PHARMACEUTICAL COMPOSITION IN THE FORM OF AN INJECTABLE AQUEOUS SOLUTION INCLUDING AT LEAST A RAPID-ACTING INSULIN ANALOG AND A GLUCAGON SUPPRESSOR WITH PRANDIAL ACTION

Abstract

A composition in the form of an injectable aqueous solution, the pH of which is from 3.0 to 4.4, in particular from 3.6 to 4.4, including at least a rapid-acting insulin analog and at least one glucagon suppressor with prandial action. The glucagon suppressor with prandial action is selected from the group consisting of an amylin analog or an amylin receptor agonist or a GLP-1 analog or a GLP-1 receptor agonist (GLP-1 RA). The glucagon suppressor with prandial action is an amylin analog or an amylin receptor agonist. The glucagon suppressor peptide with prandial action is pramlintide.

Claims

1-20. (canceled)

21. A pharmaceutical composition in the form of an injectable aqueous solution, the pH of which is from 3.0 to 4.4, comprising pramlintide and a rapid-acting insulin analog insulin lispro A21G, also called human insulin A21G, B28K, B29P.

22. The pharmaceutical composition according to claim 21, wherein it is free of zinc.

23. The pharmaceutical composition according to claim 21, wherein it comprises nicotinamide.

24. The pharmaceutical composition according to claim 21, wherein the concentration of insulin lispro A21G is from 2 to 20 mg/mL.

25. The pharmaceutical composition according to claim 21, wherein the concentration of insulin lispro A21G or lispro A21G, desB30 is 3.5 mg/mL.

26. The pharmaceutical composition according to claim 21, wherein the concentration of pramlintide is from 0.32 to 5 mg/mL.

27. The pharmaceutical composition according to claim 21, wherein the pH of the solution is from 3.7 to 4.3.

28. The pharmaceutical composition according to claim 21, for use in the treatment of diabetes, wherein it is administered as one or several boluses at mealtime.

29. The pharmaceutical composition according to claim 21, for use in the treatment of diabetes, wherein, it is administered using a pump.

30. The pharmaceutical composition according to claim 21, for use in the treatment of diabetes, wherein it is administered to improve the control of postprandial glycemia.

31. The pharmaceutical composition according to claim 21, for use in the treatment of diabetes, wherein it is administered to improve the control of postprandial glycemia and to decrease the adverse effects of pramlintide.

32. The pharmaceutical composition according to claims 21, for use in the treatment of diabetes, wherein enables to decrease the food consumption induced by insulin.

Description

[0128] In the present application, as mentioned, amylin refers to the compounds described in the Pats. US 5,124,314 and US 5,234,906. When the term “analog” is used, it refers to a peptide or a protein in which one or more constitutive amino acid residues of the primary sequence have been substituted by other amino acid residues and/or in which one or more constitutive amino acid residues have been eliminated and/or in which one or more constitutive amino acid residues have been added. The percentage of homology that is accepted for the present definition of an analog is 50%. In the case of amylin, an analog can be, for example, derived from the primary amino acid sequence of amylin by substituting one or more natural or non-natural or peptidomimetic amino acids.

[0129] In an embodiment, the invention relates to a pharmaceutical composition in the form of an injectable aqueous solution, the pH of which is between 3.0 to 4.4, in particular 3.6 to 4.4, including at least a rapid-acting insulin analog with the A21 substitution and a GLP-1 receptor agonist or a GLP-1 analog. According to an embodiment, said GLP-1 receptor agonist is exenatide. According to another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0130] In an embodiment, the invention relates to a pharmaceutical composition in the form of an injectable aqueous solution, the pH of which is between 3.7 to 4.3, including at least a rapid-acting insulin analogs with A21 substitution, and free of zinc or magnesium ions and a GLP-1 receptor agonist or a GLP-1 analog. According to an embodiment, said GLP-1 receptor agonist is exenatide. According to another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0131] In an embodiment, the invention relates to a pharmaceutical composition in the form of an injectable aqueous solution, the pH of which is between 3.8 to 4.2, including at least a rapid-acting insulin analog with the A21 substitution and a GLP-1 receptor agonist or a GLP-1 analog. According to an embodiment, said GLP-1 receptor agonist is exenatide. According to another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0132] In an embodiment, the invention relates to a pharmaceutical composition in the form of an injectable aqueous solution, the pH of which is 3.8, including at least a rapid-acting insulin analog with the A21 substitution and a GLP-1 receptor agonist or a GLP-1 analog. According to an embodiment, said GLP-1 receptor agonist is exenatide. According to another embodiment, said GLP-1 receptor agonist is lixisenatide.

[0133] Exenatide and lixisenatide, which are described in the applications US2004/0023871 and WO0104156, respectively, are generally considered to be GLP-1 receptor agonists. In an embodiment, the glucagon suppressor with prandial action is pramlintide (Symlin®).

[0134] In an embodiment, the GLP-1, GLP-1 analogs, or GLP-1 RA are referred to as “short-acting” or “prandial.” “Short-acting” or “prandial” is understood to mean GLP-1, GLP-1 analogs, or GLP-1 RA of which the apparent half-life of elimination after subcutaneous injection in humans is less than 8 hours, in particular less than 5 hours, preferably less than 4 hours or else less than 3 hours, such as, for example, exenatide or lixisenatide.

[0135] In an embodiment, the GLP-1, the GLP-1 analogs, or the GLP-1 RA are selected from the group consisting of exenatide (Byetta®), lixisenatide (Lyxumia®), the analogs or derivatives thereof and pharmaceutically acceptable salts thereof.

[0136] In an embodiment, the GLP-1, the GLP-1 analog, or GLP-1 RA is exenatide or Byetta®, analogs or derivatives thereof and pharmaceutically acceptable salts thereof.

[0137] In an embodiment, GLP-1, GLP-1 analog, or GLP-1 RA is lixisenatide or Lyxumia®, analogs or derivatives thereof and pharmaceutically acceptable salts thereof.

[0138] In an embodiment, the concentration of pramlintide is from 0.32 to 5 mg/mL.

[0139] In an embodiment, the concentration of pramlintide is from 0.5 to 1.5 mg/mL.

[0140] In an embodiment, the concentration of pramlintide is from 0.6 to 1.2 mg/mL.

[0141] In an embodiment, the concentration of pramlintide is from 0.6 to 1 mg/mL.

[0142] In an embodiment, the concentration of pramlintide is 1.0 mg/mL.

[0143] In an embodiment, the concentration of pramlintide is 0.6 mg/mL.

[0144] In an embodiment, the concentration of exenatide is from 30 to 2000 .Math.g/ml.

[0145] In an embodiment, the concentration of exenatide is from 50 to 1500 .Math.g/ml.

[0146] In an embodiment, the concentration of exenatide is from 10 to 1000 .Math.g/mL.

[0147] In an embodiment, the concentration of exenatide is from 100 to 1000 .Math.g/ml.

[0148] In an embodiment, the concentration of exenatide is from 40 to 150 .Math.g/mL.

[0149] In an embodiment, the concentration of exenatide is from 40 to 80 .Math.g/mL.

[0150] In an embodiment, the concentration of exenatide is 50 .Math.g/mL.

[0151] In an embodiment, the concentration of lixisenatide is from 80 to 3000 .Math.g/ml.

[0152] In an embodiment, the concentration of lixisenatide is from 100 to 2000 .Math.g/ml.

[0153] In an embodiment, the concentration of lixisenatide is from 150 to 1500 .Math.g/ml.

[0154] In an embodiment, the concentration of lixisenatide is from 20 to 1000 .Math.g/mL.

[0155] In an embodiment, the concentration of lixisenatide is from 80 to 160 .Math.g/mL.

[0156] In an embodiment, the concentration of lixisenatide is 100 .Math.g/mL.

[0157] In an embodiment, the concentration of pramlintide is from 0.4 to 3 mg/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0158] In an embodiment, the concentration of pramlintide is from 0.5 to 1.5 mg/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0159] In an embodiment, the concentration of pramlintide is from 0.6 to 1.2 mg/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0160] In an embodiment, the concentration of pramlintide is from 0.6 to 1 mg/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0161] In an embodiment, the concentration of pramlintide is 1.0 mg/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0162] In an embodiment, the concentration of pramlintide is 0.6 mg/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0163] In an embodiment, the concentration of exenatide is from 10 to 1000 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0164] In an embodiment, the concentration of exenatide is from 30 to 1000 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0165] In an embodiment, the concentration of exenatide is from 50 to 750 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0166] In an embodiment, the concentration of exenatide is from 50 to 500 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0167] In an embodiment, the concentration of exenatide is from 40 to 150 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0168] In an embodiment, the concentration of exenatide is from 40 to 80 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0169] In an embodiment, the concentration of exenatide is 50 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0170] In an embodiment, the concentration of lixisenatide is from 80 to 1500 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0171] In an embodiment, the concentration of lixisenatide is from 100 to 1400 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0172] In an embodiment, the concentration of lixisenatide is from 120 to 1200 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0173] In an embodiment, the concentration of lixisenatide is from 20 to 1000 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0174] In an embodiment, the concentration of lixisenatide is from 80 to 160 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0175] In an embodiment, the concentration of lixisenatide is 100 .Math.g/mL for 100 U/ml of the rapid-acting insulin with A21 substitution.

[0176] In an embodiment, the pharmaceutical composition according to the invention does not contain a long-acting insulin.

[0177] In an embodiment, the pharmaceutical composition according to the invention moreover includes buffers.

[0178] In an embodiment, the pharmaceutical composition according to the invention includes a buffer selected from the group consisting of a sodium acetate buffer and Tris.

[0179] In an embodiment, the pharmaceutical composition according to the invention moreover includes preservatives.

[0180] In an embodiment, the preservatives are selected from the group consisting of m-cresol and phenol, alone or in a mixture.

[0181] In an embodiment, the preservative is m-cresol.

[0182] In an embodiment, the concentration of m-cresol is ranging from 10 to 50 mM.

[0183] In an embodiment, the concentration of m-cresol is ranging from 10 to 40 mM.

[0184] In an embodiment, the preservative is phenol.

[0185] In an embodiment, the concentration of phenol is ranging from 10 to 60 mM.

[0186] In an embodiment, the concentration of phenol is ranging from 20 to 50 mM.

[0187] In an embodiment, the pharmaceutical composition according to the invention includes a surfactant.

[0188] In an embodiment, the surfactant is selected from the group consisting of Poloxamer 188, Tween® 20, also referred to as Polysorbate 20, and Tween® 80, also referred to as Polysorbate 80.

[0189] In an embodiment, the Tween® 20 concentration varies from 5 to 50 .Math.g/mL.

[0190] In an embodiment, the Tween® 20 concentration varies from 5 to 25 .Math.g/mL.

[0191] In an embodiment, the Tween® 20 concentration is 10 .Math.g/mL.

[0192] In an embodiment, the Tween® 20 concentration is 10 .Math.M.

[0193] The pharmaceutical composition according to the invention can moreover include additives such as tonicity agents.

[0194] In an embodiment, the tonicity agents are selected from the group consisting of glycerol, sodium chloride, mannitol and glycine.

[0195] In an embodiment, the pharmaceutical composition according to the invention moreover includes an antioxidant.

[0196] In an embodiment, the antioxidant is methionine.

[0197] In one embodiment, the pharmaceutical composition further comprises at least one absorption promoter chosen from absorption promoters, diffusion promoters or vasodilator agents, individually or in combination.

[0198] 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, niacin amide, vitamin B3 and their salts; inhibitors of pancreatic trypsin; magnesium salts; polyunsaturated fatty acids; phosphatidylcholine didecanoyl; aminopolycarboxylates; tolmetin; sodium caprate; salicylic acid; oleic acid; linoleic acid; eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); benzyl acid; donors of nitric oxide, for example, 3- (2-Hydroxy-1- (1-methylethyl) -2-nitrosohydrazino) -1-propanamine, N-ethyl-2- (1-ethyl-hydroxy 2-1-nitrosohydrazino) -ethanamine, or S-nitroso-N-acetylpenicillamine; bile acids, glycine in its bile acid conjugated form; sodium ascorbate, potassium ascorbate; sodium salicylate, potassium salicylate, acetylsalicylic acid, salicylosalicylic acid, aluminum acetylsalicylate, choline salicylate, salicylamide, lysine acetylsalicylate; exalamide; the diflunisal; ethenzamide; EDTA; alone or in combination.

[0199] 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.

[0200] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator.

[0201] In one embodiment, the pharmaceutical composition further comprises at least one vasodilator causing hyperpolarization by blocking calcium ion channels.

[0202] In one embodiment, the vasodilator agent causing hyperpolarization by blocking the ion channels of calcium is adenosine, a hyperpolarizing agent obtained from endothelium, a phosphodiesterase type 5 (PDE5) inhibitor, a potassium channel opening agent or any combination of these agents.

[0203] In one embodiment, the pharmaceutical composition further comprises at least one cAMP mediated vasodilator.

[0204] In one embodiment, the pharmaceutical composition further comprises at least one cGMP-mediated vasodilator.

[0205] In one embodiment, the pharmaceutical composition further comprises at least one vasodilating agent chosen from the group consisting of vasodilator agents that react by causing hyperpolarization by blocking calcium ion channels, cAMP-mediated vasodilator agents, and cGMP-mediated vasodilators agents.

[0206] At least one vasodilator is chosen from the group consisting of nitrogen monoxide donors, for example, nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, amyl nitrate, erythrityl, 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 trisphosphate; the GPLC; L-carnitine; arginine; prostaglandin D2; potassium salts; and in some cases, alpha1 and alpha2 receptor antagonists, e.g., prazosin, phenoxybenzamine, phentolamine, dibenamine, moxisylyte hydrochloride and tolazoline), betazole, dimaprit; beta2 receptor agonists, for example, isoproterenol, dobutamine, albuterol, terbutaline, aminophylline, theophylline, caffeine; alprostadil, ambrisentan; the cabergoline; diazoxide; dihydralazine mesilate; diltiazem hydrochloride; enoximone; flunarizine hydrochloride; Ginkgo biloba extract; levosimendan; molsidomine; the acidic oxalate of naftidrofuryl, nicorandil, pentoxifylline; phenoxybenzamine chloride; the piribedil base; the piribedil mesilate; regadenoson monohydrate; riociguat; sildenafil citrate, tadalafil, vardenafil hydrochloride trihydrate; trimetazidine hydrochloride; trinitrine; 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.

[0207] According to one embodiment, the vasodilator agent is treprostinil.

[0208] In one embodiment, the composition comprises a nicotinic compound or one of its derivatives.

[0209] In one embodiment, the composition comprises nicotinamide.

[0210] In one embodiment, the concentration of nicotinamide ranges from 10 to 210 mM

[0211] In one embodiment, the concentration of nicotinamide ranges from 10 to 160 mM.

[0212] In one embodiment, the concentration of nicotinamide ranges from 20 to 150 mM.

[0213] In one embodiment, the concentration of nicotinamide ranges from 40 to 120 mM.

[0214] In one embodiment, the concentration of nicotinamide ranges from 60 to 100 mM.

[0215] In an embodiment, the pharmaceutical composition according to the invention contains 3.5 mg/mL to 10.5 mg/mL of insulin lispro A21G, 0.4 mg/mL to 3 mg/mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerol at a pH of 3.8. This composition can moreover include polysorbate 20, in particular from 8 to 10 .Math.M, and most particularly 8 .Math.M. This composition can also include nicotinamide, in particular from 10 to 210 mM.

[0216] In an embodiment, the pharmaceutical composition according to the invention contains 3.5 mg/mL of insulin lispro A21G, 0.6 mg/mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerol, at a pH of 3.8. This composition can moreover include polysorbate 20, in particular 8 .Math.M. This composition can also include nicotinamide, in particular from 10 to 210 mM.

[0217] In an embodiment, the pharmaceutical composition according to the invention contains 3.5 mg/mL of insulin lispro A21G, 0.6 mg/mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerol, 18 mM acetate buffer at a pH of 4.0. This composition can moreover include polysorbate 20, in particular 8 .Math.M. This composition can also include nicotinamide, in particular from 10 to 210 mM.

[0218] In an embodiment, the pharmaceutical composition according to the invention contains 3.5 mg/mL of insulin lispro A21G, 0.6 mg/mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerol, 18 mM acetate buffer at a pH of 3.7. This composition can moreover include polysorbate 20, in particular 8 .Math.M. This composition can also include nicotinamide, in particular from 10 to 210 mM.

[0219] In an embodiment, the pharmaceutical composition according to the invention contains 7.0 mg/mL of insulin lispro A21G, from 0.8 to 2.0 mg/mL of pramlintide, 25 mM of m-cresol, 150 to 200 mM of glycerol, at a pH of 4.0. This composition can moreover include polysorbate 20, in particular from 8 to 10 .Math.M, and most particularly 8 .Math.M. This composition can also include nicotinamide, in particular from 10 to 210 mM.

[0220] In an embodiment, the pharmaceutical composition according to the invention contains 3.5 mg/mL to 10.5 mg/mL of insulin lispro A21G, desB30, 0.4 mg/mL to 3 mg/mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerol at a pH of 3.8. This composition can moreover include polysorbate 20, in particular from 8 to 10 .Math.M, and most particularly 8 .Math.M.

[0221] In an embodiment, the pharmaceutical composition according to the invention contains 3.5 mg/mL to 10.5 mg/mL of insulin lispro A21G, desB30, 0.4 mg/mL to 3 mg/mL of pramlintide, 25 mM of m-cresol, 184 mM of glycerol at a pH of 3.7. This composition can moreover include polysorbate 20, in particular from 8 to 10 .Math.M, and most particularly 8 .Math.M.

[0222] The pharmaceutical composition according to the invention can moreover include excipients in compliance with the Pharmacopoeias, in particular the EP and/or US Pharmacopoeias, and compatible with the insulins used at the usual concentrations.

[0223] According to an embodiment, the pharmaceutical composition can be reconstituted from a solid or lyophilized form.

[0224] The methods of administration considered are the intravenous, subcutaneous, intradermal or intramuscular route.

[0225] According to a particular embodiment, the method of administration is the subcutaneous route.

[0226] In an embodiment, the pharmaceutical composition is administered in one or several boluses at mealtime.

[0227] In an embodiment, the pharmaceutical composition is administered in 2 boluses at mealtime.

[0228] In an embodiment, the pharmaceutical composition is administered in 3 boluses at mealtime.

[0229] By “at mealtime” is meant a time ranging from 20 minutes before to 30 minutes after the beginning of the meal. In particular, from 10 minutes before to 30 minutes after the beginning of the meal. More particularly 5 minutes before to 30 minutes after the beginning of the meal. Even more particularly 5 minutes before to 20 minutes after the beginning of the meal.

[0230] In an embodiment, the pharmaceutical composition, intended to be used in a diabetes treatment method, is administered to improve the control of postprandial glycemia and to decrease the adverse effects of pramlintide.

[0231] In an embodiment, the pharmaceutical composition, intended to be used in a diabetes treatment method, enables to decrease the food consumption induced by insulin.

[0232] The transdermal, oral, nasal, vaginal, ocular, buccal, pulmonary administration routes are also considered.

[0233] The invention also relates to an implantable or transportable pump including a pharmaceutical composition according to the invention.

[0234] The invention also relates to the use of a pharmaceutical composition according to the invention which is intended to be placed in an implantable or transportable pump.

[0235] The invention also relates to the use of a pharmaceutical composition according to the invention which is intended to be placed in a pump working with a closed loop system.

[0236] The invention also relates to the use of a pharmaceutical composition according to the invention which is intended to be placed in a patch pump.

[0237] The invention also relates to a cartridge comprising the pharmaceutical composition.

[0238] The invention also relates to a vial comprising the pharmaceutical composition.

[0239] The preparation of a pharmaceutical composition according to the invention has the advantage that it can be implemented by simple solubilization in water of a rapid-acting insulin analog with A21 substitution and one glucagon suppressor with prandial action.

[0240] The preparation of a pharmaceutical composition according to the invention has the advantage that it can be implemented by simple solubilization of an amylin analog or of an amylin receptor agonist and a rapid-acting insulin analog with A21 substitution.

[0241] If necessary, the composition of the pharmaceutical composition is adjusted in terms of excipients such as glycerol, m-cresol and polysorbate 20 (Tween® 20). This addition can be carried out by addition of concentrated solutions of said excipients.

[0242] The rapid-acting insulin analogs with A21 substitution can be obtained by methods of recombinant DNA technology using bacteria such as Escherichia coli and yeasts such as Saccharomyces cerevisiae (see, for example, G. Walsh Appl. Microbiol. Biotechnol. 2005, 67, 151-159 and Kohn et al., in Peptides 2007, 28, 935-948)

[0243] . In general, a proinsulin is produced, which is then digested by enzymes such as trypsin and carboxypeptidase B to obtain the desired sequence. The rapid-acting insulin can also be produced by chemical synthesis (see for example, Liu et al., in Org. Lett. 2013, 15, 960-963).

[0244] The pharmaceutical composition in the form of an injectable aqueous solution according to the invention has a physical and chemical stability enabling the development of a liquid formulation which is stable for at least one year or even 2 years at 5° C. and for at least 2 weeks 30° C. The stability being defined according to the EP and/or US Pharmacopeia.

EXAMPLE 1

Preparation of a Pharmaceutical Composition Comprising Insulin Lispro A21G 100 U/mL (3.5 mg/mL) and of Pramlintide 0.6 mg/mL Containing M-Cresol (25 mM), Glycerol (184 mM) and Acetic Acid/Sodium Acetate Buffer (18 mM) at Acidic pH of 3.8

[0245] A concentrated solution of excipients (m-cresol, glycerol) is added to a concentrated solution of insulin lispro A21G in an acetic acid/sodium acetate buffer at pH 3.8. A concentrated solution of pramlintide (10 mg/mL at pH 3.8) is added to this concentrated solution of insulin lispro A21G and of excipients so as to obtain the intended final pharmaceutical composition. The final pH 3.8, is obtained by addition of an aqueous solution of NaOH. The solution obtained is clear and homogeneous; it is subjected to a 0.22 .Math.m filtration and stored in glass cartridges (1 mL of solution per 3 mL cartridge). The composition is detailed in table 1.

EXAMPLE 2

Preparation of a Pharmaceutical Composition Comprising Insulin Lispro A21G 100 U/mL (3.5 mg/mL) and of Pramlintide 0.6 mg/mL Containing M-Cresol (25 mM), Glycerol (184 mM), Acetic Acid/Sodium Acetate Buffer (18 mM) and Tween 20 (10 .Math.g/mL) at Acidic pH of 3.8

[0246] A concentrated solution of excipients (m-cresol, glycerol) is added to a concentrated solution of insulin lispro A21G in an acetic acid/sodium acetate buffer at pH 3.8. A concentrated solution of pramlintide (10 mg/mL at pH 3.8) and a concentrated solution of Tween 20 are added to this concentrated solution of insulin lispro A21G and of excipients so as to obtain the intended final pharmaceutical composition. The final pH 3.8, is obtained by addition of an aqueous solution of NaOH. The solution obtained is clear and homogeneous; it is subjected to a 0.22 .Math.m filtration and stored in glass vials (1.2 mL of solution per 2 ml vial). The composition is detailed in table 1.

EXAMPLE 3

Preparation of Compositions Comprising Insulin Lispro A21G and of Pramlintide or GLP-1 RA at Acidic pH

[0247] A concentrated solution of excipients (m-cresol, glycerol, optionally nicotinamide) is optionally added to a concentrated solution of insulin lispro A21G or insulin lispro A21G,desB30 optionally in an acetic acid/sodium acetate buffer at acidic pH. A concentrated solution of GLP-1 receptor agonist (5 mg/mL) or a concentrated solution of pramlintide (10 mg/mL at pH 2 - 4) and optionally a concentrated solution of Tween 20 are added to the concentrated solution of insulin A21G and of excipients so as to obtain the intended final pharmaceutical composition. The final pH, is obtained by addition of an aqueous solution of NaOH. The solution obtained is clear and homogeneous; it is subjected to a 0.22 .Math.m filtration and stored in glass cartridges (1 mL of solution per 3 ml cartridge). The compositions of insulin lispro A21G CA3-1 to CA3-17 and of insulin lispro A21G,desB30 CA4-1 to CA4-6 with pramlintide are detailed in table 1.

TABLE-US-00001 Compositions comprising Insulin A21G and glucagon suppressor at acidic pH Compo sitions Insulin A21G (U/ml) Pramlintide (mg/ml) GLP-1 RA (mg/ml) pH Excipients Visual aspect CA1 Lispro A21G (100) 0.6 3.8 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (18 mM) Clear CA2 Lispro A21G (100) 0.6 3.8 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (18 mM) Tween 20 (10 .Math.g/mL) Clear CA3-1 Lispro A21G (100) 0.6 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-2 Lispro A21G (100) 0.4 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 uq/mL) Clear CA3-3 Lispro A21G (100) 0.9 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-4 Lispro A21G (100) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-5 Lispro A21G (200) 0.8 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-6 Lispro A21G (200) 1.2 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-7 Lispro A21G (200) 1.8 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-8 Lispro A21G (100) 0.6 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Nicotinamide (170 mM) Tween 20 (10 .Math.g/mL) Clear CA3-9 Lispro A21G (100) 0.6 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Nicotinamide (40 mM) Tween 20 (10 .Math.g/mL) Clear CA3-10 Lispro A21G (100) 0.4 3.4 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-11 Lispro A21G (100) 0.6 3.4 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Nicotinamide (170 mM) Tween 20 (10 .Math.g/mL) Clear CA3-12 Lispro A21G (100) 0.6 3.7 Clear CA3-13 Lispro A21G (200) 1.2 3.7 Clear CA3-14 Lispro A21G (100) Exena tide (0.05) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-15 Lispro A21G (100) Lixise natide (0.125) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-16 Lispro A21G (100) Exena tide (0.1) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-17 Lispro A21G (100) Lixise natide (0.25) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-18 Lispro A21G (100) Exena tide (0.4) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-19 Lispro A21G (100) Lixise natide (1) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-20 Lispro A21G (200) Exena tide (0.1) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA3-21 Lispro A21G (200) Lixise natide (0.25) 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA4-1 Lispro A21G, desB30 (100) 0.4 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA4-2 Lispro A21G, desB30 (100) 0.6 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA4-3 Lispro A21G, desB30 (100) 0.9 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA4-4 Lispro A21G, desB30 (200) 0.8 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA4-5 Lispro A21G, desB30 (200) 1.2 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear CA4-6 LisproA21G, desB30 (200) 1.8 3.7 m-cresol (25 mM) glycerol (184 mM) Acetate buffer (5 mM) Tween 20 (10 .Math.g/mL) Clear

Example 4: Stability in Cartridges at 37° C. and 30° C. of a Formulation of Insulin Lispro A21G and Pramlintide

[0248] Physical and chemical stability were respectively measured as follows.

[0249] At least five 3-mL cartridges (from example CA3-1; target composition 100 U/mL insulin and 0.6 mg/mL pramlintide) filled with 1 mL of composition are placed vertically in ovens maintained at 30 and 37° C. The vials are visually inspected at least weekly for the appearance of visible particles or turbidity. This inspection is carried out according to the recommendations of the European Pharmacopoeia (EP 2.9.20) and the U.S. Pharmacopoeia (USP <790>): the vials are subjected to lighting of at least 2000 Lux and are observed against a white background and a black background.

[0250] The formulations were analysed by RP-HPLC.

[0251] The results are shown in the table 2.

TABLE-US-00002 physical and chemical stability of a composition comprising lispro A21G and pramlintide T0 4 weeks at 37° C. 6 weeks at 30° C. 12 weeks at 30° C. Appearance Clear Clear Clear Clear Insulin (mg/mL) 3.37 3.24 3.28 3.22 Pramlintide (mg/mL) 0.61 0.59 0.6 0.61

[0252] The data shows that the composition is very stable and satisfies EP requirements when stored 4 weeks at 37° C. and 12 weeks at 30° C.

Example 5: Pharmacokinetic and Pharmacodynamic Study in Pigs Pharmacokinetic and Pharmacodynamic Study in Pigs of the Composition CA3-4 Consisting of Insulin Lispro A21G (100 U/mL) Alone or CA3-1 Consisting of Insulin Lispro A21G (100 U/mL) Combined with Pramlintide (0.6 mg/mL)

[0253] Domestic pigs weighing approximately 50 kg, catheterized beforehand in the jugular, were fasted for 2.5 hours before the start of the experiment. During the hour preceding the injection of insulin, 3 blood samples were drawn to determine the baseline level of glucose and of insulin.

[0254] The injection of the formulations of insulin lispro A21G (CA3-4) or of insulin lispro A21G combined with pramlintide (CA3-1) at the dose of 0.125 U of insulin/kg and 0.75 .Math.g of pramlintide/kg is performed subcutaneously in the flank of the animal with the aid of an insulin pen (Novo, Sanofi or Eli Lilly) equipped with a 31 G needle.

[0255] In order to determine the concentrations of insulin in the plasma and glucose in the blood, blood samples are drawn at the following times: 0, 10, 20, 30, 45, 60, 90, 120, 180 and 240 minutes.

Pharmacokinetic Results of the Solution of Insulin Lispro CA3-4 and of The Solution of Insulin Lispro A21G and Pramlintide CA3-1 in Pigs

[0256] The pharmacokinetic parameters of formulations CA3-4 and CA3-1 are estimated based on baseline-adjusted insulin concentrations in the plasma. A standard non-compartmental analysis is carried out with the aid of the software Phoenix WinNonlin (version 8, Certara). The values of the parameters (mean ± standard deviation) are reported in table 3.

TABLE-US-00003 PK parameters of insulin of compositions CA3-4 and CA3-1 Comp ositio n Peptides N t.sub.max insulin (min) AUC.sub.0-30min insulin (min*mU/L) AUC.sub.0-30min insulin (% of AUC.sub.0- .sub.last) CA3-4 lispro A21G (100 U/mL) 13 46 ± 20 1602 ± 789 12 ± 5 CA3-1 lispro A21G (100 U/mL) pramlintide (0.6 mg/mL ) 14 35 ± 20 1885 ± 731 20 ± 10

Where [0257] t.sub.max corresponds to the necessary time to observe the maximum plasma concentration; [0258] AUC.sub.0-30min corresponds to the area under the curve of the plasma concentration versus time between 0 and 30 min after injection; and [0259] AUC.sub.0-last corresponds to the area under the curve of the plasma concentrations versus time between 0 and the last quantifiable concentration after injection.

[0260] The analysis of the parameters indicates that the combination of insulin lispro A21G and of pramlintide (formulation CA3-1) leads to a faster absorption of insulin compared to insulin lispro A21G alone (formulation CA3-4). Formulation CA3-1 leads to a time to plasma peak (t.sub.max) which is earlier (approximately 11 min) and to a fraction of plasma insulin exposure within 0-30 min (AUC.sub.0-30min/AUC.sub.0-last) which is significantly increased (approximately 67%, p<0.05) in comparison to formulation CA3-4.

Pharmacodynamic Results of the Solution of Insulin Lispro CA3-4 and of The Solution of Insulin Lispro A21G and Pramlintide CA3-1 in Pigs

[0261] The pharmacodynamic parameters of formulations CA3-4 and CA3-1 are estimated based on baseline-adjusted (% from baseline) glucose concentrations in the blood. A standard non-compartmental analysis is carried out with the aid of the software Phoenix WinNonlin (version 8, Certara). The values of the parameters (mean ± standard deviation) are reported in table 4.

TABLE-US-00004 Pharmacodynamic parameters of glucose of compositions CA3-4 and CA3-1 Compo sition Peptides N t.sub.min glucose (min) AUC.sub.0-60mln glucose (min*% from baseline) CA3-4 lispro A21G (100 U/mL) 14 73 ± 31 1342 ± 773 CA3-1 lispro A21G (100 U/mL) pramlintide (0.6 mg/mL) 14 51 ± 16 1622 ± 619

Where [0262] t.sub.min corresponds to the necessary time to observe the minimum blood concentration; [0263] AUC.sub.0-60min corresponds to the area between the baseline and the curve of the blood concentration versus time between 0 and 60 min after injection.

[0264] The analysis of the parameters indicates that the combination of insulin lispro A21G and of pramlintide (formulation CA3-1) leads to a faster hypoglycemic effect compared to insulin lispro A21G alone (formulation CA3-4). Formulation CA3-1 leads to time to maximum hypoglycemic effect (t.sub.min) which is significantly earlier (approximately 22 min, p<0.05) and to an early hypoglycemic effect (AUC.sub.0-60min) which is increased (approximately 21%) in comparison to formulation CA3-4.