Specification
PHARMACEUTICAL COMPOSITIONS CONTAINING INSULIN AND AN INSULINO-TROPIC PEPTIDE.
FIELD OF THE INVENTION
The present invention relates to the field of pharmaceutical compositions. More specifically the invention relates to pharmaceutical compositions comprising two different phar-maceutically active peptides.
BACKGROUND OF THE INVENTION
Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. Since the introduction of insulin in the 1920's, continuous efforts have been made to improve the treatment of diabetes mellitus. Since people suffering from diabetes are subject to chronic treatment over several decades, there is a major need for safe, convenient and life quality improving insulin formulations.
In the treatment of diabetes mellitus, many varieties of insulin formulations have been suggested and used, such as regular insulin, isophane insulin (designated NPH), insulin zinc suspensions (such as Semilente®, Lente®, and Ultralente®), and biphasic isophane insulin. Some of the commercial available insulin formulations are characterized by a fast onset of action and other formulations have a relatively slow onset but show a more or less prolonged action. Fast-acting insulin formulations are usually solutions of insulin, while retarded acting insulin formulations can be suspensions containing insulin in crystalline and/or amorphous form precipitated by addition of zinc salts alone or by addition of protamine or by a combination of both. Within the last decade a number of human insulin analogues have been developed. They are designed for particular profiles of action, i.e. fast acting or prolonged action.
Another peptide expected to become very important in the treatment of diabetes is glu-cagon-like peptide-1 (GLP-1). Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesized i.a. in the L-cells in the distal ileum, in the pancreas and in the brain. GLP-1 is an important gut hormone with regulatory function in glucose metabolism and gastrointestinal secretion and metabolism. GLP-1 stimulates insulin secretion in a glucose-dependant manner, stimulates insulin biosynthesis, promotes beta cell rescue, decreases glucagon secretion, gastric emptying and food intake. As the type 2 diabetes population is rapidly increasing in the world there is a much larger need for simpler administration of more effective drugs. A combination formulation comprising an insulin peptide and a GLP-1 peptide with a fixed ratio of the two pharmaceuticals may be a very efficacious treatment as well as one requiring less injections when administered to the same patient.
A combined treatment of insulin requiring diabetes comprising administration of insulin and GLP-1 is disclosed in WO 95/31214. Pre-mixed formulation of GLP-1 compounds and basal insulin are disclosed in WO 03/020201. Shelf stable pharmaceutical compositions comprising GLP-1, a basal insulin and surfactants are disclosed in WO 2006/051103.
BREIF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the physical stability of four pharmaceutical compositions evaluated by means of an accelerated stressed test.
Figure 2 -4 show the results from a rotation test
Figure 5 -8 show the pharmacokinetic (PK) properties of fixed combination formulations with the insulin analogue NE829-(Na-(HOOC(CH2)i4CO)-Y-Glu) desB30 human insulin and liraglutide exmanined in pigs.
SUMMARY OF THE INVENTION
One object of the present invention is to provide a shelf-stable once daily fixed combination of a GLP-1 compound and a basal insulin compound with unchanged PK/PD properties of the combined peptides compared to the individual active components.
More specifically the invention is related to a shelf-stable pharmaceutical composition comprising a fixed combination of an insulinotropic GLP-1 compound and a basal insulin which composition contains at least 5 zinc ions per 6 basal insulin molecules.
In one embodiment the pharmaceutical composition contains at least 6 zinc ions per 6 basal insulin molecules.
In another embodiment the pharmaceutical composition contains at least 7 zinc ions per 6 basal insulin molecules.
In another embodiment the pharmaceutical composition contains at least 8 zinc ions per 6 basal insulin molecules.
In a further embodiment the pharmaceutical composition contains at least 9; 10; 11; 12; 13; 14; 15; or 16 zinc ions per 6 basal insulin molecules.
In one embodiment the zinc content is between 5 and 16 zinc ions per 6 insulin molecules.
In a another embodiment the zinc content is between 5 and 15; 5 and 14; 5 and 13; 5 and 12; 5 and 11; 5 and 10; 5 and 9, 5 and 8 or between 5 and 7 zinc ions per 6 insulin molecules.
In a further embodiment the zinc content is between 6 and 16 zinc ions per 6 insulin molecules.
In a further embodiment the zinc content is between 6 and 15; 6 and 14; 6 and 13; 6 and 12; 6 and 11; 6 and 10; 6 and 9 or between 6 and 8 zinc ions per 6 insulin molecules.
In another embodiment the zinc content is between 7 and 16 zinc ions per 6 insulin molecules.
In a further embodiment the zinc content is between 7 and 14; 7 and 15; 7 and 14; 7 and 13; 7 and 12; 7 and 11; 7 to 10; or 7 and 9 zinc ions per 6 basal insulin molecules.
In another embodiment the zinc content is between 8 and 16 zinc ions per 6 insulin molecules.
In another embodiment the zinc content is between 8 and 15; 8 and 14; 8 and 13; 8 and 12; 8 and 11; or 8 and 10 zinc ions per 6 insulin molecules.
The pH of the pharmaceutical formulation will in any of the above embodiments typically be above neutral and will typically be between about 7 and about 9.
In one embodiment the pH of the pharmaceutical composition is between about pH 7.4 and about pH 9; between about pH 7.4 and about pH 8.5 or between about pH 7.4 and about pH 8.2.
In another embodiment the pH of the pharmaceutical composition is between about pH 7.5 and about pH 8.5; between about pH 7.5 and about pH 8.2 or between about 7.5 and about 7.7.
In another embodiment the pH of the pharmaceutical composition is from about pH 7.6 to about pH 8.2. In a further embodiment the pH is between about 7.7 and about 8,2.
In another embodiment the pH will be between about 7.7 and about 9.
In another embodiment the pH will be between about 7.7 and about 8.9.
In another embodiment the pH will be between about 7.7 and about 8.8.
In another embodiment the pH will be between about 7.7 and about 8.7.
In another embodiment the pH will be between about 7.7 and about 8.6.
In another embodiment the pH will be between about 7.7 and about 8.5.
In another embodiment the pH will be between about 7.7 and about 8.4.
In another embodiment the pH will be between about 7.7 and about 8.3.
In another embodiment the pH will be between about 7.7 and about 8.2.
In another embodiment the pH will be between about 7.7 and about 8.1.
In another embodiment the pH will be between about 7.7 and about 8.
The insulinotropic GLP-1 compound may in any of the above embodiments be any GLP-1 compound being effective for type 2 treatment. The term "GLP-1 peptide" as used herein means GLP-1 (7-37), a GLP-1 (7-37) analogue, a GLP-1 (7-37) derivative or a derivative of a GLP-1 (7-37) analogue. Derivatives of GLP-1 may be acylated GLP1 -compounds such as disclosed in WO 98/08871 or WO 2006/097537.
In one embodiment the GLP-1 compound is an acylated GLP-1 analogue such as Arg34, Lys26(N6-(γ-Glu(NT-hexadecanoyl)))-GLP-1 (7-37), Aib8,Lys26(OEG-OEG-gamma-Glu-C18-diacid),Arg34)GLP-1 H(7-37)-OH or (N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)-acetyl][Aib8,Arg34]GLP-1-(7-37).
In another embodiment the GLP-1 is exendin-3,exendin-4 or an exendin-4 analogue. The exendin-4-analogue may comprise from 4-10, 4-8 or 4-6 basic amino acid residues added to either the C-terminal or the N-terminal end.
The basal insulin may in any of the above embodiments be any basal insulin known for treatment of type 1 and type 2 diabetes. In one embodiment the basal insulin is an insulin acylated in E-amino group in the B29Lys in the B-chain of insulin and analogues thereof as disclosed in WO 95/07931, WO 2005/012347 and in EP2007/054444.
In another embodiment the basal insulin is a pi shifted basal insulin such as the type disclosed in US patent 5,656,722 with basic amino acid residue substitutions or additions of the insulin molecule. One example of such basal insulins is GlyA21, ArgB31, Arg B32 human insulin (insulin glargine). Another basal insulin may be an amidated insulin glargine such as the compounds disclosed in WO2008/006496 and WO2008/006497.
In one embodiment the basal insulin is NEB29-tetradecanoyl des(B30) human insulin.
In a further embodiment the basal insulin is LysB29(N£ lithocholyl-Y-Glu)-des(B30) human insulin.
In a further embodiment the basal insulin is NEB29-tetradecanoyl des(B30) human insulin or NtB29-(Na-(HOOC(CH2)i4CO)-Y-Glu) desB30 human insulin.
In a further embodiment the basal insulin is NcB29-w-carboxypentadecanoyl-γ-L-glutaylamide desB30 human insulin or NEB29- co-carboxypentadecanoyl-y - amino-butanoyl des(B30) human insulin.
In one embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin selected from the group consisting of NcB29-tetradecanoyl des(B30) human insulin, LysB29(NE lithocholyl-γ-Glu)-des(B30) human insulin, NEB29-(Na-(HOOC(CH2)i4CO)-Y-Glu) desB30 human insulin, NEB29-w-carboxypentadecanoyl-γ-L-glutaylamide desB30 human insulin and NEB29- w-carboxypentadecanoyl-y - amino-butanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains at least 5; 6; 7; 8 or 9 zinc atoms per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin selected from the group consisting of NcB29-tetradecanoyl des(B30) human insulin, LysB29(NE lithocholyl-γ-Glu)-
des(B30) human insulin, NEB29-(N°-(HOOC(CH2)14CO)-Γ-GIU) desB30 human insulin, N£B29-w-carboxypentadecanoyl-Y-L-glutaylamide desB30 human insulin and NEB29- w-carboxypentadecanoyl-Y - amino-butanoyl des(B30) human insulin together with suitable pharmaceutical^ acceptable adjuvants which composition contains between 5 and 16; 5 and 14; 5 and 12; 5 and 10; or between 5 and 8 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin selected from the group consisting of N£B29-tetradecanoyl des(B30) human insulin, LysB29(NE lithocholyl-γ-Glu)-des(B30) human insulin, N£B29-(Na-(HOOC(CH2)14CO)-Y-Glu) desB30 human insulin, N£B29-co-carboxypentadecanoyl-γ-L-glutaylamide desB30 human insulin and N£B29~ w-carboxypentadecanoyl-y - amino-butanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains between 6 and 16; 6 and 14; 6 and 12; 6 and 10; or between 6 and 8 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin selected from the group consisting of N£B29-tetradecanoyl des(B30) human insulin, LysB29(N£ lithocholyl-Y-Glu)-des(B30) human insulin, N£B29-(Na-(HOOC(CH2)14CO)-Y-Glu) desB30 human insulin, NEB29-w-carboxypentadecanoyl-Y-l—glutaylamide desB30 human insulin and NEB29-w-carboxypentadecanoyl-Y - amino-butanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains between 7 and 16; 7 and 14; 7 and 12; or between 7 and 10 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin selected from the group consisting of NEB29-tetradecanoyl des(B30) human insulin, LysB29(NE lithocholyl-Y-Glu)-des(B30) human insulin, NEB29-(Na-(HOOC(CH2)14CO)-Y-Glu) desB30 human insulin, NEB29-w-carboxypentadecanoyl-Y-L-glutaylamide desB30 human insulin and NEB29- w carboxypentadecanoyl-Y - amino-butanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains between 8 and 16; 8 and 14; 8 and 12: or between 8 and 10 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin with the formula NEB29-(NQ-(HOOC(CH2)I4CO)-Γ-GIU) desB30 human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin N£B29-(N°-
(HOOC(CH2)I4CO)-Γ-GIU) desB30 human insulin together with suitable pharmaceutical^ acceptable adjuvants which composition contains between 5 and 16 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin N£B29-(Na-(HOOC(CH2)I4CO)-Γ-GIU) desB30 human insulin together with suitable pharmaceutical^ acceptable adjuvants which composition contains between 6 and 16; 6 and 15; 6 and 14; 6 and 13; 6 and 12; 6 and 10; or between 6 and 8 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin N£B29-(N°-(HOOC(CH2)14CO)-Γ-GIU) desB30 human insulin together with suitable pharmaceutical^ acceptable adjuvants which composition contains between 7 and 16; 7 and 15; 7 and 14; 7 and 13; 7 and 12; 7 and 11; 7 and 10; 7 and 9; or between 7 and 8 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an insulinotropic GLP-1 compound and an acylated basal insulin NcB29-(Na-(HOOC(CH2)I4CO)-Γ-GIU) desB30 human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains between 8 and 16; 8 and 15; 8 and 14; 8 and 13; 8 and 12; 8 and 11; 8 and 10; or between 8 and 9 zinc ions per 6 insulin molecules.
In one embodiment the invention is related to a pharmaceutical composition containing a fixed combination of an insulinotropic GLP-1 compound and a basal insulin N£B29-tetradecanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants, wherein thel-zinc content is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 zinc ions per 6 insulin molecules.
In another embodiment the invention is related to a pharmaceutical composition containing a fixed combination of an insulinotropic GLP-1 compound and a basal insulin N£B29-tetradecanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants, wherein the zinc content is between 5 and 16; 6 and 16; between 7 and 16; or between 8 and 16 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an GLP-1 (7-37) or an analogue or derivative thereof and an acylated basal insulin selected from the group consisting of N£B29-tetradecanoyl des(B30) human insulin, LysB29(N£ lithocholyl-Y-Glu)-des(B30) human insulin, N£B29-(Na-(HOOC(CH2)14CO)-Y-Glu) desB30 human insulin, N£B29-u)-carboxypentadecanoyl-Y-L-glutaylamide desB30 human insulin and NtB29- cj-carboxypentadecanoyl-y - amino-butanoyl des(B30) human insulin together with
suitable pharmaceutical^ acceptable adjuvants which composition contains at least 5 or 6 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an GLP-1(7-37) or an analogue or derivative thereof and an acylated basal insulin selected from the group consisting of NEB29-tetradecanoyl des(B30) human insulin, LysB29(N£ lithocholyl-Y-Glu)-des(B30) human insulin, N£B29-(NQ-(HOOC(CH2)I4CO)-Γ-GIU) desB30 human insulin, N£B29-γ-carboxypentadecanoyl-Y-L-glutaylamide desB30 human insulin and N£B29- oo-carboxypentadecanoyl-Y - amino-butanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains between 5 and 16; 5 and 14; 5 and 12; 5 and 10 or between 5 and 8 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an GLP-1 (7-37) or an analogue or derivative thereof and an acylated basal insulin selected from the group consisting of N£B29-tetradecanoyl des(B30) human insulin, LysB29(N£ lithocholyl-Y-Glu)-des(B30) human insulin, N£B29-(Na-(HOOC(CH2)uCO)-Y-Glu) desB30 human insulin, N£B29-oj-carboxypentadecanoyl-Y-L-glutaylamide desB30 human insulin and NtB29_ co-carboxypentadecanoyl-Y - amino-butanoyl des(B30) human insulin together with suitable pharmaceutically acceptable adjuvants which composition contains between 6 and 16; 6 and 14; 6 and 12; or 6 and 10 zinc ions per 6 insulin molecules.
In a further embodiment the pharmaceutical composition comprises a fixed combination of an GLP-1 (7-37) or an analogue or derivative thereof and an acylated basal insulin selected from the group consisting of NtB29-tetradecanoyl des(B30) human insulin, LysB29(N£ lithocholyl-Y-Glu)-des(B30) human insulin, NtB29-(Na-(HOOC(CH2)14CO)-Y-Glu) desB30 human insulin, N£B29- as sodium phosphate, TRIS, glycine and sodium citrate.
The term "preservative" as used herein refers to a chemical compound which is added to a pharmaceutical composition to prevent or delay microbial activity (growth and metabolism).
The term "isotonicity agent" as used refers to a chemical compound in a pharmaceutical composition that serves to modify the osmotic pressure of the pharmaceutical composition so that the osmotic pressure becomes closer to that of human plasma. Isotonicity agents include NaCI, glycerol, mannitol etc.
The term "stabilizer" as used herein refers to chemicals added to peptide containing pharmaceutical compositions in order to stabilize the peptide, i.e. to increase the shelf life and/or in-ude time of such compositions. Examples of stabilizers used in pharmaceutical formulations are L-glycine, L-histidine, arginine, polyethylene glycol, and carboxymethylcellu-lose.
The term "insulin peptide" as used herein means a peptide which is either human insulin or an analog or a derivative thereof with insulin activity.
The term "human insulin" as used herein means the human hormone whose structure and properties are well known. Human insulin has two polypeptide chains that are connected by disulphide bridges between cysteine residues, namely the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges: one between the cysteines in position 6 and 11 of the A-chain, the second between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain.
The term "analogue" as used herein referring to a peptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide.
The insulin analogues will typically not comprise more than about 7 mutations, more typically not more than 5 and even more typically at the most 3 mutations compared to human insulin.
Over the years a fairly large number of modification of the insulin A- and or B-chain have been disclosed. Thus the position 28 of the B chain may be modified from the natural Pro residue to Asp, Lys, or lie and Lys in position B29 may also be modified to Pro.
Also, Asn at position A21 may be modified to Ala, Gin, Glu, Gly, His, lie. Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and in particular to Gly. Furthermore, Asn at position B3 may be modified to Lys or Asp. Further examples of insulin analogues are des(B30) human insulin, insulin analogues wherein one or both of B1 and 82 have been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-temriinal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension. Also, the natural amino acid residue in position A18 may be changed to a Gin residue or one or more of the amino acid residue in positions B26-B30 may be deleted.
The term "derivative" as used herein in relation to a parent peptide means a chemically modified parent protein or an analogue thereof, wherein at least one substituent is not present in the parent protein or an analogue thereof, i.e. a parent protein which has been co-valently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, PEGylations and the like. Examples of derivatives of human insulin are threonine methyl esterB30 human insulin and NεB29-w-tetradecanoyl des(B30) human insulin.
The term "isoelectric point" as used herein means the pH value where the overall net charge of a macromolecule such as a peptide is zero. In peptides there may be several charged groups, and at the isoelectric point the sum of all these charges is zero. At a pH above the isoelectric point the overall net charge of the peptide will be negative, whereas at pH values below the isoelectric point the overall net charge of the peptide will be positive.
The term "about" as used herein in relation to the concentration of a peptide in a pharmaceutical composition means plus or minus 10%. Hence, the concentration "about 5 mg/mL insulin" means a concentration of 4.5 mg/mL insulin to 5.5 mg/mL insulin.
The invention covers the following embodiments: Embodiment 1: A soluble pharmaceutical composition for parenteral administration which comprises an insulinotfopic GLP-1 compound , a basal insulin peptide, pharmaceutically acceptable additives and zinc, wherein the zinc content is at least 5 Zn ions per 6 insulin molecules.
Embodiment 2: A soluble pharmaceutical composition for parenteral administration according to embodiment 1, which comprises an insulinotropic GLP-1 compound , a basal insulin peptide, pharmaceutically acceptable additives and zinc, wherein the zinc content is at least 6 Zn ions per 6 insulin molecules.
Embodiment 3: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 16 zinc ions per 6 insulin molecules.
Embodiment 4: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 15 zinc ions per 6 insulin molecules.
Embodiment 5: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 14 zinc ions per 6 insulin molecules.
Embodiment 6: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 13 zinc ions per 6 insulin molecules.
Embodiment 7: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 12 zinc ions per 6 insulin molecules.
Embodiment 8: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 11 zinc ions per 6 insulin molecules.
Embodiment 9: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 10 zinc ions per 6 insulin molecules.
Embodiment 10: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 9 zinc ions per 6 insulin molecules.
Embodiment 11; The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 8 zinc ions per 6 insulin molecules.
Embodiment 12: The pharmaceutical composition according to embodiment 1, wherein the zinc content is between 5 and 7 zinc ions per 6 insulin molecules.
Embodiment 13: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 16 zinc ions per 6 insulin molecules.
Embodiment 14: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 15 zinc ions per 6 insulin molecules.
Embodiment 15: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 14 zinc ions per 6 insulin molecules.
Embodiment .16: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 13 zinc ions per 6 insulin molecules.
Embodiment 17: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 12 zinc ions per 6 insulin molecules.
Embodiment 18: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 11 zinc ions per 6 insulin molecules.
Embodiment 19: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 10 zinc ions per 6 insulin molecules.
Embodiment 20: The pharmaceutical composition according to embodiment,2, wherein the zinc content is between 6 and 9 zinc ions per 6 insulin molecules.
Embodiment 21: The pharmaceutical composition according to embodiment 2, wherein the zinc content is between 6 and 8 zinc ions per 6 insulin molecules.
Embodiment 22: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 16 zinc ions per 6 insulin molecules.
Embodiment 23: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 15 zinc ions per 6 insulin molecules.
Embodiment 24: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 14 zinc ions per 6 insulin molecules.
Embodiment 25: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 13 zinc ions per 6 insulin molecules.
Embodiment 26: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 12 zinc ions per 6 insulin molecules.
Embodiment 27: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 11 zinc ions per 6 insulin molecules.
Embodiment 28: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 10 zinc ions per 6 insulin molecules.
Embodiment 29: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 7 and 9 zinc ions per 6 insulin molecules.
Embodiment 30: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 8 and 16 zinc ions per 6 insulin molecules.
Embodiment 31: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 8 and 15 zinc ions per 6 insulin molecules.
Embodiment 32: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 8 and 14 zinc ions per 6 insulin molecules.
Embodiment 33: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 8 and 13 zinc ions per 6 insulin molecules.
Embodiment 34: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 8 and 12 zinc ions per 6 insulin molecules.
Embodiment 35: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 8 and 11 zinc ions per 6 insulin molecules.
Embodiment 36: The pharmaceutical composition according to any of the preceding embodiments, wherein the zinc content is between 8 and 10 zinc ions per 6 insulin molecules.
Embodiment 37: The pharmaceutical composition according to any of the preceding embodiments, wherein the pH of said pharmaceutical composition from about pH 7.4 to about pH 9.
Embodiment 38: The pharmaceutical composition according to any of the preceding embodiments, wherein the pH of said pharmaceutical composition from about pH 7.4 to about pH 8.2.
Embodiment 39: The pharmaceutical composition according to any of the preceding embodiments, wherein the pH of said pharmaceutical composition from about pH 7.4 to about pH 7.7.
Embodiment 40: The pharmaceutical composition according to any of the preceding embodiments, wherein the pH of said pharmaceutical composition from about pH 7.6 to about pH 8.2.
Embodiment 41: The pharmaceutical composition according to any of the preceding embodiments, wherein the pH of said pharmaceutical composition from about pH 7.7 to about pH 8.2.
Embodiment 42: The pharmaceutical composition according to any of the preceding V embodiments wherein the pH of said pharmaceutical composition from about pH 8.0 to about pH 9.
Embodiment 43: The pharmaceutical composition according to any of the preceding embodiments, wherein the insuiinotropic GLP-1 peptide is GLP-1(7-37), a GLP-1(7-37) analogue, a derivative of GLP-1 (7-37), or a derivative of a GLP-1 (7-37) analogue.
Embodiment 44: The pharmaceutical composition according to any of the preceding embodiments, wherein the derivative of GLP-1 (7-37) comprises a lysine residue.
Embodiment 45; The pharmaceutical composition according to embodiment 44, wherein said derivative of a GLP-1 (7-37) analogue is Arg34, Lys26(Nε-(Y-Glu(N"-hexadecanoyl)))-GLP-1 (7-37).
Embodiment 46: The pharmaceutical composition according to embodiment 44, wherein said derivative of a GLP-1(7-37) analogue is Aib8,Lys26(OEG-OEG-gamma-Glu-C18-diacid),Arg34)GLP-1(7-37).
Embodiment 47: The pharmaceutical composition according to embodiment 44, wherein said derivative of a GLP-1(7-37) analogue is [desaminoHis7,Arg34]GLP-1-(7-37), [Aib8Glu'22Arg26Arg34,Lys37]GLP-1-(7-37)amide.
Embodiment 48: The pharmaceutical composition according to any of the preceding embodiments 1-42, wherein the insulinotropic GLP-1 peptide is exendin-4 or an analogue thereof.
Embodiment 49: The pharmaceutical composition according to any one of the preceding embodiments, wherein the basal insulin peptide is an acylated insulin.
Embodiment 50: A pharmaceutical composition according to any one of the preceding embodiments, wherein the acylated insulin is acylated in position B29 with a lipophilic group.
Embodiment 51: The pharmaceutical composition according to embodiment 50, wherein the lipophilic group has from 8 to 40, 8 to 24, or 12-18 carbon atoms.
Embodiment 52: A pharmaceutical composition according to embodiment 50, wherein the basal insulin is selected from the group consisting of NεB29-tetradecanoyl des(B30) human insulin, NεB29-(N°-(HOOC(CH2)I4CO)-Y-GIU) desB30 human insulin, LysB29(Nε lithocholyl-v-Glu)-des(B30) human insulin, NεB29-w-carboxypentadecanoyl-y-L-glutaylamide desB30 human insulin and NεB29- -carboxypentadecanoyl-y - amino-butanoyi des(B30) human insulin.
Embodiment 53: A pharmaceutical composition according to embodiment 50, wherein the basal insulin is NεB29-tetradecanoyl des(B30) human insulin, NεB29-(N°-(HOOC(CH2)14CO)-v-Glu) desB30 human insulin.
Embodiment 54: A pharmaceutical composition according to embodiment 50, wherein the basal insulin is NεB29-(N°-(HOOC(CH2)14CO)-v-Glu) desB30 human insulin.
Embodiment 55: A pharmaceutical composition according to embodiment 50, wherein the basal insulin is NεB29-w-carboxypentadecanoyl-y-L-glutaylamide desB30 human insulin or NεB29-w-carboxypentadecanoyl-y - amino-butanoyI des(B30) human insulin.
Embodiment 56: The pharmaceutical composition according to any one of the preceding embodiments 1-42, wherein the basal insulin is NεB29-(Nα-(HOOC{CH2)i4CO)-v-Glu) desB30 human insulin and the insulinotropic GLP-1 compound is Arg34, Lys26(Nε-(Y-Glu(Nα-hexadecanoyl)))-GLP-1 (7-37).
Embodiment 57: The pharmaceutical composition according to any of the preceding embodiments, wherein the concentration of the basal insulin is in the range from 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.
Embodiment 58: The pharmaceutical formulation according to embodiment 1-36, wherein the basal insulin is insulin glargine and the insulinotropic GLP-1 compound is ZP10.Embodiment according to embodiment 57, wherein the pH is about 4.
Embodiment 59: The pharmaceutical formulation according to embodiment 58, wherein the zinc content is between 4 an 5 zinc ions per 6 molecules insulin glargine.
EXAMPLES
EXAMPLE 1
A typical formulation of the fixed combination consisting of the insulin analogue NεB29_(Na_(,HOOC(CH2)14CO)-y-Glu desB30 human insulin and the GLP-1 analogue Arg34
Lys26®(Nε-(Y-Glu(N"-hexadecanoyl)))-GLP-1(7-37), liraglutide, was prepared as follows. Appropriate stock solutions in water were prepared of the insulin analogue and the GLP-1 analogue. The peptide concentrations of both stocks were typically around 10 mM and pH was adjusted to approximately pH 7.7 for the insulin analogue and approximately pH 8.2 for the liraglutide stock solutions. These pH adjustments were done using either NaOH or HCIO4 solutions with maximalconcentrations of 1.0 N.
The following excipients were mixed from appropriate stock solutions in water in this order: 1) water 2) glycerol (isotonicity modifier) 3) insulin analogue 4) phenol. The addition of phenol before zinc was critical; otherwise a gel was formed. After 15 minutes the zinc acetate was added in three portions: Firstly, a zinc amount corresponding to a concentration of 3 zinc/6 insulin analogue molecules; secondly, a zinc amount corresponding to a concentration of 3 zinc/6 insulin analogue molecules; and thirdly any additional zinc. The solution was equilibrated 5 minutes after each addition. The pH was measured and adjusted to pH 8.2 as described above. This solution was left to equilibrate for approximately 48 hours at 4°C before an appropriate amount of the liraglutide stock solution was added. Finally, pH was measured and if necessary adjusted to pH 8.2. Following this procedure a formulation was prepared as described in the Table 1.
Table 1
(Table Removed)
1)' Corresponding to 8 Zn2+/6 insulin analogue molecules
2)' Was added in three portions: 0.441 ml, 0.440 ml, 0.293 ml, each with 5 minutes in-between
and after the last addition
3)' The solution was equilibrated at 4 °C for approximately 48 hours before the addition of
liraglutide
EXAMPLE 2
The protracting mechanism of the insulin analogue NεB29-(N°-(HOOC(CH2)I4CO)-Y-Glu) desB30 human insulin has been described to depend on the formation of self-associates in the presence of more than 3 zinc ions per 6 insulin analogue molecules. Hence, if a formulation of insulin analogue NεB29-(N°-(HOOC(CH2)I4CO)-Y-GIU) desB30 human insulin contains a significant fraction of the analogue on monomeric form, the formulation is expected to exhibit an undesirable fast-acting component. Furthermore, a high insulin analogue monomer content could compromise the physical stability of the formulation. The presence of the GLP-1 analogue liraglutide in a fixed combination with the insulin analogue NεB29-(N°-(HOOC(CH2)I4CO)-V-GIU) desB3 human insulin may perturb the insulin analogue self-association equilibrium towards the monomeric state with a simultaneously formation of a liraglutide zinc binding complex. It has been demonstrated that liraglutide forms a hep-tameric self-assembly under formulation relevant conditions, and in the presence of equimo-lar concentrations of zinc ions, a zinc binding liraglutide diheptamer is formed.
Formulations containing both the insulin analogue NεB29-(NΑ'-(HOOC(CH2)I4CO)-Y-Glu) desB30 human insulin and liraglutide were analysed by a size exclusion chromatography (SEC) method with fluorescence detection. The SEC method used a Superose 12 10/300GL column, 50 \i\ samples were injected and the flow was 0.8 ml/min. Two methods including and excluding phenol were used with the solvent 140 mM NaCI, 10 mM Tris/HCI, pH 7.7, +/- 2 mM phenol. Detection was done by one channel with absorption at 276 nm
(measuring total peptide amount) and another channel with fluorescence detection (excitation at 310 nm, emission at 380 nm), which was specific for liraglutide.
Under some conditions specific quantification was impossible due to experimental conditions. Here, the amount of insulin analogue on the monomeric form was quantified relative to the total amount of peptide.
Formulations were prepared as described in Example 1. Four formulations each with 0.3 mlVI and 0.6 mM insulin analogue, respectively, were prepared with zinc contents corresponding to 6, 8,10, and 12 zinc ions per 6 insulin analogue molecules. These eight formulations were stored at both 4°C and 37°C.
All formulation contained 0.3 or 0.6 mM insulin analogue, zinc acetate, 1.6 mM liraglutide, 50 mM phenol, 174 mM glycerol, pH 8.2.
The stored formulations were analysed using both the SEC with phenol and the SEC without phenol methods after 0, 2, 4, and 8 weeks of storage. Table 2 and Table 3 show the amounts of insulin analogue on the monomeric form. The shaded areas indicate conditions where an exact quantification was not possible. Therefore, the quantification was also done for insulin analogue monomer relative to total peptide content. This is shown in Table 4 and Table 5.
Table 2 Table 3 Table 4 Table 5 (Table Removed)
The amount of zinc binding liragiutide di-heptamer was readily measured using the fluorescence detection. Table 6 and Table 7 show the measured amounts in the analysed samples.
Table 6 Table 7 (Table Removed)
EXAMPLE 3
Following the procedure in Example 1 four fomnulations were prepared with the compositions shown in Table 8. In Formulation 3 the glycylglycine buffer was added together with the water. In Fomriulation 4 the phosphate buffer was added after the zinc acetate was added. This was done in order to minimise zinc-phosphate precipitation. The used insulin analogue was NεB29-(N°-(HOOC(CH2)I4CO)-Y-GIU) desB30 human insulin.
The physical stability of the four pharmaceutical compositions was evaluated by means of an accelerated stressed test. The stressed test was performed as a rotation test. 50 µL air was added to each of 5 cartridges (glass vials) of each fomnulation. The cartridges were rotated with a frequency of 30 rotations per minute for 4 hours daily. The inspection of the cartridges was followed daily or as required. The turbidity of the formulation was characterized by nephelometric measurement and specified in "Nephelometric Turbidity Unit" (NTU). Physical instability of the protein was characterised by high turbidity measurements.
This rotation test is shown in Figure 1. It revealed that the formulation with the insulin analogue and liraglutide combined and formulated at pH 7.7 (Formulation 3) was less stable than the insulin analogue formulated alone at pH 7.4 (Formulation 1) and probably had an unacceptable stability for further development. Formulation 4 also contained the insulin analogue and liraglutide combination but was formulated at pH 8.2. This formulation had an only marginally lower physical stability than the insulin analogue alone. Hence, this comparison illustrated that combining the insulin analogue and liraglutide without any optimisation
resulted in an unstable formulation. Increasing the pH to pH 8.2, however, resulted in much improved physical stability comparable to that of insulin analogue alone.
Table 8
(Table Removed)
EXAMPLE 4
In another rotation test performed as described in Example 3, four combination formulations containing both the insulin analogue NεB29-(N°-(HOOC(CH2)I4CO)-Y-GIU) desB30 human insulin and liraglutide and two references with the insulin analogue and liraglutide alone were tested.
The formulations were prepared as described in Example 1 and their compositions are shown in Table 9.
Table 9
(Table Removed)
The results from the rotation test are shown in Figure 2-4.
Formulation 3 contained 8 zinc/6 insulin analogue molecules and had a comparable stability to the both references with the insulin analogue (with 6 zinc/6 insulin analogue molecules) and liraglutide alone. Formulation 6 only contained 0.3 mM insulin analogue but required 10 zinc/6 insulin analogue molecules in order to exhibit a similar stability as the reference. This indicated that higher zinc content relative to the amount of insulin analogue was required in order to achieve the same stability as the insulin analogue reference alone with 6 zinc ions/6 insulin analogue molecules.
EXAMPLE 5
The pharmacokinetic (PK) properties of fixed combination formulations with the insulin analogue NεB29-(N''-(HOOC(CH2)I4CO)-Y-GIU) desB3 human insulin and liraglutide were exmanined in pigs. The appearances of the two peptides in the blood stream were measured by standard assay techniques for up to 72 hours after injection. For clarity, however, only the first 24 hours are shown for the insulin analogue. The results are shown in Figure 5 and Figure 6 as means of the six replica and shown with standard error of the mean. Three combination formulations and two references were prepared as outlined in Example 1. Their compositions are shown in Table 10. The PK appearance curve for the insulin analogue is shown in Figure 5 and the PK appearance curve for liraglutide is shown in Figure 6.
Table 10
(Table Removed)
The appearance of the insulin analogue in the combination is critically for several reasons: A fast-acting component will give rise to an unexpected early on-set of effect. An increased bioavailability will also result in a larger blood glucose lowering effect and subsequently an adjustment of the dose would be required. It is interesting to conclude that the combination formulation 3 (formulated with 8 zinc/6 insulin analogue molecules) exhibited a
very similar insulin appearance curve compared to the curve for the insulin analogue alone (Formulation 1). Hence, a combination formulation with 8 zinc/6 insulin analogue molecules exhibited very similar insulin PK properties when compared to the insulin analogue alone. All combination formulations exhibited very similar liraglutide appearance curves (Formulations 3-5).
EXAMPLE 6
The pharmacokinetic (PK) properties of other fixed combination formulations with the insulin analogue NεB29-N-(HOOC(CH2)I4CO)-Y-GIU) desB30 human insulin and liraglutide in a different ratio were likewise examined in pigs. This ratio enables a larger amount of the insulin analogue relative to liraglutide to be delivered to the patient in a single injection. An equal dose of insulin was administered to the pigs of both the insulin analogue reference (Fonnulation 1) and the combinations (Formulations 3-5). The appearances of the two peptides in the blood stream were measured by standard assay techniques for up to 72 hours after injection. For clarity, however, only the first 6 hours and 24 hours, respectively, are shown. The results are shown in Figure 7 and Figure 8 as means of the six replica and shown with standard error of the mean. Three combination formulations and two references were prepared as outlined in Example 1. Their compositions are shown in Table 11. The PK appearance curve for the insulin analogue is shown in Figure 7 and the PK appearance curve for liraglutide is shown in Figure 8.
Table 11
(Table Removed)
Compared to the insulin analogue reference (Formulation 1) the combination with 6 Zn2+/6 insulin molecules (Fonnulation 3) exhibited a minor shoulder with an earlier on-set than the main peak of the reference. Increasing the zinc content to 7 Zn2+/6 insulin molecules (Formulation 4) resulted in an insulin analogue appearance curve much more similar to that of the insulin reference (Formulation 1). Increasing the zinc content further to 8 Zn2+/6 insulin
molecules (Formulation 5) did not result in any further improvement. No statistically significant differences were found between the PK appearance curves for liraglutide in the three combinations (Fomnulations 3-5) and liraglutide alone (Formulation 2). This indicated that a zinc content of 7 Zn2+/6 insulin molecules for the 1.2 mM insulin analogue - 1.6 mM liraglutide combination was preferable in order to obtain a similar PK appearance curve for the insulin analogue in the combination compared to the analogue alone.
EXAMPLE 7
The influence of pH on the content of insulin analogue NεB29-N2α-(HOOC(CH2)14CO)-y-Glu) desB30 human insulin monomer in combinations with liraglutide was investigated. Three fonnulations were prepared as outlined in Example 1 but adjusted to pH 8.2, pH 7.7, and pH 7.4. The composition of all three formulations was: 0.6 mM insulin analogue, 0.8 mM zinc acetate (8 zinc per 6 insulin analogue molecules), 1.6 mM liraglutide, 50 mM phenol, 214 mM glycerol. After preparation the formulations were stored at 4 °C for some weeks before the first measurement at time point 0 weeks. Hereafter the formulations were stored at both 5 °C and 37 °C and analysed after further 2 and 4 weeks of storage at the two temperatures. The formulations were analysed using the SEC method described in Example 2. Both versions with and without phenol were used.
The relative contents of insulin analogue monomer compared to total insulin analogue measured by the SEC analysis with phenol and without phenol are shown in Table 12 and Table 13, respectively. In both SEC methods and at both temperatures, decreasing pH resulted in lower insulin analogue monomer content at each time point of measurement. This indicated that pH is an important factor for controlling and modulating the insulin monomer content in the combinations with liraglutide. A lower insulin monomer content could be obtained by using a pH lower than pH 8.2, e.g. pH 7.7.
Table 12 Table 13 (Table Removed)
The liraglutide diheptamer contents were also measured in a similar fashion and these results are shown in Table 14 and Table 15 for the SEC method with phenol and without phenol, respectively.
Table 14 Table 15 (Table Removed)
EXAMPLE 8
The study described in Example 7 also contained an insulin analogue NεB29-(Nα-(HOOC(CH2)14CO)-Y-Glu) desB30 human insulin - liraglutide combination formulation containing a low concentration of histidine. The amino acid was intended to act as a zinc buffer in the presence of an increased zinc content. Formulation 1 was composed of 0.6 mM insulin analogue, 0.8 mM zinc acetate (8 zinc per 6 insulin analogue molecules), 1.6 mM liraglutide, 50 mM phenol, 214 mM glycerol, with pH adjusted to pH 8.2. Formulation 2 was composed of 0.6 mM insulin analogue, 1.0 mM zinc acetate (10 zinc per 6 insulin analogue molecules), 1.6 mM liraglutide, 50 mM phenol, 214 mM glycerol, 0.5 mM histidine, pH adjusted to pH 8.2.
Both formulations were basically produced as outlined in Exannple 1, the histidine was added as the final excipient after liraglutide. The formulations were analysed as described in Example 2 and Example 7.
The relative contents of insulin analogue monomer compared to total insulin analogue measured by the SEC analysis with phenol and without phenol are shown in Table 16 and Table 17, respectively. Formulation 2 with 10 zinc/6 insulin molecules and 0.5 mM histidine present contained less insulin monomer over time at both temperatures than Formulation 1 with only 8 zinc/6 insulin molecules.
Table 16 Table 17 (Table Removed)
The liraglutide diheptamer contents were also measured in a similar fashion and these results are shown in Table 18 and Table 19 for the SEC method with phenol and without phenol, respectively.
Table 18 Table 19 (Table Removed)
EXAMPLE 9
The study described in Example 7 also contained an insulin analogue NεB29-(Nα-(HOOC(CH2)14CO)-Y-Glu) desB30 human insulin - liraglutide combination composed of 0.3 mM insulin analogue, 0.8 mM zinc acetate (16 zinc per 6 insulin analogue molecules), 1.6 mM liraglutide, 50 mM phenol, 214 mM glycerol, adjusted to pH 8.2.
The relative contents of insulin analogue monomer compared to total insulin analogue measured by the SEC analysis with phenol and without phenol (as described in Example 2) are shown in Table 20 and Table 21, respectively.
Table 20 Table 21 (Table Removed)
The liraglutide diheptamer contents were also measured in a similar fashion and these results are shown in Table 22 and Table 23 for the SEC method with phenol and without phenol, respectively.
Table 22 Table 23 (Table Removed)
EXAMPLE 10
Formulations combining insulin glargine and the exendin-4 analogue ZP10 (also known as AVE0010) were prepared. The ZP10 exendin-4 analogue is described in Thorkild-sen et al. (2003), JPET 307:490-496 and has the systematic name [des-Pro38]exendin-4-(1-39)yl-Lys-Lys-Lys-Lys-Lys-Lys-NH2. From appropriate stock solutions the following compositions were prepared by dilution in water: Formulation 1 consisted of 0.6 mM insulin glargine, 0.46 mM zinc acetate (4.6 zinc/ 6 insulin glargine), 60 µM ZP10. Formulation 2 consisted of 0.6 mM insulin glargine, 0.6 mM zinc acetate (6 zinc/ 6 insulin glargine), 60 µM ZP10. Both fomiulations were adjusted to about pH 4.0 using HCI and NaOH and stored at ambient temperature for three days. After storage both formulations were still about pH 4.0. The two formulations maintained clear solutions immediately after preparation and after the storage at ambient temperature.
WE CLAIMS:-
1. A soluble pharmaceutical composition for parenteral administration, which comprises an insulinotropic GLP-1 compound , a basal insulin peptide, pharmaceutically acceptable additives and zinc, wherein the zinc content is at least 5 or at least 6 Zn ions per 6 insulin molecules.
2. The pharmaceutical composition according to claim 1, wherein the zinc content is between
5 and 16 zinc ions per 6 insulin molecules.
3. The phannaceutical composition according to claim 1, wherein the zinc content is between
6 and 16 zinc ions per 6 insulin molecules.
4. The phamnaceutical composition according to any of the preceding claims, wherein the pH of said pharmaceutical composition from about pH 7.4 to about pH 8.2.
5. The phannaceutical composition according to any of the preceding claims, wherein the pH of said pharmaceutical composition from about pH 7.4 to about pH 7.7 or between about 7.7 and about 8.2..
6. The pharmaceutical composition according to any of the preceding claims, wherein the insulinotropic GLP-1 peptide is GLP-1 (7-37), a GLP-1 (7-37) analogue, a derivative of GLP-1(7-37), or a derivative of a GLP-1 (7-37) analogue.
7. The phamnaceutical composition according to claim 6, wherein said derivative of a GLP-1(7-37) analogue is Arg34 Lys26(N'-(Y-Glu(N"-hexadecanoyl)))-GLP-1(7-37).
8. The pharmaceutical composition according to any one of the preceding claims, wherein the basal insulin peptide is an acylated insulin.
9. A phannaceutical composition according to claim 8, wherein the acylated insulin is acylated in position B29 with a lipophilic group.
10. A pharmaceutical composition according to claim 9, wherein the basal insulin is selected
from the group consisting of NεB29-tetradecanoyl des(B30) human insulin, NεB29-(Nα-
{HOOC(CH2)i4CO)-Y-Glu) desB30 human insulin, LysB29(Nε lithocholyl-Y-Glu)-des(B30) hu
man insulin, NεB29--carboxypentadecanoyl-Y-L-glutayiamide desBSO human insulin and
N'^^®- co-carboxypentadecanoyi- - amino-butanoyi des(B30) human insulin.
11. A pharmaceutical composition according to claim 10, wherein the basal insulin is NεB29 tetradecanoyl des(B30) human insulin, NεB29-(Nα-(HOOC(CH2)I4CO)-Y-GIU) desB30 human insulin.
12. A pharmaceutical composition according to any one of the preceding claims, wherein the basal insulin is NεB29-(Nα-(HOOC(CH2)I4CO)-Y-GIU) desB30 human insulin and the insulino-tropic GLP-1 compound is Arg34 Lys26(N-(Nε-Glu(Nα-hexadecanoyl)))-GLP-1(7-37).
13. A pharmaceutical composition according to any of the preceding claims, wherein the concentration of the basal insulin is in the range from 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.
14. A pharmaceutical composition according to any of the preceding claims, wherein the basal insulin is an insulin glargine peptide or amidated insulin glargine peptide and the insuli-notropic GLP-1 compound is exendin-4 or an analogue thereof with up to 8 added basic amino acid residues.
15. A pharmaceutical composition according to any of the preceding further comprising his-tidine.