA in 2005 as a supplement to insulin therapy for the treatment of type 1 and type 2 diabetes. Given its relatively short half-life of several hours, it must be administered subcutaneously three times daily in the hour before meals to improve postprandial blood glucose control. The peptide is formulated at pH 4.0 and has been described to undergo fibrinolysis when the pH of the solution is above 5.5. Variations of this analogue are described in patent US5,686,411.

[0009] Amylin analogues or amylin receptor agonists, often referred to as "mealtime" or "short-acting," reproduce the effects of amylin while having a longer half-life. These amylin derivatives, which control blood glucose during meals, have a half-life of less than 8 hours. This half-life is the apparent elimination half-life following subcutaneous injection in humans. The half-life of these amylin analogues or amylin receptor agonists can be less than 5 hours, particularly less than 4 hours, and even less than 3 hours.

[0010] However, compositions containing amylin or amylin analogues (and especially pramlintide) can cause certain adverse effects in patients. In particular, these compositions can cause nausea in patients.

[0011] Another physiological peptide, GLP-1, has also been described as playing a similar role to amylin in inhibiting postprandial glucagon secretion. GLP-1 is also known to act as an insulin secretagogue, and is therefore particularly effective when used as insulin supplementation, especially in patients with type 2 diabetes. These effects are glucose-dependent, which minimizes the risk of hypoglycemia. To date, GLP-1 RAs have only been approved for use in patients with type 2 diabetes.

[0012] Similarly, due to its extremely short half-life, human GLP-1 cannot be used for therapeutic purposes. Various GLP-1 derivatives, GLP-1 receptor agonists (called GLP-1 RAs), or GLP-1 analogs reproduce the effects of GLP-1 while having longer half-lives. These GLP-1 derivatives can be divided into three groups based on their respective half-lives: those with short-acting or prandial effects, used to control blood glucose during meals (half-life less than 8 hours); those with 24-hour effects, covering the needs throughout the day (half-life greater than 8 or 10 hours); and those with 14-hour effects, covering the needs throughout the week (half-life greater than 48 hours). For prandial GLP-1 RAs, the two peptides approved to date are exenatide (…). ASTRA-ZENECA (applied twice daily) and lixisenatide ( SANOFI (administered once daily). Like Pramlinitide, both of these GLP-1 RAs are formulated at a pH close to 4 and must be administered one hour before a meal.

[0013] For insulin-dependent patients, one of the main difficulties in using these different short-acting glucagon inhibitor compounds is related to the additional number of injections, which can be 1 to 3 times daily in addition to the 2 to 4 injections of insulin. Therefore, it is important to be able to combine prandial insulin with glucagon inhibitors that have a prandial effect in solution, so that these insulin-complementary compounds can be used without complicating the patient's treatment. Furthermore, this allows for a more precise mimicking of physiological effects, as both hormones are secreted in response to meals to improve postprandial blood glucose control, particularly better control of postprandial hyperglycemia, and thus better treatment of diabetes.

[0014] However, prandial insulin and these target peptides are incompatible in aqueous solutions. In fact, prandial insulin has optimal chemical stability at a pH close to 7, while amylin or GLP-1 derivatives are physically and chemically unstable at pH close to physiological levels.

[0015] This difficulty has led to the design of pumps containing two reservoirs for keeping prandial insulin separate from amylin derivatives. Roche's US Patent US2016 / 001002 describes such a pump, enabling the co-administration of amylin and prandial insulin using a single medical device. However, for the sake of using existing medical devices that are simpler and / or have a lower risk of failure, the ability to mix these hormones in an aqueous solution would be preferred.

[0016] Another solution to the problem of mixing these hormones in aqueous solutions lies in using organic solvents instead of water. XERIS's patent application WO2013067022 describes a composition of amylin and insulin contained in a solution of an organic solvent. However, in the treatment of chronic diseases such as diabetes, the use of organic solvents (e.g., DMSO) raises safety concerns for patients. Furthermore, organic solvents can be problematic for injection devices, particularly because they dissolve some of the components of the injection device. Therefore, it is desirable to develop these combinations in aqueous solution form.

[0017] The stability issues in aqueous solutions are also circumvented by preparing solid mixtures. Patent application EP2060268 from NOVONORDISK describes formulations of insulin and pramlintide in nebulized powder form for nasal administration. However, the preferred and most commonly used route of administration to date is the subcutaneous route, which requires readily available aqueous solutions.

[0018] Another approach involves combining prandial insulin with the amylin derivative at a physically stable pH. Biodel's patent application US20090192075 describes a liquid composition comprising human insulin, pramlintide, and a zinc chelating agent at pH 4.0. This application describes the rapid action of human insulin due to the presence of the zinc chelating agent. However, human insulin is known to be chemically unstable at acidic pH, and this technique does not meet the standards of the EP and / or US Pharmacopoeia.

[0019] An alternative approach involves modifying the structure of prandial insulin to improve its stability at acidic pH. Application WO2007104786 illustrates compositions comprising rapid-acting insulin analogs A21G, B28D, desB30 and A21G, B28E, desB30 that are soluble at acidic pH.

[0020] Application WO2007104786 also proposes rapid-acting insulin compositions, particularly B28D (insulin aspart), in the presence of surfactants (and especially glycerophosphate derivatives, more particularly dimyristoyl glycérophosphoglycérol (DMPG)) at neutral pH, which results in much greater stability as measured by ThT than rapid-acting insulin analogs A21G, B28D, desB30 and A21G, B28E, desB30 at acidic pH.

[0021] Prior art compositions comprising a combination of prandial insulin and pramlintide have most often described different types of prandial insulin; however, examples involve compositions comprising so-called rapid-acting prandial insulin analogs, which are considered to be more effective than human insulin.

[0022] Surprisingly, the applicant has demonstrated that a composition containing human insulin A21G in aqueous solution at pH 3.5 to 4.4, in combination with a glucagon inhibitor having a mealtime effect, enables better control of postprandial blood glucose than so-called rapid-acting mealtime insulin analogs. The human insulin A21G is referred to as "conventional," meaning that this insulin differs from human insulin only in that the asparagine residue at position 21 of the A chain is replaced with a glycine residue, which is not as rapid as so-called "rapid-acting" insulin analogs.

[0023] Furthermore, the applicant unexpectedly demonstrated that the composition containing human insulin A21G in aqueous solution at a pH of 3.5 to 4.4 in combination with a glucagon inhibitor having a mealtime effect exhibits physical and chemical stability that is compatible with pharmaceutical requirements and greater than that of solutions proposed in the prior art.

[0024] The acquisition of compositions in injectable aqueous solutions that exhibit better control and improved physical and chemical stability for postprandial hyperglycemia compared to those described in the prior art is remarkable, as it is well known to those skilled in the art that, in the case of combinations, it is difficult to predict the pharmacokinetic and physical and chemical stability properties of the combined products.

[0025] In addition, there is a search for compositions that can reduce or even eliminate all or some of the adverse effects that may be produced by the active substance.

[0026] The present invention relates to compositions in the form of injectable aqueous solutions having a pH of 3.5 to 4.4, comprising at least so-called conventional human insulin A21G and at least one glucagon inhibitor having a mealtime effect.

[0027] According to one implementation, glucagon inhibitors with prandial action are amylin analogs or amylin receptor agonists, GLP-1 analogs or GLP-1 receptor agonists (also known as GLP-1RAs).

[0028] The applicant has observed that the formulations of the present invention with a pH of 3.5 to 4.4 have pharmacokinetic properties compatible with use during meals and enable better control of postprandial blood glucose.

[0029] Furthermore, the applicant has demonstrated that these formulations result in a slower absorption of pramlindide. This is particularly evident in the significantly delayed peak plasma absorption of pramlindide compared to administration of pramlindide alone. max ) and / or significantly reduced early plasma pralaminide exposure (AUC) 0-30min ) is a characteristic.

[0030] This slowing down allows for the reduction or even elimination of the adverse effects of pramlinide, particularly in terms of nausea.

[0031] The present invention also relates to the use of an injectable aqueous solution of a composition comprising at least human insulin A21G and, in particular, a glucagon inhibitor having a mealtime effect, at a pH of 3.5 to 4.4 for improving postprandial blood glucose control.

[0032] The present invention also relates to compositions according to the invention intended for use in the treatment of diabetes, characterized in that they are administered by bolus injection before meals.

[0033] The present invention also relates to compositions according to the invention intended for use in the treatment of diabetes, characterized in that they are administered to improve control of postprandial blood glucose.

[0034] The present invention also relates to compositions according to the invention intended for use in the treatment of diabetes, characterized in that they are administered to improve control of postprandial blood glucose and reduce the adverse effects of pramlintide.

[0035] The present invention also relates to compositions according to the invention intended for use in the treatment of diabetes, characterized in that they enable the reduction of insulin-induced food intake.

[0036] According to one implementation plan, the reduction in food intake occurs over a period of four hours from the time of injection.

[0037] According to one implementation plan, the reduction in food intake occurs over a period of time from injection to 3 hours after injection.

[0038] According to one implementation plan, the reduction in food intake occurs over a period of time from injection to two hours after injection.

[0039] According to one implementation plan, the reduction in food intake involves the period from injection to one hour after injection.

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

[0041] The requirements for obtaining injectable drug formulations for the treatment of diabetes are particularly:

[0042] - A physically and chemically stable aqueous liquid formulation that is at least 2 weeks or even 1 month at 30°C (multiple uses) and at least 1 year or even 2 years at 5°C.

[0043] - Compatibility with antimicrobial preservatives.

[0044] Similarly, formulations of human insulin A21G with pH 3.5 to 4.4 containing GLP-1 analogs or GLP-1 receptor agonists (also known as GLP-1 RAs, such as exenatide or lixisenatide) have physical and chemical stability, enabling the development of liquid formulations that are stable at 30°C for at least 2 weeks or even 1 month and at 5°C for at least 1 year or even 2 years.

[0045] Regarding stability, a conventional method for measuring the stability of proteins or peptides involves measuring fibril formation using thioflavine T (also known as ThT). This method allows for the measurement of the hysteresis time prior to fibril formation by measuring the increase in fluorescence under conditions of temperature and agitation that can accelerate this phenomenon. The compositions according to the invention exhibit a significantly longer hysteresis time prior to fibril formation than those described in the literature. The compositions according to the invention demonstrate physical and chemical stability that is much greater than that described in the prior art using commercial mealtime insulin.

[0046] In one embodiment, the formulation according to the invention exhibits a hysteresis time of at least 8 hours as measured by ThT.

[0047] In one embodiment, the formulation according to the invention exhibits a hysteresis time of at least 10 hours as measured by ThT.

[0048] In one embodiment, the formulation according to the invention exhibits a hysteresis time of at least 15 hours as measured by ThT.

[0049] In one embodiment, the formulation according to the invention exhibits a hysteresis time of at least 20 hours as measured by ThT.

[0050] In one embodiment, the formulation according to the invention exhibits a hysteresis time of at least 25 hours as measured by ThT.

[0051] In one embodiment, the present 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 amylin analog. According to one embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0052] In one embodiment, the present 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 amylin analog. According to one embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0053] In one embodiment, the present 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 amylin analog. According to one embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0054] In one embodiment, the present 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 amylin analog. According to one embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0055] In one embodiment, the present 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 GLP-1 analogue. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide.

[0056] In one embodiment, the present invention relates to an injectable solution composition having a pH of 3.5 to 4.2, comprising at least human insulin A21G and a GLP-1 receptor agonist or GLP-1 analog. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide.

[0057] In one embodiment, the present 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 GLP-1 analog. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide.

[0058] In one embodiment, the present 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 GLP-1 analog. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide.

[0059] In one embodiment, the present 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 amylin analog, and a GLP-1 receptor agonist or a GLP-1 analog. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide. According to yet another embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0060] In one embodiment, the present 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 GLP-1 analog. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide. According to yet another embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0061] In one embodiment, the present 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 amylin analog, and at least one GLP-1 receptor agonist or GLP-1 analog. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide. According to yet another embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0062] In one embodiment, the present 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 amylin analog, and at least one GLP-1 receptor agonist or GLP-1 analog. According to one embodiment, the GLP-1 receptor agonist is exenatide. According to another embodiment, the GLP-1 receptor agonist is lixisenatide. According to yet another embodiment, the amylin receptor agonist or amylin analog is pramlintide.

[0063] It is particularly advantageous to combine human insulin A21G in aqueous solution with amylin analogs, amylin receptor agonists or GLP-1 and GLP-1 analogs or GLP-1 receptor agonists, because this so-called “triple” combination specifically enables the enhancement of the effect of each hormone and the reduction of the dose of each.

[0064] The composition according to the invention, in the form of an injectable aqueous solution, is a clear solution. "Clear solution" should be understood to mean a composition that meets the standards described in the United States Pharmacopeia and the European Pharmacopeia for injectable solutions. In the United States Pharmacopeia, a solution is... <1151> The definition provided in the section refers to injectable drugs. <1> (Refer to USP 35) <788> And according to USP 35 <788> The provisions of the law, and in accordance with USP 38 <787> , <788> and <790> (As specified in USP 38 (effective August 1, 2014)). In the European Pharmacopoeia, injectable solutions must meet the standards given in sections 2.9.19 and 2.9.20.

[0065] In this application, as described above, amylin refers to the compounds described in patents US 5,124,314 and US 5,234,906. When the term "analyte" is used, it refers to a peptide or protein in which one or more constituent amino acid residues of its primary sequence have been replaced by other amino acid residues and / or one or more constituent amino acid residues have been eliminated and / or one or more constituent amino acid residues have been added. The accepted percentage of homology for this analog definition is 50%. In the case of amylin, an analog may be derived from the primary amino acid sequence of amylin, for example, by substituting one or more natural or non-natural or peptide-like amino acids.

[0066] Exenatide and lixisenatide, described in applications US2004 / 0023871 and WO0104156 respectively, are generally considered GLP-1 receptor agonists. In one embodiment, the glucagon inhibitor with prandial action is pramlintide, marketed by AstraZeneca AB.

[0067] In one embodiment, GLP-1, GLP-1 analogs, or GLP-1 RAs are referred to as “short-acting” or “mealtime.” “Short-acting” or “mealtime” should be understood to mean GLP-1, GLP-1 analogs, or GLP-1 RAs, such as exenatide or lixisenatide, with an apparent elimination half-life of less than 8 hours, particularly less than 5 hours, preferably less than 4 hours, or less than 3 hours after subcutaneous injection in humans.

[0068] In one embodiment, GLP-1, a GLP-1 analog, or GLP-1 RA is selected from exenatide (…). ASTRA-ZENECA), lixisenatide ( SANOFI), its analogues or derivatives and their medicinal salts.

[0069] In one embodiment, GLP-1, a GLP-1 analog, or a GLP-1 RA is exenatide or Its analogues or derivatives and their medicinal salts.

[0070] In one embodiment, GLP-1, a GLP-1 analog, or a GLP-1 RA is lixisenatide or Its analogues or derivatives and their medicinal salts.

[0071] In one embodiment, the concentration of human insulin A21G is 240 to 3000 μM or 40 to 500 U / mL.

[0072] In one embodiment, the concentration of human insulin A21G is 600 μM or 100 U / mL.

[0073] In one embodiment, the concentration of human insulin A21G is 1200 μM or 200 U / mL.

[0074] In one embodiment, the concentration of human insulin A21G is 1800 μM or 300 U / mL.

[0075] In one embodiment, the concentration of human insulin A21G is 2400 μM or 400 U / mL.

[0076] In one implementation, the concentration of human insulin A21G is 3000 μM or 500 U / mL.

[0077] In this application, 100 U / mL of human insulin A21G corresponds to 3.5 mg / mL.

[0078] In one embodiment, the concentration of pramlintide is 0.32 to 5 mg / mL.

[0079] In one embodiment, the concentration of pramlinide is 0.4 to 3 mg / mL.

[0080] In one embodiment, the concentration of pramlinide is 0.5 to 2 mg / mL.

[0081] In one embodiment, the concentration of pramlintide is 0.5 to 1.5 mg / mL.

[0082] In one embodiment, the concentration of pramlinide is 0.6 to 1 mg / mL.

[0083] In one embodiment, the concentration of pramlintide is 1.0 mg / mL.

[0084] In one embodiment, the concentration of pramlinide is 0.6 mg / mL.

[0085] In one embodiment, the concentration of exenatide is 10 to 1000 μg / mL.

[0086] In one embodiment, the concentration of exenatide is 10 to 500 μg / mL.

[0087] In one embodiment, the concentration of exenatide is 20 to 400 μg / mL.

[0088] In one embodiment, the concentration of exenatide is 20 to 300 μg / mL.

[0089] In one embodiment, the concentration of exenatide is from 30 to 300 μg / mL. In another embodiment, the concentration of exenatide is from 30 to 150 μg / mL.

[0090] In one embodiment, the concentration of exenatide is 40 to 150 μg / mL.

[0091] In one embodiment, the concentration of exenatide is 40 to 80 μg / mL.

[0092] In one implementation, the concentration of exenatide is 50 μg / mL.

[0093] In one embodiment, the concentration of liximab is 20 to 1000 μg / mL.

[0094] In one embodiment, the concentration of liximab is 20 to 800 μg / mL.

[0095] In one embodiment, the concentration of liximab is 40 to 600 μg / mL.

[0096] In one embodiment, the concentration of liximab is 60 to 600 μg / mL.

[0097] In one embodiment, the concentration of liximab is 60 to 300 μg / mL.

[0098] In one embodiment, the concentration of liximab is 80 to 300 μg / mL.

[0099] In one embodiment, the concentration of liximab is 80 to 160 μg / mL.

[0100] In one embodiment, the concentration of liximab is 100 μg / mL.

[0101] In one embodiment, the concentration of exenatide, its analogues or derivatives and pharmaceutically acceptable salts is from 0.01 to 1.0 mg / 100U insulin.

[0102] In one embodiment, the concentration of exenatide, its analogues or derivatives and pharmaceutically acceptable salts is 0.01 to 0.5 mg / 100U insulin.

[0103] In one embodiment, the concentration of exenatide, its analogues or derivatives and pharmaceutically acceptable salts is 0.02 to 0.4 mg / 100U insulin.

[0104] In one embodiment, the concentration of exenatide, its analogues or derivatives and pharmaceutically acceptable salts is 0.03 to 0.3 mg / 100U insulin.

[0105] In one embodiment, the concentration of exenatide, its analogues or derivatives and pharmaceutically acceptable salts is 0.03 to 0.2 mg / 100U insulin.

[0106] In one embodiment, the concentration of exenatide, its analogues or derivatives and pharmaceutically acceptable salts is 0.03 to 0.15 mg / 100U insulin.

[0107] In one embodiment, the concentration of exenatide, its analogues or derivatives and pharmaceutically acceptable salts is 0.05 mg / 100 U insulin.

[0108] In one embodiment, the concentration of lixisenatide, its analogues or derivatives, and its pharmaceutically acceptable salts is from 0.01 to 1 mg / 100 U insulin.

[0109] In one embodiment, the concentration of liximab, its analogues or derivatives, and its pharmaceutically acceptable salts is 0.01 to 0.5 mg / 100U insulin.

[0110] In one embodiment, the concentration of liximab, its analogues or derivatives, and its pharmaceutically acceptable salts is 0.02 to 0.4 mg / 100U insulin.

[0111] In one embodiment, the concentration of liximab, its analogues or derivatives, and its pharmaceutically acceptable salts is 0.03 to 0.3 mg / 100U insulin.

[0112] In one embodiment, the concentration of liximab, its analogues or derivatives, and its pharmaceutically acceptable salts is from 0.04 to 0.2 mg / 100U insulin.

[0113] In one embodiment, the concentration of liximab, its analogues or derivatives, and its pharmaceutically acceptable salts is 0.04 to 0.15 mg / 100U insulin.

[0114] In one embodiment, the concentration of lixisenatide, its analogues or derivatives, and its pharmaceutically acceptable salts is 0.1 mg / 100 U insulin.

[0115] In one embodiment, the composition according to the invention is prepared by mixing a solution of an amylin analog or amylin receptor agonist with a solution of GLP-1, a GLP-1 analog or a GLP-1 receptor agonist RA at a volume ratio of 10 / 90 to 90 / 10.

[0116] In one embodiment, the composition according to the invention further comprises a zinc salt with an insulin concentration of 0 to 800 μM / 100 U.

[0117] In one embodiment, the composition according to the invention further comprises a zinc salt at a concentration of 0 to 500 μM / 100 U insulin.

[0118] In one embodiment, the composition according to the invention further comprises a zinc salt at a concentration of 100 to 500 μM / 100U insulin.

[0119] In one embodiment, the composition according to the invention further comprises a zinc salt at a concentration of 200 to 400 μM / 100U insulin.

[0120] In one embodiment, the composition according to the invention further comprises a zinc salt at a concentration of 300 μM / 100 U insulin.

[0121] In one embodiment, the composition according to the invention further comprises a buffer.

[0122] In one embodiment, the composition according to the invention comprises a buffer selected from sodium acetate buffer and Tris buffer.

[0123] In one embodiment, the composition according to the invention further comprises a preservative.

[0124] In one embodiment, the preservative is selected from m-cresol and phenol, either alone or in mixture.

[0125] In one embodiment, the concentration of the preservative is 10 to 50 mM.

[0126] In one embodiment, the concentration of the preservative is 10 to 40 mM.

[0127] In one embodiment, the composition of the present invention further comprises a surfactant.

[0128] In one embodiment, the surfactant is selected from poloxamer 188, 20 (also known as polysorbate 20) and 80 (also known as polysorbate 80).

[0129] In one implementation scheme The concentration of 20 is 5 to 50 μg / mL.

[0130] In one implementation scheme The concentration of 20 is 5 to 25 μg / mL.

[0131] In one implementation scheme The concentration of 20 is 10 μM.

[0132] Furthermore, the compositions according to the invention may contain additives, such as detonic agents.

[0133] In one embodiment, the tensioning agent is selected from glycerol, sodium chloride, mannitol, and glycine.

[0134] In one embodiment, the composition according to the invention further comprises an antioxidant.

[0135] In one implementation, the antioxidant is methionine.

[0136] In one embodiment, the pharmaceutical composition further comprises at least one absorption enhancer selected from absorption enhancers, diffusion enhancers, or vasodilators, either alone or in mixtures.

[0137] Absorption enhancers include, but are not limited to, alone or in mixtures: surfactants, such as bile salts, fatty acid salts, or phospholipids; nicotinic agents, such as nicotinamides, nicotinic acid, niacine, niacinamide, vitamin B3 and its salts; pancreatic trypsin inhibitors; magnesium salts; polyunsaturated fatty acids; phosphatidylcholine didecyl; amino polycarboxylate; tolmétine; sodium decanoate; salicylic acid; oleic acid; linoleic acid; eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA). Benzoic acid; nitric oxide donors, such as 3-(2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazyl)-1-propane, N-ethyl-2-(1-ethylhydroxy-2-1-nitrosohydrazyl)ethylamine, or S-nitroso-N-acetylpenicillamine; bile acids, glycine in bile acid conjugated form; sodium ascorbate, potassium ascorbate; sodium salicylate, potassium salicylate, acetylsalicylic acid, salicylsalicylic acid, aluminum acetylsalicylate, salicylcholine, salicylamide, lysine acetylsalicylic acid; esaxamide; diflunisal; ethylsalicylic acid; EDTA.

[0138] In one embodiment, the pharmaceutical composition further comprises at least one diffusion promoter. Examples of diffusion promoters include, but are not limited to, glycosaminoglycans, such as hyaluronidase.

[0139] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator.

[0140] In one embodiment, the pharmaceutical composition further includes at least one vasodilator that induces hyperpolarization by blocking calcium ion channels.

[0141] In one implementation, the vasodilator that induces hyperpolarization by blocking calcium ion channels is adenosine, an endothelial-derived hyperpolarizer, a type 5 phosphodiesterase (PDE5) inhibitor, a potassium channel opener, or any combination of these agents.

[0142] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator mediated by AMPc.

[0143] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator mediated by GMPc.

[0144] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator selected from the group consisting of: vasodilators that act by inducing hyperpolarization through blocking calcium ion channels, vasodilators mediated by AMPc, and vasodilators mediated by GMPc.

[0145] The at least one vasodilator is selected from the group consisting of, alone or in combination, the following: nitric oxide donors, such as nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, amyl nitrate, erythritol tetranitrate, and nitroprussiate; prostacyclin and its analogues, such as eprostacyclin sodium, iloprost, eprostacyclin, treprostacyclin, or celecoxib; histamine, 2-methylhistamine, 4-methylhistamine; 2-(2-pyridyl)ethylamine, 2-(2-thiazolyl)ethylamine; papaverine, papaverine hydrochloride; minoxidil; dipyridamole. Moxicillin, hydralazine; adenosine, adenosine triphosphate; uridine triphosphate; GPLC; L-carnitine; arginine; prostaglandin D2; potassium salts; and in some cases, α1 and α2 receptor antagonists, such as prazosin, phenoxybenzamine, phentolamine, dibenzamine, moxicillin hydrochloride, and tolazoline; betazol, dimaprit; β2 receptor agonists, such as isoproterenol, dobutamine, salbutamol, terbutaline, aminophylline, theophylline, caffeine; alprostadil, ambesentan; cabergoline; diazoxide; dihydralazine mesylate; diltiazem hydrochloride Eloxicam; Flunarizine hydrochloride; Ginkgo biloba extract; Levosimendan; Madomin; Naftidrofuryl oxalate; Nicorandil; Pentoxoxothecobalamin; Phenoxybenzamine hydrochloride; Pirbedil base; Pirbedil mesylate; Regadson monohydrate; Riociguat; Sildenafil citrate, Tadalafil, Vardenafil hydrochloride trihydrate; Trimetazidine hydrochloride; Trinitrin; Verapamil hydrochloride; Endothelin receptor antagonists, such as avanafil and bosentan monohydrate. Monohydrae; and calcium channel inhibitors, such as amlodipine, areadipine, azledipine, barnidipine, benidipine, cilnidipine, clovidipine, isadipine, ivodidipine, felodipine, lacidipine, lecanidipine, manidipine, nicardipine, nifedipine, nivardipine, nimodipine, nisodipine, nifedipine, and pradipine.

[0146] In one embodiment, the composition according to the invention comprises 3.5 mg / mL to 10.5 mg / mL human insulin A21G at pH 4.0, 0.6 mg / mL to 3 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 300 to 900 μM zinc. The composition may also contain polysorbate 20, particularly 8 to 10 μM, and most particularly 8 μM.

[0147] In one embodiment, the composition according to the invention comprises 3.5 mg / mL human insulin A21G at pH 4.0, 0.6 to 1 mg / mL pramlintide, 25 to 30 mM m-cresol, and 150 to 200 mM glycerol. The composition may also contain 300 μM zinc. The composition may also contain polysorbate 20, particularly 8 to 10 μM, and most particularly 8 μM.

[0148] In one embodiment, the composition according to the invention comprises 3.5 mg / mL human insulin A21G at pH 4.0, 0.6 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 300 μM zinc. The composition may also contain polysorbate 20, particularly 10 μM.

[0149] In one embodiment, the composition according to the invention comprises 3.5 mg / mL human insulin A21G at pH 4.0, 0.6 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 300 μM zinc. The composition may also contain polysorbate 20, particularly 8 μM.

[0150] In one embodiment, the composition according to the invention comprises 3.5 mg / mL human insulin A21G at pH 4.0, 1.0 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 300 μM zinc. The composition may also contain polysorbate 20, particularly 8 μM.

[0151] In one embodiment, the composition according to the invention comprises 7.0 mg / mL human insulin A21G at pH 4.0, 1.2 to 2.0 mg / mL pramlintide, 25 mM m-cresol, and 150 to 200 mM glycerol. The composition may also contain 600 μM zinc. The composition may also contain polysorbate 20, particularly 8 to 10 μM, and most particularly 8 μM.

[0152] In one embodiment, the composition according to the invention comprises 7.0 mg / mL human insulin A21G at pH 4.0, 1.2 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 600 μM zinc. The composition may also contain polysorbate 20, particularly 10 μM.

[0153] In one embodiment, the composition according to the invention comprises 7.0 mg / mL human insulin A21G at pH 4.0, 1.2 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 600 μM zinc. The composition may also contain polysorbate 20, particularly 8 μM.

[0154] In one embodiment, the composition according to the invention comprises 7.0 mg / mL human insulin A21G at pH 4.0, 2.0 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 600 μM zinc. The composition may also contain polysorbate 20, particularly 8 μM.

[0155] In one embodiment, the composition according to the invention comprises 10.5 mg / mL human insulin A21G at pH 4.0, 1.8 to 3 mg / mL pramlintide, 25 mM m-cresol, and 150 to 200 mM glycerol. The composition may also contain 900 μM zinc. The composition may also contain polysorbate 20, particularly 8 to 10 μM, and most particularly 8 μM.

[0156] In one embodiment, the composition according to the invention comprises 10.5 mg / mL human insulin A21G at pH 4.0, 1.8 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 900 μM zinc. The composition may also contain polysorbate 20, particularly 10 μM.

[0157] In one embodiment, the composition according to the invention comprises 10.5 mg / mL human insulin A21G at pH 4.0, 1.8 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 900 μM zinc. The composition may also contain polysorbate 20, particularly 8 μM.

[0158] In one embodiment, the composition according to the invention comprises 10.5 mg / mL human insulin A21G at pH 4.0, 3 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The composition may also contain 900 μM zinc. The composition may also contain polysorbate 20, particularly 8 μM.

[0159] The compositions of the present invention may also contain all excipients that conform to the pharmacopoeia (especially the EP and / or US pharmacopoeia) and are compatible with insulin used at normal concentrations.

[0160] According to one embodiment, the composition can be in solid or lyophilized form. The composition can then be used to reconstitute solutions or formulations.

[0161] The administration methods to be considered are intravenous, subcutaneous, intradermal, or intramuscular.

[0162] According to a specific implementation plan, the method of administration is subcutaneous.

[0163] It also considered transdermal, oral, nasal, vaginal, ocular, oral, and pulmonary administration routes.

[0164] The present invention also relates to implantable or transportable pumps comprising compositions according to the present invention.

[0165] The present invention also relates to the use of compositions according to the invention intended for placement in implantable or transportable pumps.

[0166] This invention also relates to single-dose formulations.

[0167] In one embodiment, the formulation is in the form of an injectable solution.

[0168] The preparation of the composition according to the invention has the following advantages: it can be achieved by simply mixing an aqueous solution of an amylin analog or amylin receptor agonist and human insulin A21G in an aqueous solution or in lyophilized form.

[0169] If necessary, in the presence of excipients (e.g., glycerin, m-cresol, zinc chloride, and polysorbate 20) 20) Adjust the composition of the mixture. This addition can be made by adding a concentrated solution of the excipient.

[0170] In one embodiment, the composition is characterized in that its solubility at pH 4.0 and its physical stability as measured by ThT are greater than those of a reference composition containing an amylin analog or amylin receptor agonist and commercially available mealtime insulin.

[0171] ThT is measured according to the schemes described in some embodiments.

[0172] In one embodiment, the composition is characterized in that its solubility at pH 4.0 and its physical stability as measured by ThT are greater than those of a reference composition containing GLP-1, a GLP-1 analogue, or a GLP-1 receptor agonist and commercially available mealtime insulin.

[0173] Insulin and insulin analogs can be obtained using recombinant DNA techniques with bacteria (e.g., *Escherichia coli*) and yeasts (e.g., *Saccharomyces cerevisiae*) (see, for example, G. Walsh Appl. *Microbiol. Biotechnol.* 2005, 67, 151-159). Typically, proinsulin is produced and then digested with enzymes (e.g., trypsin and carboxypeptidase B) to obtain the desired sequence.

[0174] To produce human insulin A21G, proinsulin is encoded such that glycine is located at A21, and the desired insulin is obtained after digestion with trypsin and carboxypeptidase B. The procedure is described by Kohn et al. in Peptides 2007, 28, 935-948.

[0175] The present invention also relates to a method for obtaining human insulin A21G, comprising at least one step of reacting human insulin A21G, B31R, B32R (glargine insulin) with rat carboxypeptidase B at an insulin / carboxypeptidase ratio of 500 to 2000 at a pH of 7.5 to 8.5 and a temperature of 20 to 30°C for 10 to 20 hours. The product can then be purified. This purification can be performed by liquid chromatography.

[0176] Therefore, human insulin A21G can be obtained by digesting glargine insulin with carboxypeptidase B to remove two arginine residues. After enzymatic digestion, human insulin A21G is purified by chromatography and subsequently separated by lyophilization or crystallization using conventional methods. Attached image description:

[0177] Figure 1 The fibrillation hysteresis time was determined graphically.

[0178] Figure 1 This is a graphical representation of fibrillation lag time based on virtual instances. On the horizontal axis, time is displayed in minutes, and on the vertical axis, fluorescence ThT is displayed in arbitrary units (unitéarbitraire, au), with LT representing lag time.

[0179] Figure 2 Plasma concentrations of pramlinide and insulin after administration of formulation A21-8 (mean ± standard deviation)

[0180] The square represents the concentration of insulin, and the triangle represents the concentration of pramlinide.

[0181] On the horizontal axis, the time after injection is shown in minutes; on the left vertical axis, the baseline-corrected insulin concentration is shown in pmol / L, and the baseline-corrected pramlinpeptide concentration is shown in pmol / L.

[0182] Figure 3 Blood glucose levels (mean ± standard deviation) after administration of formulation A21-8.

[0183] On the horizontal axis, the time after injection is shown in minutes; and on the vertical axis, blood glucose is shown as a percentage of the baseline level.

[0184] Figure 4 Concentrations of pramlinide (mean ± standard deviation) after administration of formulation A21-9 (curve plotted with squares) and PRAM (curve plotted with triangles).

[0185] On the horizontal axis, the time after injection is shown in minutes; and on the vertical axis, the baseline-corrected pramlintide concentration is shown in pmol / L (subtracting the pre-administration concentration). Example

[0186] Example 1. Preparation of human insulin A21G

[0187] 5 g of insulin glargine (Gan & Lee Pharmaceuticals) was mixed with carboxypeptidase B (reference 08039852001; Sigma-Aldrich) at pH 8.0 (pH adjusted by adding Tris buffer) and incubated at 25°C for 17 hours, resulting in an insulin glargine concentration of approximately 4 mg / mL. The enzyme / insulin glargine ratio was 1 / 500. The mixture was then purified by liquid chromatography, dialyzed against 0.01 N hydrochloric acid, and subsequently lyophilized. The product was human insulin A21G with a purity of 98% and a yield of approximately 90%. The molecular weight of insulin was determined to be 5752 Da by mass spectrometry (Maldi-Tof). Human insulin A21G can also be obtained using recombinant techniques as described by Kohn et al. (Peptides 2007, 28, 935-948).

[0188] Example 2. A combination of prandial insulin and pramlinide, exenatide or lixisenatide at acidic pH.

[0189] Preparation of a solution containing human insulin A21G 100 U / mL (3.5 mg / mL) and pramlintide 1 mg / mL at an acidic pH of 3.5 or 4.0, consisting of m-cresol (25 mM), glycerol (184 mM), and zinc chloride (300 μM).

[0190] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin A21G (300 U / mL at pH 3.5). A concentrated solution of pramlintide (Ambiopharm) (10 mg / mL at pH 4) and a concentrated solution of zinc chloride were added to the concentrated solution of human insulin A21G and excipients to obtain the desired final composition. The final pH (i.e., 3.5 or 4.0) was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0191] Preparation of a solution containing human insulin A21G 100 U / mL and pramlintide 0.6 mg / mL at an acidic pH of 4.0, consisting of m-cresol (25 mM), glycerol (184 mM), zinc chloride (300 μM).

[0192] Prepare this solution in the same manner as the solution described above.

[0193] Preparation of a solution containing human insulin A21G 100 U / mL and pramlinpeptide 1 mg / mL at an acidic pH of 4.0, consisting of m-cresol (25 mM) and glycerol (184 mM).

[0194] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin A21G (800 U / mL at pH 3.5). A concentrated solution of pramlintide (10 mg / mL at pH 4) was added to the human insulin and excipient solutions to obtain the desired final composition. The final pH (i.e., 4.0) was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0195] Preparation of a pH 4 solution containing m-cresol (25 mM), glycerol (184 mM), Tween 20 (10 μg / mL), human insulin A21G 100 U / mL, and pramlintide 1 mg / mL.

[0196] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin A21G (300 U / mL at pH 3.5). A concentrated solution of pralamin (10 mg / mL at pH 4) and Tween 20 was added to the concentrated solution of human insulin A21G and excipients to obtain the desired final composition. The final pH was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0197] Preparation of a pH 4 solution containing m-cresol (25 mM), glycerol (184 mM), zinc chloride (300 μM), Tween 20 (10 μg / mL), human insulin A21G 100 U / mL, and pramlintide 1 mg / mL.

[0198] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin A21G (300 U / mL at pH 3.5). A concentrated solution of pramlintide (10 mg / mL at pH 4), a concentrated solution of zinc chloride, and a concentrated solution of Tween 20 were added to the concentrated solution of human insulin A21G and excipients to obtain the desired final composition. The final pH was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0199] Preparation of a solution containing 100 U / mL human insulin A21G and 50 μg / mL exenatide at an acidic pH, consisting of m-cresol (25 mM), glycerol (184 mM), and zinc chloride (300 μM).

[0200] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin A21G (300 U / mL at pH 3.5). A concentrated solution of exenatide (Bachem) (10.5 mg / mL at pH 4) and a concentrated solution of zinc chloride were added to the concentrated solution of human insulin A21G and excipients to obtain the desired final composition. The final pH was adjusted to the desired value of 4.0 by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0201] Preparation of a solution containing 100 U / mL human insulin A21G and 100 μg / mL lixisenatide at an acidic pH. (25 mM mC, 184 mM glycerol, and 300 μM zinc chloride)

[0202] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin A21G (230 U / mL at pH 3.5). Concentrated solutions of liximabide (Ambiopharm) (10.5 mg / mL at pH 4) and zinc chloride were added to the concentrated solutions of human insulin A21G and excipients to obtain the desired final composition. The final pH was adjusted to the desired value of 4.0 by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0203] Preparation of a solution containing m-cresol (25 mM), glycerol (184 mM), and Tween 20 (10 μg / mL) human insulin A21G 100 U / mL, exenatide 50 μg / mL, and pramlintide 0.6 mg / mL at an acidic pH of 4.0.

[0204] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin A21G (300 U / mL at pH 3.5). Concentrated solutions of pramlintide (Ambiopharm) (10 mg / mL at pH 4), exenatide (Bachem) (10.5 mg / mL at pH 4), and Tween 20 were added to the concentrated solutions of human insulin A21G and excipients to obtain the desired final composition. The final pH was adjusted to the desired value of 4.0 by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0205] Preparation of a solution containing 100 U / mL human insulin and 1 mg / mL pramlinpeptide at an acidic pH of 3.5 or 4.0, consisting of m-cresol (25 mM), glycerol (184 mM), and zinc chloride (300 μM).

[0206] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of human insulin (Amphastar Pharmaceuticals) (800 U / mL at pH 3.5). A concentrated solution of pramlintide (10 mg / mL at pH 4) and a concentrated solution of zinc chloride were added to the concentrated solution of human insulin and excipients to obtain the desired final composition. The final pH (i.e., 3.5 or 4.0) was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0207] Preparation of a solution of insulin aspart at 100 U / mL and pramlintide at 1 mg / mL containing m-cresol (25 mM), glycerol (184 mM), and zinc chloride (300 μM) at an acidic pH of 3.5 or 4.0.

[0208] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of insulin aspart (HEC Pharmaceuticals) (500 U / mL at pH 3). A concentrated solution of pramlintide (10 mg / mL at pH 4) and a concentrated solution of zinc chloride were added to the concentrated solution of insulin aspart and excipients to obtain the desired final composition. The final pH (i.e., 3.5 or 4.0) was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. After pH adjustment, the solution adjusted to pH 4.0 was cloudy. The solution adjusted to pH 3.5 was clear. It was then filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0209] Preparation of a solution of lispro insulin 100 U / mL and pramlintide 1 mg / mL containing m-cresol (25 mM), glycerol (184 mM), and zinc chloride (300 μM) at an acidic pH of 3.5 or 4.0.

[0210] A concentrated solution of excipients (m-cresol, glycerol) was added to a concentrated solution of lispro insulin (Gan & Lee Pharmaceuticals) (650 U / mL at pH 3). A concentrated solution of pramlintide (10 mg / mL at pH 4) and a concentrated solution of zinc chloride were added to the concentrated solution of lispro insulin and excipients to obtain the desired final composition. The final pH (i.e., 3.5 or 4.0) was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial).

[0211] Commercial products are present at acidic pH values ​​of 3.0, 3.5, or 4.0. Preparation of solutions of insulin glutathione 100 U / mL and pramlinpeptide 1 mg / mL with excipients (29 mM cresol, 50 mM Tris, 86 mM zinc chloride and 8.15 μM Tween 20).

[0212] Commercial insulin glutalis was prepared by adding an aqueous HCl solution. The pH of the solution was adjusted to pH 2.5. This solution was added to pramlinpeptide powder to obtain a solution containing 100 U / mL insulin and 1 mg / mL pramlinpeptide. The final pH was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. After pH adjustment, the solutions adjusted to pH 3.5 and 4.0 were cloudy. The solution adjusted to pH 3.0 was clear. It was filtered through a 0.22 μm filter and stored in glass vials (1 mL solution per vial). After several hours of storage, the solution became cloudy and non-uniform.

[0213] Preparation of a 1 mg / mL solution of pramlinpeptide containing m-cresol (20 mM), mannitol (236 mM), and acetate / sodium acetate buffer (30 mM) at pH 4.0.

[0214] A 10 mg / mL concentrated solution of pramlinpeptide was prepared by dissolving the powdered form purchased from Ambiopharm. This solution was then added to a concentrated solution of excipients (m-cresol, mannitol, and acetate / sodium acetate buffer) to obtain the desired final composition. The final pH was adjusted to 4.0 ± 0.2 by adding NaOH / HCl.

[0215] Preparation of a pH 4 solution containing m-cresol (25 mM), glycerol (184 mM), acetate / sodium acetate buffer (18 mM), and Tween 20 (8 μM) of human insulin A21G 100 U / mL and pramlinpeptide 0.6 mg / mL.

[0216] A concentrated solution of glycerol and m-cresol was added to a concentrated solution of human insulin A21G in an acetate / sodium acetate buffer at pH 4 (300 U / mL). Finally, a concentrated solution of ambiopharm (10 mg / mL at pH 4) and Tween 20 were added to the concentrated solution of human insulin A21G and excipients to obtain the desired final composition. The final pH was adjusted to the desired value by adding an aqueous solution of NaOH or HCl. The resulting solution was clear and homogeneous; it was filtered through a 0.22 μm filter.

[0217] The compositions prepared above are listed in Table 1 below:

[0218] Table 1: Combinations of insulin and / or glucagon inhibitors

[0219]

[0220]

[0221]

[0222] Example 3. Study on the compatibility of prandial insulin with pramlintide under acidic pH.

[0223] Visual appearance of solutions of insulin and pramlinpeptide at acidic pH.

[0224] After the solutions in the container have stabilized for 2 to 3 hours, they are observed at ambient temperature. Table 2 shows the visual appearance of the insulin and pramlinpeptide solutions described above.

[0225] Table 2. Visual appearance of solutions of insulin and pramlintide.

[0226] Composition Visual appearance A21-1 clarify A21-2 clarify A21-3 clarify RHI-1 clarify RHI-2 clarify ASP-1 turbid ASP-2 clarify GLU-1 turbid GLU-2 turbid GLU-3 turbid LIS-1 clarify LIS-2 clarify

[0227] Of the insulins evaluated, only human insulin, lispro insulin, and human insulin A21G resulted in homogeneous and clear formulations with pramlintide at pH 4, demonstrating the substance's solubility. Aspart insulin and glutalis insulin were not suitable for obtaining clear formulations with pramlintide at pH 4.0.

[0228] Example 4. Study on fibrillation hysteresis time.

[0229] principle

[0230] Poor stability of peptides can lead to the formation of amyloid fibrils, which are defined as ordered macromolecular structures. These fibrils can cause gel formation within the sample.

[0231] The fluorescence assay for thiosulfate T (ThT) is used to analyze the physical stability of formulations. Thisulfate T is a small probe molecule that exhibits a characteristic fluorescence signature when it binds to amyloid fibrils (Naiki et al. (1989) Anal. BioChem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284).

[0232] This method enables monitoring of fibril formation at low ThT concentrations in undiluted formulations. This monitoring was conducted under accelerated stability conditions: with stirring and at 37°C.

[0233] Experimental conditions

[0234] Samples were prepared just before measurement. The preparation of each composition is described in the relevant examples. Thiamine T was added to the composition from the concentrated stock solution so that dilution of the composition was negligible. The concentration of thiosulfate T in the composition was 40 μM. 150 μL of the composition was introduced into the wells of a 96-well plate. Each composition was analyzed in triplicate on the same plate. The plate was sealed with a transparent membrane to prevent evaporation of the composition.

[0235] The board was then placed inside the housing of the board reader (EnVision 2104 Multilabel, Perkin Elmer). The temperature was set to 37°C, and transverse stirring was performed at 960 rpm and an amplitude of 1 mM.

[0236] The fluorescence intensity in each well was read out over time using an excitation wavelength of 442 nm and an emission wavelength of 482 nm.

[0237] The fibrillation process is manifested by a strong increase in fluorescence after a delay known as the hysteresis time.

[0238] For each well, the delay is graphically defined as the intersection of the baseline of the fluorescence signal and the ramp of the fluorescence-to-time curve, determined during the initial strong increase in fluorescence. Figure 1 As shown. The plotted hysteresis time values ​​correspond to the average hysteresis time of the three orifices.

[0239] Then, in the presence of ThT, the clarified solutions of pramlinpeptide and insulin at pH 3.5 and 4.0 of the aforementioned examples were subjected to fibrillation tests.

[0240] The lag times reported in Table 3 correspond to the average of the three measurements; the uncertainty interval corresponds to the standard deviation between these three results.

[0241] Table 3. Lag time of insulin and pramlintide solutions.

[0242] Composition Lag time (h) A21-1 13.7+ / -0.8 A21-2 10.4+ / -1.8 A21-3 15.6+ / -5.3 RHI-1 5.0+ / -0.7 RHI-2 1.7+ / -0 ASP-2 2.0+ / -0 LIS-1 1.8+ / -0.1 LIS-2 4.3+ / -0.4

[0243] Surprisingly, the fibrinolysis lag time of formulations containing human insulin A21G was much longer than that of commercially available insulins tested at pH 3.5 or pH 4.0, especially longer than that of rapid-acting insulin analogs lispro and aspart.

[0244] Example 5. Study on fibrillation hysteresis time in the presence of Tween 20

[0245] Table 4 shows the hysteresis times of solutions of human insulin A21G and pramlintide at pH 4 in the presence of Tween 20.

[0246] Table 4. Lag time of solutions of human insulin A21G and pramlintide in the presence of Tween 20.

[0247] Composition Lag time (h) A21-4 38.1+ / -8.8 A21-5 48.3+ / -9.2

[0248] Therefore, the physical stability was improved in the presence of 10 μg / mL Tween 20.

[0249] Example 6. Physical stability of the formulation under static conditions at 30°C

[0250] Place glass vials containing 1 mL of the composition in an oven maintained at 30°C. Visually inspect the vials for the appearance of visible particles or turbidity. This examination is performed according to the recommendations of the European Pharmacopoeia (EP 2.9.20): expose the vials to light at a minimum of 2000 lux and observe them against both white and black backgrounds. These results are consistent with those of the United States Pharmacopeia (USP). <790> Consistent.

[0251] Table 5. Physical stability of insulin and pramlinpeptide solutions under static conditions at 30°C.

[0252] Composition Visual appearance after 4 weeks A21-4 clarify A21-5 clarify A21-2 clarify A21-1 clarify A21-3 clarify ASP-2 turbid

[0253] Insulin aspart formulated with pramlinide at pH 3.5 is less stable than human insulin A21G formulated with pramlinide at pH 3.5 or 4.0.

[0254] Example 7. Study on the physical stability of human insulin A21G with exenatide and lixisenatide.

[0255] Formulations A21-6 and A21-7 were placed in a container and subsequently kept at 30°C for 4 weeks. The fibrillation hysteresis time of the temporarily formulated formulations was measured and is listed in Table 6.

[0256] Table 6: Hysteresis time and physical stability of human insulin A21G solutions with exenatide or lixisenatide at 30°C under static conditions.

[0257] Composition Lag time (h) Visual appearance after 4 weeks at 30°C A21-6 18.1+ / -5.6 clarify A21-7 33.0+ / -9.7 clarify

[0258] Example 8. Chemical stability of formulations of human insulin A21G and pramlintide

[0259] All formulations were prepared at pH 4.0 or pH 3.5 and contained 100 U / mL human insulin A21G, 1 mg / mL pramlintide, 25 mM m-cresol, and 184 mM glycerol. The formulations were stored in glass cylinders and kept at 30°C under static conditions. Human insulin A21G and pramlintide were determined by reversed-phase liquid chromatography (HPLC). The results are shown in Table 7.

[0260] Table 7: Changes in insulin concentration (a) (U / mL) and pramlinide concentration (b) (mg / mL)

[0261]

[0262] Formulations containing human insulin A21G and pramlintide exhibited good chemical stability after 4 weeks at 30°C. Pramlintide formulations containing commercial insulin degraded rapidly at pH 4.0, and even more rapidly at pH 3.5.

[0263] Example 9. Pharmacokinetic and Pharmacodynamic Studies in Dogs

[0264] Pharmacokinetic and pharmacodynamic studies in dogs of a combination consisting of human insulin A21G (100 U / mL, i.e., 3.5 mg / mL) and pramlintide (0.6 mg / mL). The formulation tested was at pH 4.0 and contained 25 mM m-cresol and 184 mM glycerol (formulation A21-8).

[0265] Four animals, which had been fasting for approximately 18 hours, received subcutaneous injections of 0.2 U / kg insulin and 0.12 μg / kg pramlintide in the neck. One hour prior to injection, one or more blood samples were drawn to determine baseline levels of glucose, insulin, and pramlintide. Blood samples were then drawn over a 5-hour period following administration. Blood glucose levels were measured using a glucometer. Plasma insulin and pramlintide levels were determined by ELISA testing.

[0266] Pharmacokinetic parameters of formulation A21-8 were assessed based on baseline-corrected plasma concentrations of insulin and pramlinide. Standard non-compartmental analyses were performed using Phoenix WinNonlin software (version 7, Certara). Parameter values ​​(mean ± standard deviation) are recorded in Tables 8 and 9 below:

[0267] Table 8: PK parameters of total insulin analogs

[0268]

[0269] Table 9: PK parameters of pramlin peptide

[0270]

[0271] Figure 2 The mean pharmacokinetic (PK) profiles of total insulin (squares) and pramlinpeptide (triangles) in plasma are shown in the figure.

[0272] Figure 3 The average blood glucose profile is shown as a percentage of baseline levels.

[0273] Both pramlintide and human insulin A21G exhibited mealtime absorption kinetics, resulting in early hypoglycemic activity, followed by a return to near-baseline blood glucose levels 5 hours after administration. These pharmacokinetic and pharmacodynamic results clearly demonstrate that formulation A21-8 is compatible with mealtime administration.

[0274] Example 10: Pharmacokinetic Study of Pramlinide in Pigs

[0275] Pharmacokinetic studies of a combination of human insulin A21G (3.5 mg / mL, equivalent to 100 U / mL insulin) and pramlintide (0.6 mg / mL) in pigs.

[0276] Before the experiment began, domestic pigs weighing approximately 50 kg with a catheter pre-inserted in their jugular vein were fasted for 2.5 hours. During that hour before insulin injection, three blood samples were drawn to determine baseline glucose and insulin levels.

[0277] Using an insulin pen (Novo, Sanofi, or Eli Lilly) equipped with a 31G needle, an insulin formulation of human insulin A21G (A21-9) or pramlintide (PRAM) is injected subcutaneously into the flank of an animal at a dose of 0.2 U / kg insulin and 1.2 μg / kg pramlintide.

[0278] To determine the concentration of pramlinitide in plasma, blood samples were drawn at the following times: 4, 8, 12, 16, 20, 30, 40, 50, 60, 70, 80, 100, 120, 150, and 180 minutes. After each draw, the catheter was flushed with a diluted heparin solution.

[0279] Pharmacokinetic results of human insulin A21G, pramlinide solution A21-9, and pramlinide solution PRAM in pigs

[0280] Results from three studies conducted on the same herd of pigs were synthesized to compare the pharmacokinetics of pramlinineptide between formulation A21-9 and formulation PRAM. Pharmacokinetic parameters of formulations A21-9 and PRAM were assessed based on baseline-corrected plasma pramlinineptide concentrations. Standard non-compartmental analyses were performed using Phoenix WinNonlin software (version 7, Certara). Parameter values ​​(mean ± standard deviation) are listed in the table below.

[0281] Table 10: PK parameters of pramlinpeptide in compositions A21-9 and PRAM

[0282]

[0283] Among them, t max == Time required to observe maximum plasma concentration; AUC 0-30min =Area under the plasma concentration versus time curve from 0 to 30 minutes after injection; AUC 0-t =0 to the area under the plasma concentration versus time curve from the final quantifiable concentration after injection

[0284] The pharmacokinetic results of pramlinide obtained using formulations A21-9 and PRAM are shown in... Figure 4Analysis of these spectra and parameters showed that the combination of human insulin A21G and pramlintide (formulation A21-9, curve plotted as a square) resulted in a significantly slower absorption of pramlintide compared to pramlintide alone (formulation PRAM, curve plotted as a triangle). Formulation A21-9 resulted in a significantly slower plasma peak (t...) compared to formulation PRAM. max Significantly delayed (approximately 18 min, p < 0.05) and early plasma exposure (AUC) of pramlinitide. 0-30min The total plasma exposure (AUC) of pramlinitide was significantly reduced (approximately 43%, p < 0.05). On the other hand, the total plasma exposure of pramlinitide between the two formulations was significantly reduced (approximately 43%, p < 0.05). 0-t The results appear similar, indicating comparable bioavailability.

[0285] Example 11. Feeding study in rats after injection of the control composition and after injection of a composition containing human insulin A21G and / or pramlintide.

[0286] The study was conducted on a group of 40 male Sprague Dawley rats at least 6 weeks old.

[0287] Except for a 6-hour fasting period prior to subcutaneous injection of the compositions described in the table below, rats had free access to food and water.

[0288] Table 11. Compositions injected into rats and number of rats treated

[0289]

[0290] The control composition was a salt solution, i.e., an aqueous solution containing 150 mM NaCl.

[0291] Composition R is a commercially available solution of human insulin marketed by ELI LILLY. This product is 100 U / mL human insulin. The excipients for R are glycerol, m-cresol, sodium hydroxide for pH adjustment (pH 7.0-7.8), hydrochloric acid, and water.

[0292] Food (approximately 100g per rat) was distributed at t0, just before injection. Food intake (cumulative average) was measured at 1, 2, and 3 hours after t0, or at t+1h, t+2h, and t+3h.

[0293] The results are shown in the table below:

[0294] Table 12: Food intake 1, 2 and 3 hours after injection

[0295] Composition Comparison Humulin PRAM A21-9 Food intake (g) at t+1h 3.8 4.7 1.5 3.2 Food intake (g) at t+2h 4.4 5.3 3.3 4.4 Food intake (g) at t+3h 5.9 6.9 4.2 5.3

[0296] These results indicate that the combination of insulin A21G and pramlintide, composition A21-9, enables not only a reduction in food intake induced by insulin injection, but also a restriction of food intake to levels less than or equal to those of the control group receiving injections of the control composition (saline solution).

Claims

1. Composition in the form of an injectable aqueous solution, having a pH of 3.5 to 4.4, comprising at least the so-called regular human insulin A21G, which differs from human insulin only in that the asparagine residue in position 21 of the A chain is replaced by a glycine residue, and pramlintide, wherein the concentration of said human insulin A21G is 2 to 20 mg / mL and the concentration of pramlintide is 0.6 to 1 mg / mL.

2. The composition of claim 1, wherein The concentration of human insulin A21G is 3.5 mg / mL.

3. The composition according to claim 1 or 2, characterized in that The pH of the solution is 3.8 to 4.

2.

4. The composition according to claim 1 or 2, characterized in that The pH of the solution is 4.

0.

5. The composition according to claim 1 or 2, characterized in that It further comprises a zinc salt.

6. The composition according to claim 1 or 2, characterized in that It further comprises m-cresol.

7. The composition according to claim 1 or 2, characterized in that It also comprises 20 excipient.

8. The composition according to claim 1 or 2, characterized in that It further comprises the poloxamer 188 excipient.

9. The composition according to claim 1 or 2, characterized in that It further comprises methionine.

10. The composition according to claim 1 or 2, intended for use in a method for the treatment of diabetes, characterized in that it is administered as a bolus before a meal.

11. The composition according to claim 1 or 2, intended for use in a method for the treatment of diabetes, characterized in that it is administered to improve control of postprandial glycemia.

12. The composition according to claim 1 or 2, intended for use in a method for the treatment of diabetes, characterized in that it is administered to improve control of postprandial glycemia and reduce adverse effects of pramlintide.

13. The composition according to claim 1 or 2, intended for use in a method for the treatment of diabetes, characterized in that it enables reduction of food intake induced by insulin.

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