Pharmaceutical compositions comprising a GLP-1 peptide or exendin-4 and a basal insulin peptide

Abstract

Pharmaceutical compositions for parenteral administration comprising a basal insulin peptide and an insulinotropic GLP-1 peptide and comprising at least 6 zinc atoms per 6 insulin molecules.

Description

[0001] This application is a divisional application of PCT application filed on November 14, 2008, with international application number PCT / EP2008 / 065 601, national phase application number 200880116227.5, and invention title "Pharmaceutical composition comprising GLP-1 peptide or sarcanthus exopeptide-4 and basal insulin peptide". Technical Field

[0002] This invention relates to the field of pharmaceutical compositions. More specifically, this invention relates to pharmaceutical compositions containing two different pharmaceutically active peptides. Background Technology

[0003] Diabetes mellitus is a metabolic disease characterized by partial or complete loss of glucose utilization. Since the introduction of insulin in the 1920s, continuous efforts have been made to improve the treatment of diabetes. Because diabetic patients require decades of chronic treatment, there is a significant demand for safe, convenient insulin formulations that improve quality of life.

[0004] In the treatment of diabetes, many different insulin formulations have been proposed and used, such as regular insulin, protamine zinc insulin (denoted as NPH), and insulin zinc suspension (e.g., and And biphasic protamine zinc insulin. Some commercially available insulin formulations are characterized by rapid onset of action, while others have a relatively slower onset of action but show more or less prolonged efficacy. Rapid-acting insulin formulations are usually solutions of insulin, while slow-acting insulin can be suspensions containing crystalline and / or amorphous forms of insulin precipitated by adding zinc salts alone, or by adding protamine, or by adding a combination of both zinc salts and protamine. Many insulin analogs have been developed over the past decade. They are designed for specific therapeutic ranges, namely rapid-acting or long-acting.

[0005] Another peptide expected to be very important in diabetes treatment is glucagon-like peptide-1 (GLP-1). Human GLP-1 is a 37-amino acid peptide derived from preproglucagon, which is synthesized particularly in L cells of the terminal ileum, pancreas, and brain. GLP-1 is an important gastrointestinal hormone that plays a regulatory role in glucose metabolism and gastrointestinal secretion and metabolism. GLP-1 stimulates insulin secretion in a glucose-dependent manner, stimulates insulin biosynthesis, promotes β-cell rescue, reduces glucagon secretion, gastric emptying, and food intake. With the rapid increase in the global population with type 2 diabetes, there is a greater need for drugs that are easier to administer and more effective. Combination formulations that include insulin peptides and GLP-1 peptides in a fixed ratio of the two drugs may be very effective therapeutic agents and require smaller injection doses when administered to the same patient.

[0006] WO95 / 31214 discloses a combination therapy for insulin-requiring diabetes, comprising the administration of insulin and GLP-1. WO03 / 020201 discloses a premixed formulation of a GLP-1 compound and basal insulin. WO2006 / 051103 discloses a storage-stable pharmaceutical composition comprising GLP-1, basal insulin, and a surfactant. Attached Figure Description

[0007] Figure 1 The physical stability of four pharmaceutical compositions evaluated using accelerated stress testing is shown.

[0008] Figure 2-4 The results of the rotation test are shown.

[0009] Figure 5-8 The following is a diagram showing the insulin analog N tested in pigs. εB29 -(N α -(HOOC(CH2) 14 Pharmacokinetic (PK) performance of a fixed-combination formulation of human insulin and liraglutide (CO)-γ-Glu)desB30. Summary of the Invention

[0010] One object of the present invention is to provide a storage-stable, once-daily fixed combination of GLP-1 compounds and basal insulin compounds, which has unchanged PK / PD performance compared to a single active component.

[0011] More specifically, the present invention relates to a storage-stable pharmaceutical composition comprising a fixed combination of an insulin-stimulating GLP-1 compound and basal insulin, the composition containing at least 5 zinc ions / 6 basal insulin molecules.

[0012] In one embodiment, the pharmaceutical composition contains at least 6 zinc ions / 6 basal insulin molecules.

[0013] In another embodiment, the pharmaceutical composition contains at least 7 zinc ions / 6 basal insulin molecules.

[0014] In another embodiment, the pharmaceutical composition contains at least 8 zinc ions / 6 basal insulin molecules.

[0015] In yet another embodiment, the pharmaceutical composition contains at least 9, 10, 11, 12, 13, 14, 15, or 16 zinc ions / 6 basal insulin molecules.

[0016] In one implementation, the zinc content is 5-16 zinc ions / 6 insulin molecules.

[0017] In another embodiment, the zinc content is 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, or 5-7 zinc ions / 6 insulin molecules.

[0018] In yet another implementation, the zinc content is 6-16 zinc ions / 6 insulin molecules.

[0019] In yet another implementation, the zinc content is 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, or 6-8 zinc ions / 6 insulin molecules.

[0020] In another implementation, the zinc content is 7-16 zinc ions / 6 insulin molecules.

[0021] In yet another implementation, the zinc content is 7-14, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, or 7-9 zinc ions / 6 basal insulin molecules.

[0022] In another embodiment, the zinc content is 8-16 zinc ions / 6 insulin molecules.

[0023] In another embodiment, the zinc content is 8-15, 8-14, 8-13, 8-12, 8-11, or 8-10 zinc ions / 6 insulin molecules.

[0024] The pH of the drug formulation is typically above neutral in any of the above embodiments, usually from about 7 to about 9.

[0025] In one embodiment, the pH of the pharmaceutical composition is from about pH 7.4 to about pH 9, from about pH 7.4 to about pH 8.5, or from about pH 7.4 to about pH 8.2.

[0026] In another embodiment, the pH of the pharmaceutical composition is about pH 7.5 to about pH 8.5, about pH 7.5 to about pH 8.2, or about 7.5 to about 7.7.

[0027] In another embodiment, the pH of the pharmaceutical composition is from about pH 7.6 to about pH 8.2. In yet another embodiment, the pH is from about 7.7 to about 8.2.

[0028] In another embodiment, the pH is approximately 7.7 to approximately 9.

[0029] In another embodiment, the pH is approximately 7.7 to approximately 8.9.

[0030] In another embodiment, the pH is approximately 7.7 to approximately 8.8.

[0031] In another embodiment, the pH is approximately 7.7 to approximately 8.7.

[0032] In another embodiment, the pH is approximately 7.7 to approximately 8.6.

[0033] In another embodiment, the pH is approximately 7.7 to approximately 8.5.

[0034] In another embodiment, the pH is approximately 7.7 to approximately 8.4.

[0035] In another embodiment, the pH is approximately 7.7 to approximately 8.3.

[0036] In another embodiment, the pH is approximately 7.7 to approximately 8.2.

[0037] In another embodiment, the pH is approximately 7.7 to approximately 8.1.

[0038] In another embodiment, the pH is approximately 7.7 to approximately 8.

[0039] The insulin-stimulating GLP-1 compound in any of the above embodiments can be any GLP-1 compound effective for the treatment of type 2 diabetes. As used herein, the term "GLP-1 peptide" refers to GLP-1(7-37), a GLP-1(7-37) analog, a GLP-1(7-37) derivative, or a derivative of a GLP-1(7-37) analog. A derivative of GLP-1 can be an acylated GLP-1 compound as disclosed in WO 98 / 08871 or WO 2006 / 097537.

[0040] In one embodiment, the GLP-1 compound is an acylated GLP-1 analog, such as Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37), Aib8,Lys26(OEG-OEG-γ-Glu-C18-diacid),Arg34)GLP-1 H(7-37)-OH or (N-ε26-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxy-heptadecylamino)butyrylamino]ethoxy}ethoxy)acetamido]ethoxy}ethoxy)-acetyl][Aib8,Arg34]GLP-1-(7-37).

[0041] In another embodiment, GLP-1 is exendin-3, exendin-4, or an exendin-4 analog. The exendin-4 analog may comprise 4-10, 4-8, or 4-6 basic amino acid residues added to the C-terminus or N-terminus.

[0042] In any of the above embodiments, the basal insulin may be any basal insulin known for the treatment of type 1 and type 2 diabetes. In one embodiment, the basal insulin is insulin with the ε-amino group of the B29Lys of the B chain of the insulin and its analogs disclosed in WO 95 / 07931, WO 2005 / 012347 and EP2007 / 054444 acylated.

[0043] In another embodiment, the basal insulin is a p1-shifted basal insulin, such as the type disclosed in US Patent 5,656,722, wherein the insulin molecule has a substitution or addition of a basic amino acid residue. An example of such basal insulin is GlyA21, ArgB31, Arg B32 human insulin (insulin glargine). Another basal insulin could be amidated glargine insulin, such as the compounds disclosed in WO2008 / 006496 and WO2008 / 006497.

[0044] In one implementation scheme, basal insulin is N εB29 -Tetradecanoyldes (B30) human insulin.

[0045] In yet another implementation plan, basal insulin is Lys B29 (N ε Lithochoyl-γ-Glu-des(B30) human insulin.

[0046] In yet another implementation plan, basal insulin is N εB29 -Tetradecanoyldes (B30) Human insulin or N εB29 -(Nα-(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin.

[0047] In yet another implementation plan, basal insulin is N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine (desB30) human insulin or N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin.

[0048] In one embodiment, the pharmaceutical composition comprises a fixed combination of an insulin-stimulating GLP-1 compound and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine (desB30) human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing at least 5, 6, 7, 8 or 9 zinc atoms / 6 insulin molecules.

[0049] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of an insulin-stimulating GLP-1 compound and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine (desB30) human insulin and N εB29-ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 5-16, 5-14, 5-12, 5-10 or 5-8 zinc ions / 6 insulin molecules.

[0050] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of an insulin-stimulating GLP-1 compound and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine (desB30) human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 6-16, 6-14, 6-12, 6-10 or 6-8 zinc ions / 6 insulin molecules.

[0051] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of an insulin-stimulating GLP-1 compound and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine (desB30) human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 7-16, 7-14, 7-12 or 7-10 zinc ions / 6 insulin molecules.

[0052] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of an insulin-stimulating GLP-1 compound and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, LysB29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine (glutaylamide) desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 8-16, 8-14, 8-12 or 8-10 zinc ions / 6 insulin molecules.

[0053] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of an insulin-stimulating GLP-1 compound and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, said acylated basal insulin having the formula N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin, the composition containing at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 zinc ions / 6 insulin molecules.

[0054] In yet another embodiment, the pharmaceutical composition comprises an insulin-stimulating GLP-1 compound and acylated basal insulin N. εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a suitable pharmaceutically acceptable adjuvant, the composition containing 5-16 zinc ions / 6 insulin molecules.

[0055] In yet another embodiment, the pharmaceutical composition comprises an insulin-stimulating GLP-1 compound and acylated basal insulin N. εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a suitable pharmaceutically acceptable adjuvant, the composition containing 6-16, 6-15, 6-14, 6-13, 6-12, 6-10 or 6-8 zinc ions / 6 insulin molecules.

[0056] In yet another embodiment, the pharmaceutical composition comprises an insulin-stimulating GLP-1 compound and acylated basal insulin N. εB29 (N α -(HOOC(CH2) 14A fixed combination of CO)-γ-Glu)desB30 human insulin and a suitable pharmaceutically acceptable adjuvant, the composition containing 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10 or 7-8 zinc ions / 6 insulin molecules.

[0057] In yet another embodiment, the pharmaceutical composition comprises an insulin-stimulating GLP-1 compound and acylated basal insulin N. εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a suitable pharmaceutically acceptable adjuvant, the composition containing 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10 or 8-9 zinc ions / 6 insulin molecules.

[0058] In one embodiment, the present invention relates to a compound containing insulin-stimulating GLP-1 and basal insulin N. εB29 -Tetradecanoyldes (B30) human insulin fixed combination and pharmaceutical composition of suitable pharmaceutical adjuvant, wherein the zinc content is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 zinc ions / 6 insulin molecules.

[0059] In another embodiment, the present invention relates to a compound containing insulin-stimulating GLP-1 and basal insulin N. εB29 -Tetradecanoyldes (B30) human insulin fixed combination and pharmaceutical composition of suitable pharmaceutical adjuvant, wherein the zinc content is 5-16, 6-16, 7-16 or 8-16 zinc ions / 6 insulin molecules.

[0060] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of GLP-1 (7-37) or its analogues or derivatives and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing at least 5 or 6 zinc ions / 6 insulin molecules.

[0061] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of GLP-1 (7-37) or its analogues or derivatives and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 5-16, 5-14, 5-12, 5-10 or 5-8 zinc ions / 6 insulin molecules.

[0062] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of GLP-1 (7-37) or its analogues or derivatives and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 6-16, 6-14, 6-12 or 6-10 zinc ions / 6 insulin molecules.

[0063] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of GLP-1 (7-37) or its analogues or derivatives and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2)14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 7-16, 7-14, 7-12 or 7-10 zinc ions / 6 insulin molecules.

[0064] In yet another embodiment, the pharmaceutical composition comprises a fixed combination of GLP-1 (7-37) or its analogues or derivatives and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N... εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 8-16, 8-12 or 8-10 zinc ions / 6 insulin molecules.

[0065] In one embodiment, the present invention relates to a compound containing GLP-1 (7-37) or its analogues or derivatives and N εB29 -Tetradecanoyldes (B30) human insulin fixed combination and pharmaceutical composition of suitable pharmaceutical adjuvant, wherein the zinc content is at least 5, 6, 7, 8, 9 or 10 zinc ions / 6 insulin molecules.

[0066] In one embodiment, the present invention relates to a compound containing GLP-1 (7-37) or its analogues or derivatives and N εB29 -Tetradecanoyldes (B30) human insulin fixed combination and pharmaceutical composition with suitable pharmaceutical adjuvant, wherein the zinc content is 6-16, 7-16, 8-16, 8-14, 8-12 or 8-10 zinc ions / 6 insulin molecules.

[0067] In yet another embodiment, the pharmaceutical composition includes Arg 34 Lys 26 (N ε -(γ-Glu(N αA fixed combination of -hexadecyl)))-GLP-1(7-37) and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 zinc ions / 6 insulin molecules.

[0068] In yet another embodiment, the pharmaceutical composition includes Arg 34 Lys 26 (N ε -(γ-Glu(N α A fixed combination of -hexadecyl)))-GLP-1(7-37) and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 5-16, 5-14, 5-12 or 5-10 zinc ions / 6 insulin molecules.

[0069] In yet another embodiment, the pharmaceutical composition includes Arg 34 Lys 26 (N ε -(γ-Glu(N α A fixed combination of -hexadecyl)))-GLP-1(7-37) and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N εB29-Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 6-16, 6-14, 6-12 or 6-10 zinc ions / 6 insulin molecules.

[0070] In yet another embodiment, the pharmaceutical composition includes Arg 34 Lys 26 (N ε -(γ-Glu(N α A fixed combination of -hexadecyl)))-GLP-1(7-37) and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 7-16, 7-14, 7-12 or 7-10 zinc ions / 6 insulin molecules.

[0071] In yet another embodiment, the pharmaceutical composition includes Arg 34 Lys 26 (N ε -(γ-Glu(N α A fixed combination of -hexadecyl)))-GLP-1(7-37) and acylated basal insulin, and a suitable pharmaceutically acceptable adjuvant, wherein the acylated basal insulin is selected from N εB29 -Tetradecanoyldes(B30) human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, NεB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin, the composition containing 8-16, 8-14, 8-12 or 8-10 zinc ions / 6 insulin molecules.

[0072] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a pharmaceutical composition with suitable pharmaceutically acceptable adjuvants, wherein the zinc content is at least 5 zinc ions / 6 insulin molecules.

[0073] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a pharmaceutical composition with suitable pharmaceutically adjuvants, wherein the zinc content is at least 6 zinc ions / 6 insulin molecules.

[0074] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO).γ-Glu)desB30 human insulin and a pharmaceutical composition with suitable pharmaceutically adjuvants, wherein the zinc content is at least 7 zinc ions / 6 insulin molecules.

[0075] In yet another embodiment, the present invention relates to Arg-containing...34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-l(7-37) with N εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a pharmaceutical composition with suitable pharmaceutically adjuvants, wherein the zinc content is at least 8 zinc ions / 6 insulin molecules.

[0076] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a pharmaceutical composition with suitable pharmaceutically adjuvants, wherein the zinc content is at least 9 zinc ions / 6 insulin molecules.

[0077] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 -(N α -(HOOC(CH2) 14 A fixed combination of CO)-γ-Glu)desB30 human insulin and a pharmaceutical composition with suitable pharmaceutically adjuvants, wherein the zinc content is at least 10 zinc ions / 6 insulin molecules.

[0078] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin fixed combination and pharmaceutical composition with suitable pharmaceutical adjuvant, wherein the zinc content is 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9 or 5-8 zinc ions / 6 insulin molecules.

[0079] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 (N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin fixed combination and pharmaceutical composition with suitable pharmaceutically adjuvant, wherein the zinc content is 6-16, 6-15, 6-14, 6-13, 6-12, 6-11 or 6-10 zinc ions / 6 insulin molecules.

[0080] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin fixed combination and pharmaceutical composition with suitable pharmaceutical adjuvant, wherein the zinc content is 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9 or 7-8 zinc ions / 6 insulin molecules.

[0081] In yet another embodiment, the present invention relates to Arg-containing... 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) with N εB29 (N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin fixed combination and pharmaceutical composition with suitable pharmaceutically adjuvant, wherein the zinc content is 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10 or 8-9 zinc ions / 6 insulin molecules.

[0082] In yet another embodiment, the present invention relates to a pharmaceutical composition comprising a fixed combination of insulin glargine and a salicornis exopeptide-4 analog (e.g., a salicornis exopeptide-4 analog comprising 4-6 additional Lys- or Arg-amino acid residues) and a suitable pharmaceutically acceptable adjuvant, wherein the zinc content is at least greater than 5, 6, 7, or 8 zinc ions / 6 insulin molecules. In this embodiment, insulin glargine may also be amidated insulin glargine as disclosed in WO2008 / 006496 and WO2008 / 006497.

[0083] In any of the above embodiments, the pH can be 7.4, 7.5, 7.6, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5. In another embodiment, the pH is 7.4-8.2 or 7.5-8. In yet another embodiment, the pH is 7.6-8.

[0084] If the GLP-1 compound is Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37), then the pH is typically from about 7.7 to about 8.2.

[0085] Another aspect of the invention relates to a method for preparing a pharmaceutical composition according to any of the above embodiments, the method comprising dissolving the basal insulin, mixing it with a preservative and an isotonic modifier, then adding zinc, and finally mixing it with the dissolved insulin-stimulating peptide.

[0086] One embodiment of the method according to the invention includes the following steps: 1) dissolving basal insulin in an aqueous solution of buffer and isotonic agent; 2) optionally adding zinc in steps; 3) adjusting the pH; 4) storing the solution at a temperature between about 4-5°C and ambient temperature; and 5) adding a GLP-1 compound.

[0087] In another aspect, the present invention relates to a method for treating hyperglycemia, the method comprising administering an effective amount of the pharmaceutical composition according to any of the above embodiments to a mammal in need of such treatment via gastrointestinal administration.

[0088] In another aspect, the present invention relates to a method for treating obesity, β-cell deficiency, IGT, or dyslipidemia, the method comprising administering an effective amount of the pharmaceutical composition according to any of the above embodiments to a mammal in need of such treatment via gastrointestinal administration.

[0089] Another aspect of the invention relates to the use of a storage-stable pharmaceutical composition for the treatment of hyperglycemia via parenteral administration, said pharmaceutical composition comprising a mixture of an insulin-stimulating GLP-1 compound and basal insulin, and containing at least 6 zinc atoms / 6 insulin molecules.

[0090] In another aspect, the present invention relates to a method for weight loss, the method comprising administering an effective amount of a pharmaceutical composition comprising a mixture of an insulin-stimulating GLP-1 compound and basal insulin, said pharmaceutical composition containing at least 6 zinc atoms / 6 insulin molecules. Detailed Implementation

[0091] This invention relates to the administration of a fixed mixture of basal insulin and an insulin-stimulating GLP-1 compound to patients with type 2 diabetes. When the GLP-1 compound and basal insulin are combined in a single formulation, patients receive one less injection compared to the need for two separate injections when the two components are administered separately. More importantly, numerous positive synergistic effects are expected when basal insulin analogs and GLP-1 compounds are administered simultaneously.

[0092] In such a mixture, the individual active components, namely basal insulin and GLP-1 compounds, should maintain their attractive pharmacokinetic profiles known from monotherapy. For example, basal insulin analogs such as insulin N... εB29 (N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin has an attractive distribution suitable for once-daily dosing. Similarly, GLP-1 analog Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) is administered once daily. Therefore, insulin N εB29 -(N α -(HOOC(CH2) 14 The combination of CO)-γ-Glu)desB30 human insulin and liraglutide constitutes an attractive combination of basal insulin and GLP-1 analogs.

[0093] However, acylated insulin analogs such as N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin and GLP-1 compounds such as Arg 34 Lys 26 (N ε -(γ-Glu(Nα Direct mixing of -hexadecyl)))-GLP-1(7-37) results in a physically unstable formulation and altered PK properties of acylated insulin. However, commercially available formulations must possess high physical stability. Furthermore, if the PK properties of basal insulin are altered too much, it may lead to a loss of its 24-hour coverage.

[0094] Most basal insulins are formulated with zinc to achieve satisfactory pharmacokinetic (PK) and physical stability. For example, it has been shown that N... εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin binds more zinc than traditional insulin with 2-3 zinc ions / 6 zinc ions. N εB29 -(N α -(HOOC(CH2) 14 Human insulin (CO)-γ-Glu)desB30 requires an additional 2-3 zinc ions (totaling 5-6 zinc ions / 6 insulin molecules) for optimal once-daily distribution.

[0095] We have discovered that Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) has a low-affinity zinc bond, and N εB29 -(N α -(HOOC(CH2) 14 The physical stability and PK properties of CO)-γ-Glu)desB30 human insulin are both affected by the above-mentioned factors in mixtures of these two components. In Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) and N εB29 -(N α -(HOOC(CH2) 14 In a mixture of CO)-γ-Glu)desB30 human insulin, Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) neutralizes insulin N by binding with these additional zinc ions. εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 competes with human insulin.

[0096] We have found that adding additional zinc ions to N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 Human Insulin and Arg 34 Lys 26 (N ε -(γ-Glu(N α The physical stability of the mixture of -hexadecyl)))-GLP-1(7-37) was improved, and the N εB29 (N α -(HOOC(CH2) 14 The p-p-p-p-p-glucose distribution of human insulin (CO)-γ-Glu)desB30 tends to be that of insulin N alone. εB29 -(N α -(HOOC(CH2) 14 The desired distribution of CO)-γ-Glu)desB30 human insulin under conditions of 5-6 zinc ions / insulin molecules.

[0097] As used herein, the term "GLP-1 compound" refers to GLP-1 (7-37), its insulin-stimulating analogs, and insulin-stimulating derivatives. GLP-1 compounds also include sarcoptide exopeptide compounds such as sarcoptide exopeptide-3 and sarcoptide exopeptide-4, and their analogs such as ZP10 or ZP10A, which are sarcoptide exopeptide-4 analogs with six additional lysine residues at the C-terminus of the molecule; see Cristian Thorkildsen et al., The Journal of Pharmacology and Experimental Therapeutics, Vol. 307, No. 2 (2003), 490-496.

[0098] One embodiment of the present invention includes a pharmaceutical composition according to any of the above embodiments, wherein the insulin-stimulating GLP-1 peptide is acylated salicornic exopeptide-4 or an acylated salicornic exopeptide-4 analog, such as [N-ε(17-carboxyheptadecanoic acid)20salicornic exopeptide-4(1-39)-amide and N-ε32-(17-carboxy-heptadecanoic acid)[Lys32]salicornic exopeptide-4(1-39)amide.

[0099] A simple system is used to describe fragments and analogues of GLP-1. Thus, for example, Gly... 8-GLP-1(7-37) represents a GLP-1(7-37) analogue formally derived from GLP-1(7-37) by substituting the 8-position naturally occurring amino acid residue (Ala) with Gly. Similarly, Lys 34 (N ε (-Tetradecanoyl)-GLP-1(7-37) represents GLP-1(7-37), wherein the ε-amino group of the Lys residue at position 34 is tetradecanoylated. PCT disclosures WO 98 / 08871 and WO 99 / 43706 disclose stable derivatives of GLP-1 analogs having lipophilic substituents. These stable derivatives of GLP-1 analogs have prolonged therapeutic distribution compared to the corresponding GLP-1 analogs.

[0100] The term "insulin-stimulating" as used herein for peptides or compounds refers to the ability to stimulate insulin secretion in response to elevated plasma glucose levels. Insulin-stimulating peptides and compounds are agonists of the GLP-1 receptor. The insulin-stimulating properties of compounds can be determined by in vitro or in vivo assays known in the art.

[0101] In one embodiment of the invention, GLP-1 has an Arg residue at position 34. In another embodiment, GLP-1 has a Glu residue at position 22. In another embodiment of the invention, GLP-1 has an L-histidine residue at position 8. In another embodiment of the invention, GLP-1 has a Val residue at position 8. In another embodiment of the invention, a derivative of the GLP-1(7-37) analog is a GLP-1(7-37)-amide.

[0102] In one embodiment of the invention, the GLP-1(7-37) analogue is selected from GLP-1(7-36) amides, Arg 34 -GLP-1(7-37), Gly 8 -GLP-1(7-36)-amide, Gly 8 -GLP-1(7-37), Val 8 -GLP-1(7-36)-amide, Val 8 -GLP-1(7-37), Val 8 Asp 22 -GLP-1(7-36)-amide, Val 8 Asp 22 -GLP-1(7-37), Val 8 Glu 22 -GLP-1(7-36)-amide, Val 8 Glu 22 -GLP-1(7-37), Val8 Lys 22 -GLP-1(7-36)-amide, Val 8 Lys 22 -GLP-1(7-37), Val 8 Arg 22 -GLP-1(7-36)-amide, Val 8 Arg 22 -GLP-1(7-37), Val 8 His 22 -GLP-1(7-36)-amide, Val 8 His 22 -GLP-1(7-37), Val 8 Trp 19 Glu 22 -GLP-1(7-37), Val 8 Glu 22 Val 25 -GLP-1(7-37), Val 8 Tyr 16 Glu 22 -GLP-1(7-37), Val 8 Trp 16 Glu 22 -GLP-1(7-37), Val 8 Leu 16 Glu 22 -GLP-1(7-37), Val 8 Tyr 18 Glu 22 -GLP-1(7-37), Val 8 Glu 22 His 37 -GLP-1(7-37), Val 8 Glu 22 Ile 33 -GLP-1(7-37), Val 8 Trp 16 Glu 22 Val 25 Ile 33 -GLP-1(7-37), Val (这里似乎有重复,按照要求保留原文) 8 Trp 16 Glu 22 Ile 33 -GLP-1(7-37), Val 8 Glu 22 Val 25 Ile 33-GLP-1(7-37), Val 8 Trp 16 Glu 22 Val 25 -GLP-1(7-37).

[0103] In another embodiment of the invention, the insulin-stimulating peptide is a derivative of GLP-1 (7-37) or a derivative of a GLP-1 (7-37) analog (having a lysine residue, such as one lysine), wherein a lipophilic substituent is optionally linked to the ε-amino group of the lysine via a spacer group. In one embodiment of the invention, the lipophilic substituent has 8-40 carbon atoms, preferably 8-24 carbon atoms, for example 12-18 carbon atoms. In another embodiment of the invention, the spacer group is present and selected from amino acids, such as β-Ala, L-Glu, and aminobutyryl. In another embodiment of the invention, the insulin-stimulating peptide is a dipeptide aminopeptidase IV-protected GLP-1 compound. In another embodiment of the invention, the insulin-stimulating peptide is a plasma-stable GLP-1 compound. In another embodiment of the invention, the derivative of the GLP-1 (7-37) analog is Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37). A non-limiting example of a GLP-1 derivative is deamino-His 7 Arg 26 Lys 34 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37), deamino-His 7 Arg 26 Lys 34 (N ε -Octanyl)-GLP-1(7-37), Arg 26，34 Lys 38 (N ε -(ω-carboxypentadecanyl))-GLP-1(7-38), Arg 26，34 Lys 36 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-36) and Arg 34 Lys 26 (N ε -(γ-Glu(N α-Hexadecyl)))-GLP-1(7-37). A non-limiting example of a GLP-1 derivative is deamino-His 7 Arg 26 Lys 34 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37), deamino-His 7 Arg 26 Lys 34 (Nε-octanoyl)-GLP-1(7-37), Arg 26，34 Lys 38 (N ε -(ω-carboxypentadecanyl))-GLP-1(7-38), Arg 26，34 Lys 36 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-36) and Arg 34 Lys 26 (N ε -(γ-Glu(N α -hexadecyl)))-GLP-1(7-37).

[0104] In another embodiment, the GLP-1 analogue is a further extended analogue of the type disclosed in WO 2006 / 097537, such as Aib8, Lys26 (OEG-OEG-γ-Glu-C18-diacid), Arg34)GLP-1 (7-37), N-ε 26 -(17-Carboxyheptadecanoyl)-[Aib8,Arg34]GLP-1-(7-37)-peptide, N-ε 26 -(19-Carboxynonadecanoyl)-[Aib8,Arg34]GLP-1-(7-37)-peptide, N-ε 26 -(4-{[N-(2-carboxyethyl)-N-(15-carboxypentadecanoyl)amino]methyl}benzoyl)[Arg34]GLP-1-(7-37),N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetamido)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)peptide.

[0105] In one embodiment, the GLP-1 derivative is Aib8, Lys26 (OEG-OEG-γ-Glu-C18-diacid), Arg34)GLP-1(7-37). Its potency is higher than Arg. 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37) requires a lower dose once daily compared to once-weekly dosing, thus requiring a lower concentration of the GLP-1 compound in its combination formulation with basal insulin.

[0106] The basal insulin may be the acylated soluble insulin disclosed in WO 95 / 07931, WO 2005 / 012347 or EP2007 / 054444.

[0107] Acylated basal insulin can be selected from the following list:

[0108] N εB29 -Decidecyldes (B30) human insulin,

[0109] N εB29 -Tetradecanoyldes (B30) human insulin,

[0110] N εB29 - Decanoyl des(B30) human insulin,

[0111] N εB29 -Lanoyldes (B30) human insulin,

[0112] N εB29 (N α -(HOOC(CH2) 14 CO)-γ-Glu)des(B30) human insulin;

[0113] N εB29 (N α -(HOOC(CH2) 15 CO)-γ-Glu)des(B30) human insulin;

[0114] N εB29 (N α -(HOOC(CH2) 16 CO)-γ-Glu)des(B30) human insulin;

[0115] N εB29 (N α -(HOOC(CH2) 17 CO)-γ-Glu)des(B30) human insulin;

[0116] N εB29 (N α -(HOOC(CH2) 18 CO)-γ-Glu)des(B30) human insulin;

[0117] N εB29 (N α -(HOOC(CH2) 16 CO)-γ-Glu-N α -(γ-Glu))des(B30) human insulin;

[0118] N εB29 (N α -(Asp-OC(CH2) 16 CO)-γ-Glu)des(B30) human insulin;

[0119] N εB29 (N α -(Glu-OC(CH2) 14 CO)-γ-Glu)des(B30) human insulin;

[0120] N εB29 (N α -(Glu-OC(CH2) 14 CO-)des(B30) human insulin;

[0121] N εB29 (N α -(Asp-OC(CH2) 16 CO-)des(B30) human insulin;

[0122] N εB29 (N α -(HOOC(CH2) 16 CO)-α-Glu-N α -(β-Asp)des(B30) human insulin;

[0123] N εB29 (N α -(Gly-OC(CH2) 13 CO)-γ-Glu)des(B30) human insulin;

[0124] N εB29 (N α -(Sar-OC(CH2) 13 CO)-γ-Glu)des(B30) Human Insulin

[0125] N εB29-ω-carboxy-pentadecanoyl-γ-L-glutamine desB30 human insulin

[0126] N εB29 -ω-carboxy-pentadecanoyl-γ-amino-butyryl desB30 human insulin

[0127] N εB29 -ω-carboxy-tetradecanoyl-γ-L-glutamine desB30 human insulin

[0128] N εB29 -ω-carboxy-tridecyl-γ-L-glutamine desB30 human insulin

[0129] N εB29 -ω-Carboxy-pentadecanoyl-β-alanyldesB30 human insulin

[0130] N εB29 -ω-carboxy-pentadecanoyl-γ-L-asparagine desB30 human insulin

[0131] N εB29 -ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human insulin

[0132] N εB29 -ω-Carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human insulin

[0133] N εB29 -10-(4-Carboxyphenoxy)-decanoyl-γ-L-glutamine desB30 human insulin,

[0134] N εB29 -4-[11-(4-carboxyphenyl)undecanoylamino]butyryldesB30 human insulin,

[0135] N εB29 -(3-(3-{4-[3-(7-carboxyheptanylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamine)desB30 human insulin,

[0136] N εB29 -ω-carboxy-tridecyl-γ-amino-butyryl desB30 human insulin

[0137] N εB29 -ω-carboxy-undecanoyl-γ-amino-butyryl desB30 human insulin

[0138] N εB29 -ω-carboxy-tetradecanoyl-γ-amino-butyryl desB30 human insulin

[0139] N εB29 -{4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyryl}desB30 insulin,

[0140] N εB29 -{4-[(14-carboxy-tetradecanoylamino)-methyl]-benzoyl}desB30 insulin,

[0141] N εB29 -[16-(4-carboxy-phenoxy)hexadecanoyl]desB30 insulin,

[0142] N εB29 -{4-[(15-carboxypentadecanoyl)benzoyl]-desB30 human insulin and

[0143] N εB29 -{4-[(15-carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30 insulin.

[0144] The concentration of the GLP-1 compound in the pharmaceutical composition according to the present invention may be about 1 to 25 mg / mL, about 2 to about 15 mg / mL, about 2 to about 10 mg / mL, about 2 to about 8 mg / mL, or about 2 to about 6 mg / mL.

[0145] The basal insulin concentration will be approximately 1 to approximately 25, approximately 1.5 to approximately 15, approximately 1.5 to approximately 12; approximately 1.5 to 8; approximately 2 to approximately 15, approximately 2 to approximately 10, approximately 2 to approximately 8; 2 to approximately 7; and approximately 3 to 6 mg / mL.

[0146] In one implementation, the basal insulin concentration is 2 mg / mL.

[0147] In another implementation, the basal insulin concentration is 4 mg / mL.

[0148] In another implementation, the basal insulin concentration is 8 mg / mL.

[0149] In one embodiment of the invention, Arg 34 Lys 26 (N ε -(γ-Glu(N α The concentration of α-hexadecyl)))-GLP-1(7-37) is approximately 2 mg / mL to approximately 10 mg / mL, and the concentration of basal insulin is approximately 3 mg / mL to approximately 5 mg / mL.

[0150] In another embodiment of the invention, Arg 34Lys 26 (N ε -(γ-Glu(N α The concentration of α-hexadecyl)))-GLP-1(7-37) is approximately 2 mg / mL to approximately 10 mg / mL, and the concentration of basal insulin is approximately 7 mg / mL to approximately 9 mg / mL.

[0151] When mixing this combination formulation, the order in which each component is added is important. As a first step, mix water, a buffer (if not a phosphate buffer), and an isotonic modifier. Then, add an appropriate amount of the basal insulin analog raw material solution and mix gently. Next, add a preservative such as phenol and gently mix the mixture for approximately 15–60 minutes. As a next step, add a portion of zinc in an amount corresponding to a concentration of 3 zinc ions / 6 insulin molecules. Then gently mix the mixture for at least 5 minutes. As a next step, add the remaining zinc ions to reach the desired concentration. Then, if the pH is outside the desired pH range, adjust the pH. Finally, add an appropriate amount of the GLP-1 compound raw material solution to reach the desired final concentration and adjust the pH to the desired value.

[0152] In one embodiment of the method for preparing the pharmaceutical composition, a preservative is added before zinc is added to the mixture. Therefore, in one embodiment, the order in which the components are added is: 1) basal insulin, 2) preservative, 3) zinc, and 4) GLP-1 compound. In another embodiment, zinc is added in several steps, for example, up to 3-4 steps.

[0153] Physical stability was evaluated using the Thioflavin T fibrillation test and the rotation test of the insulin penfill.

[0154] The zinc binding of the GLP-1 compound was studied using 1D H-NMR.

[0155] The self-association equilibrium of insulin in the presence or absence of GLP-1 compounds and in the presence of increased zinc concentration was studied using size exclusion chromatography with fluorescence detection (for identifying GLP-1 compounds in the chromatogram).

[0156] The PK performance of the combined formulation was evaluated using a standard appearance pig model.

[0157] In addition to the active ingredient, the pharmaceutical compositions of the present invention also contain conventional pharmaceutically acceptable adjuvants, such as isotonic agents, buffers, preservatives, and stabilizers.

[0158] Pharmaceutical preservatives are selected from phenol, o-cresol, m-cresol, p-cresol, methylparaben, propylparaben, 2-phenoxyethanol, butylparaben, 2-phenylethanol, benzyl alcohol, chlorobutanol, thimerosal, bronopol, benzoic acid, imidurea, chlorhexidine, sodium dehydroacetate, chlorocresol, ethylparaben, benzyl chloride, chlorophenoxypropyl-1,2-diol, or mixtures thereof.

[0159] In one embodiment, the preservative is a mixture of phenol and m-cresol. In another embodiment, the preservative is phenol. The use of preservatives in pharmaceutical compositions is well known to those skilled in the art. For convenience, please refer to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0160] Formulations according to the present invention generally include isotonic agents, such as mannitol, sorbitol, glycerol, propylene glycol, or mixtures thereof. In one embodiment, the isotonic agent is not a salt. The use of isotonic agents in pharmaceutical compositions is well known to those skilled in the art. For convenience, please refer to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0161] The pharmaceutical composition according to the invention may further include a buffer. This buffer may be selected from zwitterionic buffers, glycyl-glycine, TRIS, N-di(hydroxyethyl)glycine (bicine), HEPES, MOBS, MOPS, TES, and mixtures thereof. Other suitable buffers are sodium acetate, sodium carbonate, citrate, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine (tricine), malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof.

[0162] The pharmaceutical formulations according to the present invention preferably do not contain zinc-binding buffers, such as phosphate buffers.

[0163] The composition may also include a zinc chelating compound, which can act as a zinc buffer for free zinc ions in the formulation. This results in an increased zinc concentration with less liraglutide diheptamer formation. Such a zinc chelating compound may include (but is not limited to) histidine, imidazole, citrate, or derivatives thereof.

[0164] In one implementation, the zinc chelating agent is histidine.

[0165] In another embodiment, the zinc chelating agent is imidazole.

[0166] The addition of this zinc chelating agent is preferably less than 10 mM, more preferably less than 5 mM, more preferably less than 2 mM, more preferably less than 1 mM, and more preferably less than 0.5 mM.

[0167] As used herein, the term "storage-stable pharmaceutical composition" refers to a pharmaceutical composition that is stable for at least the time required by regulatory authorities for therapeutic proteins. Preferably, the storage-stable pharmaceutical composition is stable at 5°C for at least one year. Stability includes both chemical and physical stability.

[0168] As used herein, the term "basal insulin" refers to insulin with a time-action duration of more than 8 hours in a standard diabetes model. Preferably, basal insulin has a time-action duration of at least 9 hours. Preferably, basal insulin has a time-action duration of at least 10 hours. Preferably, basal diet-related insulin has a time-action duration of 9-15 hours. Preferably, the time-action duration of diet-related insulin is similar to that of commercially available NPH insulin and N... εB29 -Tetradecanoyldes (B30) is found to have a timing effect in pharmaceutical compositions of human insulin.

[0169] The term "effective dose" as used here refers to a dose that is sufficient to make treatment effective for the patient compared to no treatment.

[0170] The term "medicinal" as used here means that the medication is suitable for normal use, i.e., it has not produced any adverse events for the patient.

[0171] As used herein, the term "buffer" refers to a chemical compound in a composition that reduces the tendency of the composition's pH to change over time due to chemical reactions. Buffers include chemicals such as sodium phosphate, TRIS, glycine, and sodium citrate.

[0172] The term "preservative" as used herein refers to compounds added to a pharmaceutical composition to prevent or delay microbial activity (growth and metabolism).

[0173] The term "isotonic agent" refers to a chemical compound in a pharmaceutical composition that alters the osmotic pressure of the composition to approximate that of human blood plasma. Isotonic agents include NaCl, glycerol, and mannitol.

[0174] As used herein, the term "stabilizer" refers to a chemical added to a peptide-containing pharmaceutical composition to stabilize the peptide (i.e., to increase the shelf life and / or shelf life of the composition). Examples of stabilizers used in pharmaceutical formulations include L-glycine, L-histidine, arginine, polyethylene glycol, and carboxymethyl cellulose.

[0175] The term "insulin peptide" as used herein refers to a peptide of human insulin or an insulin analog or derivative having insulin activity.

[0176] The term "human insulin" as used herein refers to the human hormone whose structure and properties are well known. Human insulin has two polypeptide chains, chain A and chain B, linked by disulfide bridges between cysteine ​​residues. Chain A is a 21-amino acid peptide, and chain B is a 30-amino acid peptide. These two chains are connected by three disulfide bridges: one between cysteine ​​residues at positions 6 and 11 of chain A, a second between cysteine ​​residues at positions 7 of chain A and 7 of chain B, and a third between cysteine ​​residues at positions 20 of chain A and 19 of chain B.

[0177] The term "analyte" as used herein refers to a modified peptide in which one or more amino acid residues have been substituted with other amino acid residues and / or one or more amino acid residues have been deleted from the peptide and / or one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can occur at the N-terminus and / or C-terminus of the peptide.

[0178] Insulin analogs generally have no more than about 7 mutations compared to human insulin, more often no more than 5 mutations, and more often up to 3 mutations.

[0179] Over the years, a considerable number of modifications to the insulin A and B chains have been disclosed. Therefore, position 28 of the B chain can be changed from the natural Pro residue to Asp, Lys, or Ile, and the Lys residue at position B29 can also be changed to Pro.

[0180] Furthermore, the Asn at position A21 can be changed to Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val, especially to Gly, Ala, Ser, or Thr, particularly Gly. Additionally, the Asn at position B3 can be changed to Lys or Asp. Other examples of insulin analogs are des(B30) human insulin, where one or both of B1 and B2 have been deleted; insulin analogs where the A chain and / or B chain has an N-terminal elongation; and insulin analogs where the A chain and / or B chain has a C-terminal elongation. Also, the native amino acid residue at position A18 can be changed to a Gln residue, or one or more amino acid residues from positions B26-B30 can be deleted.

[0181] The term "derivative" used here to refer to a chemically modified parent protein or its analogue, wherein at least one substituent is not present in the parent protein or its analogue, i.e., the parent protein has been covalently modified. Typical modifications include amides, carbohydrates, alkyl groups, acyl groups, esters, PEGylation, etc. An example of a derivative of human insulin is threonine methyl ester. B30 Human insulin and N εB29 -Tetradecanoyldes (B30) human insulin.

[0182] The term "isoelectric point" as used here refers to the pH value at which the total net charge of macromolecules such as peptides is 0. A peptide can contain several charged groups, and at the isoelectric point, the sum of all these charges is 0. Above the isoelectric point, the total net charge of the peptide will be negative, while below the isoelectric point, the total net charge will be positive.

[0183] The term "about" used here to refer to the concentration of peptides in a pharmaceutical composition means plus or minus 10%. Therefore, a concentration of "about 5 mg / mL insulin" means a concentration of insulin from 4.5 mg / mL to 5.5 mg / mL.

[0184] The present invention includes the following embodiments:

[0185] Implementation Scheme 1: A soluble pharmaceutical composition for parenteral administration, comprising an insulin-stimulating GLP-1 compound, a basal insulin peptide, a pharmaceutically acceptable additive, and zinc, wherein the zinc content is at least 5 Zn ions / 6 insulin molecules.

[0186] Implementation Scheme 2: A soluble pharmaceutical composition for parenteral administration according to Implementation Scheme 1, comprising an insulin-stimulating GLP-1 compound, a basal insulin peptide, a pharmaceutically acceptable additive, and zinc, wherein the zinc content is at least 6 zinc ions / 6 insulin molecules.

[0187] Implementation Scheme 3: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-16 zinc ions / 6 insulin molecules.

[0188] Implementation Scheme 4: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-15 zinc ions / 6 insulin molecules.

[0189] Implementation Scheme 5: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-14 zinc ions / 6 insulin molecules.

[0190] Implementation Scheme 6: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-13 zinc ions / 6 insulin molecules.

[0191] Implementation Scheme 7: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-12 zinc ions / 6 insulin molecules.

[0192] Implementation Scheme 8: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-11 zinc ions / 6 insulin molecules.

[0193] Implementation Scheme 9: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-10 zinc ions / 6 insulin molecules.

[0194] Implementation Scheme 10: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-9 zinc ions / 6 insulin molecules.

[0195] Implementation Scheme 11: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-8 zinc ions / 6 insulin molecules.

[0196] Implementation Scheme 12: The pharmaceutical composition according to Implementation Scheme 1, wherein the zinc content is 5-7 zinc ions / 6 insulin molecules.

[0197] Implementation Scheme 13: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-16 zinc ions / 6 insulin molecules.

[0198] Implementation Scheme 14: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-15 zinc ions / 6 insulin molecules.

[0199] Implementation Scheme 15: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-14 zinc ions / 6 insulin molecules.

[0200] Implementation Scheme 16: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-13 zinc ions / 6 insulin molecules.

[0201] Implementation Scheme 17: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-12 zinc ions / 6 insulin molecules.

[0202] Implementation Scheme 18: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-11 zinc ions / 6 insulin molecules.

[0203] Implementation Scheme 19: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-10 zinc ions / 6 insulin molecules.

[0204] Implementation Scheme 20: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-9 zinc ions / 6 insulin molecules.

[0205] Implementation Scheme 21: The pharmaceutical composition according to Implementation Scheme 2, wherein the zinc content is 6-8 zinc ions / 6 insulin molecules.

[0206] Implementation Scheme 22: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-16 zinc ions / 6 insulin molecules.

[0207] Implementation Scheme 23: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-15 zinc ions / 6 insulin molecules.

[0208] Implementation Scheme 24: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-14 zinc ions / 6 insulin molecules.

[0209] Implementation Scheme 25: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-13 zinc ions / 6 insulin molecules.

[0210] Implementation Scheme 26: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-12 zinc ions / 6 insulin molecules.

[0211] Implementation Scheme 27: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-11 zinc ions / 6 insulin molecules.

[0212] Implementation Scheme 28: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-10 zinc ions / 6 insulin molecules.

[0213] Implementation Scheme 29: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 7-9 zinc ions / 6 insulin molecules.

[0214] Implementation Scheme 30: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 8-16 zinc ions / 6 insulin molecules.

[0215] Implementation Scheme 31: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 8-15 zinc ions / 6 insulin molecules.

[0216] Implementation Scheme 32: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 8-14 zinc ions / 6 insulin molecules.

[0217] Implementation Scheme 33: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 8-13 zinc ions / 6 insulin molecules.

[0218] Implementation Scheme 34: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 8-12 zinc ions / 6 insulin molecules.

[0219] Implementation Scheme 35: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 8-11 zinc ions / 6 insulin molecules.

[0220] Implementation Scheme 36: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the zinc content is 8-10 zinc ions / 6 insulin molecules.

[0221] Implementation Scheme 37: A pharmaceutical composition according to any of the foregoing embodiments, wherein the pH of the pharmaceutical composition is from about pH 7.4 to about pH 9.

[0222] Implementation Scheme 38: A pharmaceutical composition according to any of the foregoing embodiments, wherein the pH of the pharmaceutical composition is from about pH 7.4 to about pH 8.2.

[0223] Implementation Scheme 39: A pharmaceutical composition according to any of the foregoing embodiments, wherein the pH of the pharmaceutical composition is from about pH 7.4 to about pH 7.7.

[0224] Implementation Scheme 40: A pharmaceutical composition according to any of the foregoing embodiments, wherein the pH of the pharmaceutical composition is from about pH 7.6 to about pH 8.2.

[0225] Implementation Scheme 41: A pharmaceutical composition according to any of the foregoing embodiments, wherein the pH of the pharmaceutical composition is from about pH 7.7 to about pH 8.2.

[0226] Implementation Scheme 42: A pharmaceutical composition according to any one of the preceding V implementation schemes, wherein the pH of the pharmaceutical composition is from about pH 8.0 to about pH 9.

[0227] Implementation Scheme 43: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the insulin-stimulating GLP-1 peptide is GLP-1(7-37), a GLP-1(7-37) analog, a derivative of GLP-1(7-37), or a derivative of a GLP-1(7-37) analog.

[0228] Implementation Scheme 44: A pharmaceutical composition according to any of the foregoing embodiments, wherein the derivative of said GLP-1(7-37) includes lysine residues.

[0229] Implementation Scheme 45: The pharmaceutical composition according to Implementation Scheme 44, wherein the derivative of the GLP-1 (7-37) analog is Arg 34 Lys 26 (N ε -(γ-Glu(N α -hexadecyl)))-GLP-1(7-37).

[0230] Implementation Scheme 46: The pharmaceutical composition according to Implementation Scheme 44, wherein the derivative of the GLP-1 (7-37) analog is Aib8, Lys26 (OEG-OEG-γ-Glu-C18-diacid), Arg34)GLP-1 (7-37).

[0231] Implementation Scheme 47: The pharmaceutical composition according to Implementation Scheme 44, wherein the derivative of the GLP-1 (7-37) analog is [deaminoHis] 7 Arg 34 GLP-1-(7-37), [Aib 8 Glu 22 Arg 26 Arg 34 Lys 37 GLP-1-(7-37)amide.

[0232] Implementation Scheme 48: A pharmaceutical composition according to any one of the aforementioned Implementation Schemes 1-42, wherein the insulin-stimulating GLP-1 peptide is lizard exopeptide-4 or an analogue thereof.

[0233] Implementation Scheme 49: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the basal insulin peptide is acylated insulin.

[0234] Implementation Scheme 50: A pharmaceutical composition according to any of the foregoing implementation schemes, wherein the acylated insulin is acylated at position B29 with a lipophilic group.

[0235] Implementation Scheme 51: The pharmaceutical composition according to Implementation Scheme 50, wherein the lipophilic group has 8-40, 8-24 or 12-18 carbon atoms.

[0236] Implementation Scheme 52: The pharmaceutical composition according to Implementation Scheme 50, wherein the basal insulin is selected from N εB29 -Tetradecanoyldes (B30) Human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin, Lys B29 (N ε Lithochoyl-γ-Glu)-des(B30) human insulin, N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin and N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin.

[0237] Implementation Scheme 53: The pharmaceutical composition according to Implementation Scheme 50, wherein the basal insulin is N εB29 -Tetradecanoyldes (B30) Human insulin, N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin.

[0238] Implementation Scheme 54: The pharmaceutical composition according to Implementation Scheme 50, wherein the basal insulin is N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin.

[0239] Implementation Scheme 55: The pharmaceutical composition according to Implementation Scheme 50, wherein the basal insulin is N εB29 -ω-Carboxypentadecanyl-γ-L-glutamine desB30 human insulin or N εB29 -ω-Carboxypentadecanoyl-γ-amino-butyryl des(B30) human insulin.

[0240] Implementation Scheme 56: A pharmaceutical composition according to any one of the aforementioned Implementation Schemes 1-42, wherein the basal insulin is N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 is human insulin, while the insulin-stimulating GLP-1 compound is Arg 34 Lys 26 (Nε -(γ-Glu(N α -hexadecyl)))-GLP-1(7-37).

[0241] Implementation Scheme 57: A pharmaceutical composition according to any of the foregoing embodiments, wherein the concentration of basal insulin is in the range of about 1.5 to about 8 mg / mL, and the concentration of the GLP-1 compound is in the range of about 2 to about 10 mg / mL.

[0242] Implementation Scheme 58: A pharmaceutical formulation according to Implementation Schemes 1-36, wherein the basal insulin is glargine insulin, and the insulin-stimulating GLP-1 compound ZP10 is also described. According to Implementation Scheme 57, the pH is approximately 4.

[0243] Implementation Plan 59: The drug formulation according to Implementation Plan 58, wherein the zinc content is 4-5 zinc ions / 6 molecules of glargine insulin.

[0244] Example

[0245] Example 1

[0246] The preparation of insulin analog N is shown below. εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin and GLP-1 analog Arg 34 Lys 26 (N ε -(γ-Glu(N α -Hexadecyl)))-GLP-1(7-37), a typical formulation of a fixed combination of liraglutide. Appropriate starting material solutions of insulin analogs and GLP-1 analogs are prepared in water. The peptide concentration of both starting material solutions is generally about 10 mM, with the pH adjusted to approximately pH 7.7 for the insulin analog and approximately pH 8.2 for the liraglutide starting material solution. These pH adjustments are performed using a NaOH or HClO4 solution with a maximum concentration of 1.0 N.

[0247] The following excipients were mixed with the appropriate aqueous solution of the raw material in the following order: 1) water 2) glycerol (isotonic regulator) 3) insulin analog 4) phenol. Adding phenol before zinc is crucial; otherwise, a gel will form. After 15 minutes, zinc acetate was added in three portions: first, an amount corresponding to a concentration of 3 zinc / 6 insulin analog molecules; second, an amount corresponding to 3 zinc / 6 insulin analog molecules; and third, any additional zinc. After each addition, the solution was equilibrated for 5 minutes. The pH was measured and adjusted to pH 8.2 as described above. The solution was equilibrated at 4°C for approximately 48 hours, and then the appropriate amount of liraglutide raw material solution was added. Finally, the pH was measured and adjusted to pH 8.2 if necessary. Following this step, the formulation as described in Table 1 was prepared.

[0248] Table 1

[0249]

[0250]

[0251] 1) Corresponding to 8 Zn 2+ / 6 insulin analog molecules

[0252] 2) Add in three portions: 0.441ml, 0.440ml, and 0.293ml, with 5 minutes between each addition and after the last addition.

[0253] 3) The solution was equilibrated at 4°C for approximately 48 hours, and then liraglutide was added.

[0254] Example 2

[0255] Insulin analog N εB29 -(N α -(HOOC(CH2) 14 The prolongation mechanism of CO)-γ-Glu)desB30 human insulin has been described as depending on the formation of self-associations in the presence of more than 3 zinc ions / 6 insulin analog molecules. Therefore, if insulin analog N εB29 -(N α -(HOOC(CH2) 14 If a formulation of human insulin (CO)-γ-Glu)desB30 contains a significant portion of the analogue in monomeric form, it is expected to exhibit an undesirable rapid onset of action. Furthermore, a high content of insulin analogue monomers can affect the physical stability of the formulation. (This is in contrast to insulin analogue N...) εB29 -(N α -(HOOC(CH2) 14The presence of the GLP-1 analog liraglutide in the fixed combination of CO)-γ-Glu)desB30 human insulin may disrupt the self-association equilibrium of insulin analogs toward a monomeric state with a simultaneously formed zinc-bound liraglutide complex. Liraglutide has been shown to form heptamer self-assemblies under formulation-related conditions, and in the presence of equimolar concentrations of zinc ions, to form zinc-bound liraglutide diheptamers.

[0256] Contains insulin analog N εB29 -(N α -(HOOC(CH2) 14 The formulations of β-glucose (CCO)-γ-Glu)desB30 human insulin and liraglutide were analyzed using size exclusion chromatography (SEC) with fluorescence detection. The SEC method used a Superose 12 10 / 300GL column, injecting 50 μl of sample at a flow rate of 0.8 mL / min. Two methods were used, one with and one without phenol, and solvents included 140 mM NaCl, 10 mM Tris / HCl, pH 7.7, and + / - 2 mM phenol. Detection was performed in one channel with absorption at 276 nm (measuring total peptide content) and another channel using liraglutide-specific fluorescence detection (excitation at 310 nm, emission at 380 nm).

[0257] Under certain conditions, specific quantification is impossible due to experimental limitations. Here, the amount of monomeric insulin analogue is quantified relative to the total peptide amount.

[0258] Formulations were prepared as described in Example 1. Four formulations, each containing 0.3 mM and 0.6 mM insulin analogs, were prepared, with zinc contents corresponding to 6, 8, 10, and 12 zinc ions / 6 insulin analog molecules, respectively. These eight formulations were stored at 4°C and 37°C.

[0259] All formulations contain 0.3 or 0.6 mM insulin analog, zinc acetate, 1.6 mM liraglutide, 50 mM phenol, 174 mM glycerol, and pH 8.2.

[0260] The stored formulations were analyzed simultaneously using SEC with and without phenol after 0, 2, 4, and 8 weeks of storage. Tables 2 and 3 show the amounts of insulin analogs in monomeric form. Shaded areas indicate conditions under which precise quantification could not be performed. Therefore, quantification of insulin analog monomers relative to total peptide content was also performed. This is shown in Tables 4 and 5.

[0261] Table 2

[0262]

[0263] Table 3

[0264]

[0265] Table 4

[0266]

[0267] Table 5

[0268]

[0269] The amount of zinc-bound liraglutide biseptamer can be easily measured using fluorescence detection. Tables 6 and 7 show the measurements in the analyzed samples.

[0270] Table 6

[0271]

[0272] Table 7

[0273]

[0274] Example 3

[0275] Four formulations having the compositions shown in Table 8 were prepared following the procedure in Example 1. In formulation 3, glycylglycine buffer was added together with water. In formulation 4, phosphate buffer was added after zinc acetate. This was done to minimize zinc phosphate precipitation. The insulin analog used was N... εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin.

[0276] The physical stability of the four pharmaceutical compositions was evaluated by accelerated stress testing. The stress test was performed as a rotation test. 50 μL of air was added to each of five tubes (glass vials) of each formulation. The tubes were rotated at a frequency of 30 rpm for 4 hours daily. The tubes were examined periodically or as needed. The turbidity of the formulations was characterized by nephelometric measurement and specified in turbidimetric units (NTU). The physical instability of the proteins was characterized by high turbidity measurements.

[0277] The rotational assay is shown in Table 1. The results showed that the formulation containing the combination of insulin analog and liraglutide, prepared at pH 7.7 (Formulation 3), had lower stability than the insulin analog prepared alone at pH 7.4 (Formulation 1), and its stability may be unacceptable for further development. Formulation 4 also contained the combination of insulin analog and liraglutide, but was prepared at pH 8.2. The physical stability of this formulation was only slightly lower than that of insulin analog alone. Therefore, this comparison illustrates that the combination of insulin analog and liraglutide without any optimization resulted in an unstable formulation. However, increasing the pH to 8.2 yielded a significantly improved physical stability comparable to that of insulin analog alone.

[0278] Table 8

[0279] Insulin analogues (mM) 0.6 na 0.6 0.6 Zinc acetate (mM) 0.6 na 0.6 0.6 Liraglutide (mM) na 1.6 1.6 1.6 Phenol (mM) 16 58 40 40 m-Cresol (mM) 16 na na na Glycerin (mM) 174 174 174 174 NaCl (mM) 10 na 10 10 Phosphate (mM) na 8 na 8 Glycylglycine (mM) na na 8 na pH 7.4 7.7 7.7 8.2

[0280] Example 4

[0281] In another rotational test as described in Example 3, the test was conducted with insulin analog N. εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin and liraglutide four combination formulations, as well as two reference formulations containing only insulin analogs and liraglutide.

[0282] The formulations were prepared as described in Example 1, and their composition is shown in Table 9.

[0283] Table 9

[0284]

[0285] The results of the rotation test are in Figure 2-4 As shown in the image.

[0286] Formulation 3 contains 8 zinc / 6 insulin analog molecules and exhibits stability comparable to both reference formulations containing only insulin analogs (6 zinc / 6 insulin analog molecules) and liraglutide. Formulation 6 contains only 0.3 mM insulin analog but requires 10 zinc / 6 insulin analog molecules to achieve similar stability to the reference formulations. This indicates that a higher zinc content than that required for insulin analogs is needed to obtain the same stability as formulations containing 6 zinc / 6 insulin analog molecules.

[0287] Example 5

[0288] Insulin analog N εB29 (N α -(HOOC(CH2) 14The pharmacokinetic (PK) performance of a fixed-combination formulation of human insulin (CO)-γ-Glu)desB30 and liraglutide was examined in pigs. The presence of both peptides in the bloodstream was measured using standard assays over a period of up to 72 hours post-injection. However, for clarity, only the first 24 hours are shown for the insulin analog. Results are presented as the average of six replicates. Figure 5 and Figure 6 The values ​​are shown in Table 10, along with the standard error of the average. Three combination formulations and two references were prepared as described in Example 1. Their compositions are shown in Table 10. The PK appearance curves of the insulin analogs are shown in Table 10. Figure 5 The figure shows the p-value curve of liraglutide in... Figure 6 As shown in the image.

[0289] Table 10

[0290]

[0291] The presence of insulin analogs in this combination is crucial for several reasons: the rapidly acting component produces an unexpectedly early onset of efficacy. Increased bioavailability also leads to a greater reduction in blood glucose, subsequently requiring dose adjustment. Interestingly, it was concluded that combination formulation 3 (formulated with 8 zinc / 6 insulin analog molecules) exhibited a very similar insulin presence curve compared to the curves of individual insulin analogs (formulation 1). Therefore, combination formulations with 8 zinc / 6 insulin analog molecules showed very similar insulin pharmacokinetic performance when compared to individual insulin analogs. All combination formulations exhibited very similar liraglutide presence curves (formulations 3-5).

[0292] Example 6

[0293] Insulin analogs with different ratios N εB29 -(N α -(HOOC(CH2) 14 The pharmacokinetic (PK) performance of other fixed-combination formulations of human insulin (CO)-γ-Glu)desB30 and liraglutide was also examined in pigs. This ratio allows for the delivery of a larger amount of insulin analog relative to liraglutide to the patient in a single injection. Equal doses of insulin were administered to pigs with insulin analog reference (formulation 1) and combinations (formulations 3-5). The presence of both peptides in the bloodstream over a period of up to 72 hours post-injection was measured using standard assay techniques. However, for clarity, only the first 6 hours and 24 hours are shown separately. Results are presented as the average of six replicates. Figure 7 and Figure 8 The values ​​are shown in Table 11, along with the standard error of the average. Three combination formulations and two references were prepared as described in Example 1. Their compositions are shown in Table 11. The PK appearance curves of the insulin analogs are shown in Table 11. Figure 7 The figure shows the p-value curve of liraglutide in... Figure 8 As shown in the image.

[0294] Table 11

[0295]

[0296] Compared to the insulin analog reference (formulation 1), it has 6 Zn 2+ The combination of 6 insulin molecules (formulation 3) showed a secondary shoulder peak that appeared earlier than the main peak of the reference. Increasing the zinc content to 7 Zn... 2+ The presence of 6 insulin molecules (Formulation 4) resulted in an insulin analog abundance curve that more closely resembled the curve of the insulin reference (Formulation 1). Further increasing the zinc content to 8 Zn... 2+ No further improvement was achieved with 6 insulin molecules (formulation 5). No statistically significant differences were found in the liraglutide PK presence curves between the three combinations (formulations 3-5) and liraglutide alone (formulation 2). This indicates that the 7 Zn molecules used in the 1.2 mM insulin analog-1.6 mM liraglutide combination... 2+ The zinc content of 6 insulin molecules is preferred so that the insulin analogs in the combination obtain a PK presence curve similar to that of the individual analogs.

[0297] Example 7

[0298] The study investigated the pH effect of insulin analogs N in combination with liraglutide. εB29 -(N α -(HOOC(CH2) 14 The effect of CO)-γ-Glu)desB30 human insulin monomer content. Three formulations were prepared as described in Example 1, but adjusted to pH 8.2, pH 7.7, and pH 7.4. All three formulations consisted of: 0.6 mM insulin analog, 0.8 mM zinc acetate (8 zinc / 6 insulin analog molecules), 1.6 mM liraglutide, 50 mM phenol, and 214 mM glycerol. After preparation, the formulations were stored at 4°C for several weeks, followed by the first measurement at time point 0. Subsequently, the formulations were stored at 5°C and 37°C, and analyzed at these temperatures after an additional 2 and 4 weeks of storage. The formulations were analyzed using the SEC method described in Example 2. Both methods with and without phenol were used.

[0299] The relative contents of insulin analog monomers compared to total insulin analogs, as measured by SEC analysis with and without phenol, are shown in Tables 12 and 13, respectively. In both SEC methods and at both temperatures, decreasing the pH resulted in lower insulin analog monomer contents at each measurement time point. This indicates that pH is an important factor in controlling and regulating insulin monomer contents in combination with liraglutide. Lower insulin monomer contents can be obtained by using pH values ​​below 8.2, such as pH 7.7.

[0300] Table 12

[0301]

[0302] Table 13

[0303]

[0304] The content of liraglutide biseptamer was also measured in a similar manner, and the results obtained by the SEC method using phenol and without phenol are shown in Tables 14 and 15, respectively.

[0305] Table 14

[0306]

[0307] Table 15

[0308]

[0309] Example 8

[0310] The study described in Example 7 also included insulin analogs N containing low concentrations of histidine. εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin-liraglutide combination formulation. This amino acid is intended to act as a zinc buffer in the presence of increased zinc content. Formulation 1 consists of 0.6 mM insulin analog, 0.8 mM zinc acetate (8 zinc / 6 insulin analog molecules), 1.6 mM liraglutide, 50 mM phenol, and 214 mM glycerol, with the pH adjusted to pH 8.2. Formulation 2 consists of 0.6 mM insulin analog, 1.0 mM zinc acetate (10 zinc / 6 insulin analog molecules), 1.6 mM liraglutide, 50 mM phenol, 214 mM glycerol, and 0.5 mM histidine, with the pH adjusted to pH 8.2. Both formulations were prepared substantially as described in Example 1, with histidine added as a final excipient after liraglutide. The formulations were analyzed as described in Examples 2 and 7.

[0311] The relative contents of insulin analog monomers compared to total insulin analogs, as measured by SEC analysis with and without phenol, are shown in Tables 16 and 17, respectively. Formulation 2, with 10 zinc / 6 insulin molecules and 0.5 mM histidine, contained fewer insulin monomers over time at both temperatures compared to formulation 1, which had only 8 zinc / 6 insulin molecules.

[0312] Table 16

[0313]

[0314] Table 17

[0315]

[0316] The content of liraglutide biseptamer was also measured in a similar manner, and the results obtained by the SEC method using phenol and without phenol are shown in Tables 18 and 19, respectively.

[0317] Table 18

[0318]

[0319] Table 19

[0320]

[0321] Example 9

[0322] The study described in Example 7 also included insulin analog N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin-liraglutide combination, which consists of 0.3 mM insulin analog, 0.8 mM zinc acetate (16 zinc / 6 insulin analog molecules), 1.6 mM liraglutide, 50 mM phenol and 214 mM glycerol, adjusted to pH 8.2.

[0323] The relative contents of insulin analog monomers compared to total insulin analogs, as measured by SEC analysis with and without phenol (as described in Example 2), are shown in Tables 20 and 21, respectively.

[0324] Table 20

[0325]

[0326] Table 21

[0327]

[0328] The content of liraglutide biseptamer was also measured in a similar manner, and the results obtained by the SEC method using phenol and without phenol are shown in Tables 22 and 23, respectively.

[0329] Table 22

[0330]

[0331] Table 23

[0332]

[0333] Example 10

[0334] Formulations were prepared combining insulin glargine and the venom exopeptide-4 analog ZP10 (also known as AVE0010). ZP10, the venom exopeptide-4 analog, is described in Thorkildsen et al., (2003), JPET 307:490-496, and has the systematic name [des-Pro38]venom exopeptide-4-(1-39)yl-Lys-Lys-Lys-Lys-Lys-Lys-NH2. The following compositions were prepared by diluting appropriate starting material solutions in water: Formulation 1 consisted of 0.6 mM insulin glargine, 0.46 mM zinc acetate (4.6 zinc / 6 insulin glargine), and 60 μM ZP10. Formulation 2 consisted of 0.6 mM insulin glargine, 0.6 mM zinc acetate (6 zinc / 6 insulin glargine), and 60 μM ZP10. Both formulations were adjusted to approximately pH 4.0 using HCl and NaOH and stored at ambient temperature for 3 days. After storage, both formulations remained at approximately pH 4.0. Both formulations remained clear solutions both after preparation and after storage at ambient temperature.

Claims

1. A soluble pharmaceutical composition for parenteral administration, comprising an insulin-stimulating GLP-1 compound, a basal insulin peptide, a pharmaceutically acceptable additive, and zinc, wherein the basal insulin peptide is N εB29 -(N α -(HOOC(CH2) 14 CO)-γ-Glu)desB30 human insulin, and the insulin-stimulating GLP-1 compound is Arg 34 Lys 26 (N ε -(γ-Glu(N α -hexadecyl)))-GLP-1(7-37), wherein the zinc content is 8-9 Zn ions / 6 insulin molecules, and the pH of the soluble pharmaceutical composition is 8 to 8.3, wherein the concentration of the basal insulin peptide is in the range of 2 to 6 mg / mL, and the concentration of the insulin-stimulating GLP-1 compound is in the range of 2 to 8 mg / mL.

2. The pharmaceutical composition according to claim 1, wherein the concentration of the basal insulin peptide is in the range of 3 to 5 mg / mL.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is 8 to 8.

2.

4. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is 8.2 or 8.

3.

5. The pharmaceutical composition according to claim 1 or 2, further comprising histidine.

6. A method for preparing a pharmaceutical composition according to any one of claims 1-5, comprising dissolving the basal insulin peptide, mixing it with a preservative and an isotonic modifier, then adding zinc, and finally mixing it with a dissolved insulin-stimulating GLP-1 compound.

7. A method for preparing a pharmaceutical composition according to any one of claims 1-5, comprising the following steps: 1) dissolving the basal insulin peptide in an aqueous solution of a buffer and an isotonic agent; 2) optionally adding zinc in steps; 3) adjusting the pH; 4) storing the solution at a temperature of 4-5°C; and 5) adding the insulin-stimulating GLP-1 compound.

8. A method for preparing a pharmaceutical composition according to any one of claims 1-5, comprising the following steps: 1) dissolving the basal insulin peptide in an aqueous solution of a buffer and an isotonic agent; 2) optionally adding zinc in steps; 3) adjusting the pH; 4) storing the solution at ambient temperature; and 5) adding the insulin-stimulating GLP-1 compound.

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