Therapeutic peptide formulations

Abstract

Stable pharmaceutical formulations for treating dual GLP-1 receptor / glucagon receptor agonists and methods of using such stable pharmaceutical formulations.

Description

[0001] This invention pertains to the medical field. More particularly, this invention relates to pharmaceutical formulations comprising therapeutic peptides suitable for subcutaneous (“SQ”), intramuscular (“IM”), and / or intraperitoneal (“IP”) administration. Still more particularly, this invention relates to pharmaceutical formulations of dual glucagon-like peptide (GLP-1) receptor and glucagon (Gcg) receptor agonist peptides. These pharmaceutical formulations comprising dual GLP-1 receptor / Gcg receptor agonists are intended for the treatment of at least type 2 diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), and / or non-alcoholic steatohepatitis (NASH).

[0002] Pharmaceutical formulations requiring dual GLP-1 / glucagon receptor agonists are used to treat patients with at least type 2 diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), and / or non-alcoholic steatohepatitis (NASH). Administration of such therapeutic peptides via sigmata (SQ), intraperitoneal (IP), and / or intramedullary (IM) administration is common and advantageous. These routes of administration allow for the delivery of the therapeutic peptide over a short timeframe and allow patients to self-administer the peptide without visiting healthcare professionals. Pharmaceutical formulations require a specific concentration of the dual GLP-1 / glucagon receptor agonist peptide to enable delivery to the patient via sigmata, IP, and / or IM. These pharmaceutical formulations with a specific concentration of the dual GLP-1 / glucagon receptor agonist peptide must maintain the peptide's physical and chemical stability. However, formulating therapeutic peptides into liquid formulations suitable for SQ, IM, and / or IP administration is both challenging and unpredictable.

[0003] The challenges and unpredictability associated with formulating therapeutic peptides into liquid pharmaceutical preparations suitable for SQ, IM, and / or IP administration are partly due to the numerous properties that pharmaceutical preparations must possess to be therapeutically feasible. The preparation must provide stability to the therapeutic peptide in solution while maintaining the functional characteristics essential for its therapeutic efficacy. Furthermore, liquid pharmaceutical preparations must be safe to administer to patients and well-tolerated, and suitable for preparation and storage.

[0004] U.S. Patent No. 9,938,335 generally describes a dual GLP-1 / glucagon receptor agonist peptide administered via a parenteral route. The compound described in Example 2 of U.S. Patent No. 9,938,335 has the sequence provided in SEQ ID NO: 1 (hereinafter referred to as Compound 1). Compound 1 is currently being evaluated for the treatment of patients with type 2 diabetes. Compound 1 is a synthetic peptide consisting of 34 amino acid residues, one non-coding amino acid (aminoisobutyric acid (Aib)), a C-terminal amide, and a C20 fatty acid moiety covalently linked to lysine 20 in the sequence. The covalent linker comprises γ-glutamic acid and two PEG units. Therapeutically, this peptide is a gastrin-like acylated peptide with dual agonist activity of human glucagon-like peptide (GLP-1) and glucagon (Gcg). It independently binds to and activates the glucagon-like peptide receptor (GLP-1R) and glucagon receptor (GcgR) on the surface of susceptible cells.

[0005] Surprisingly, the compounds described in U.S. Patent No. 9,938,335, particularly compound 1, were found to have suboptimal solubility at lower pH values ​​(e.g., pH 5.0–6.5). It was also found that the compounds described in U.S. Patent No. 9,938,335, particularly compound 1, exhibit suboptimal stability in certain formulations at pH values ​​of 7.0–8.5. Pharmaceutical formulations comprising dual GLP-1 / glucagon receptor agonist peptide compounds having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 are needed to avoid these observed problems.

[0006] The pharmaceutical formulations provided herein meet the above requirements. More specifically, the pharmaceutical formulations provided herein are suitable for dual GLP-1 / glucagon receptor agonist peptide administration in SQ, IM, and / or IP treatments, while retaining the functional characteristics of the peptides essential for therapeutic efficacy.

[0007] Therefore, a pharmaceutical preparation is provided, which comprises:

[0008] (i) Compounds of the following formula:

[0009] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-

[0010] Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-

[0011] Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly

[0012] in

[0013] Xaa2 is Aib;

[0014] Xaa28 is either Glu or Ser;

[0015] The 20-position Lys is chemically modified by conjugating the ε-amino group of the Lys side chain to the C14-C24 fatty acid via a linker between Lys at position 20 and the C14-C24 fatty acid, wherein the linker is ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)t, where t is 1 or 2; and

[0016] The C-terminal amino acid may optionally be amidated (SEQ ID NO: 5).

[0017] Or its pharmaceutically acceptable salt;

[0018] (ii) Buffers;

[0019] (iii) Tension-inducing agents; and

[0020] (iii) Antioxidants

[0021] The pH of the formulation is 7.8-9.0.

[0022] Preliminary formulation studies revealed that the compound described herein exhibited suboptimal solubility at pH 5.0–6.0. ​​These studies indicated that the compound should be formulated at approximately pH 7.0 or higher to achieve solubility suitable for SC, IM, and / or IP administration. However, further formulation studies surprisingly revealed significant stability issues with the compound described herein in the pH range of 7.0–8.5. Further investigation was conducted to understand these stability issues. Surprisingly, at least two mechanisms were found that could contribute to the stability problems. First, the compound is thought to be prone to fibrillation due to its sequence similarity to natural human glucagon. The studies described herein showed that the compound suffered significant fibrillation at pH values ​​below 7.8. Second, the compound is thought to be prone to oxidation at certain amino acid residues, particularly histidine at position 1 (His, H) and tryptophan at position 25 (Trp, W). The studies described herein showed that the compound is prone to oxidation. These confirmed reasons led to the formulation of the compound as described above to address the stability issues. Formulation of the compound in the pH range of 7.8–9.0 prevented fibrillation. The presence of antioxidants significantly reduces or eliminates aggregates originating from compound oxidation.

[0023] In a further embodiment of the present invention, the C14-C24 fatty acid is selected from the following saturated monoacids or saturated diacids: myristic acid (tetradecanoic acid) (C14 monoacid), tetradecanoic acid (C14 diacid), palmitic acid (hexadecanoic acid) (C16 monoacid), hexadecanoic acid (C16 diacid), heptadecanoic acid (heptadecanoic acid) (C17 monoacid), heptadecanoic acid (C17 diacid), stearic acid (octadecanoic acid) (C18 monoacid), octadecanoic acid (C18 diacid) ), nonadecanoic acid (nonadecanoic acid) (C19 monoacid), nonadecanedioic acid (C19 diacid), arachidic acid (eicosanoic acid) (C20 monoacid), eicosanedioic acid (C20 diacid), eicosanoic acid (eicosanoic acid) (C21 monoacid) acid), behenic acid (C21 diacid), behenic acid (behenic acid) (C22), behenic acid (C22 diacid), tetracosanoic acid (cosanoic acid) (C24 monoacid), and behenic acid (C24 diacid).

[0024] Preferably, the C14-C24 fatty acid is octadecanoic acid.

[0025] Optionally, and preferably, the C14-C24 fatty acid is eicosanoic acid.

[0026] In a preferred embodiment of the invention, the C-terminal amino acid is amidated.

[0027] In a further embodiment of the present invention, the compound is selected from:

[0028] (a) Compounds of the following formula:

[0029] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0030] Where Xaa2 is Aib;

[0031] By reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 CO2H conjugation is used to chemically modify the 20-position Lys; and

[0032] The C-terminal amino acid is amidated (SEQ ID NO: 1) (Compound 1).

[0033] Or its pharmaceutically acceptable salt;

[0034] (b) Compounds of the following formula:

[0035] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser Ser-Gly

[0036] Where Xaa2 is Aib;

[0037] By reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2) 18 CO2H conjugation is used to chemically modify the 20-position Lys; and

[0038] The C-terminal amino acid is amidated (SEQ ID NO: 2) (hereinafter referred to as compound 2).

[0039] Or its pharmaceutically acceptable salt;

[0040] (c) Compounds of the following formula:

[0041] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0042] Where Xaa2 is Aib;

[0043] By reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 16 CO2H conjugation is used to chemically modify the 20-position Lys; and

[0044] The C-terminal amino acid is amidated (SEQ ID NO: 3) (hereinafter referred to as compound 3).

[0045] or its pharmaceutically acceptable salt; and

[0046] (d) Compounds of the following formula:

[0047] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Ser-Gly-Gly-Pro-Ser-Ser-Gly

[0048] Where Xaa2 is Aib;

[0049] By reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2) 16 CO2H conjugation is used to chemically modify the 20-position Lys; and

[0050] The C-terminal amino acid is amidated (SEQ ID NO: 4) (hereinafter referred to as compound 4).

[0051] Or its pharmaceutically acceptable salt.

[0052] In a preferred embodiment of the present invention, the compound has the following formula:

[0053] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0054] Where Xaa2 is Aib;

[0055] By reacting the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 CO2H conjugation is used to chemically modify the 20-position Lys; and

[0056] The C-terminal amino acid is amidated (SEQ ID NO: 1) (Compound 1).

[0057] Or its pharmaceutically acceptable salt.

[0058] In a further embodiment of the invention, the formulation comprises 1 mg / mL to 100 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0059] Preferably, the formulation comprises 5 mg / mL to 90 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0060] More preferably, the formulation comprises 10 mg / mL to 80 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0061] Further preferably, the formulation comprises 20 mg / mL to 70 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0062] Further preferably, the formulation comprises 30 mg / mL to 60 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0063] Further preferably, the formulation comprises 40 mg / mL to 50 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0064] Alternatively, the formulation may contain 1 mg / mL to 50 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0065] Alternatively, the formulation may contain 2 mg / mL to 45 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0066] Further alternatives include formulations containing 3 mg / mL to 40 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0067] Further alternatives include formulations containing 4 mg / mL to 35 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0068] Further alternatives include formulations containing 5 mg / mL to 30 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0069] Further alternatively, the formulation may contain 6 mg / mL to 25 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0070] Further alternatively, the formulation may contain 7 mg / mL to 20 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0071] Further alternatively, the formulation may contain 8 mg / mL to 15 mg / mL of the compound or a pharmaceutically acceptable salt thereof.

[0072] Alternatively, preferably, the formulation contains 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL 15mg / mL, 16mg / mL, 17mg / mL, 18mg / mL, 19mg / mL, 20mg / mL, 21mg / mL, 22mg / mL, 23mg / mL, 24mg / mL, 25mg / mL, 26mg / mL , 27mg / mL, 28mg / mL, 29mg / mL, 30mg / mL, 31mg / mL, 32mg / mL, 33mg / mL, 34mg / mL, 35mg / mL, 36mg / mL, 37mg / mL, 38mg / mL Compounds or pharmaceutically acceptable salts thereof, at concentrations of 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, or 100 mg / mL.

[0073] In a further embodiment of the invention, the buffer is selected from phosphate buffers and tris(hydroxymethyl)aminomethane (or 2-amino-2-hydroxymethyl-prop-1,3-diol)[(HOCH2)3CNH2]) buffers.

[0074] In a further embodiment of the invention, the formulation comprises a buffer of 1 mM to 20 mM.

[0075] Preferably, the formulation contains a buffer of 3 mM to 18 mM.

[0076] More preferably, the formulation contains a buffer of 5 mM to 15 mM.

[0077] Further preferably, the formulation contains 8 mM-12 mM of buffer.

[0078] More preferably, the formulation contains a buffer of 9 mM to 11 mM.

[0079] In a further embodiment of the invention, the formulation comprises 1 mM of buffer, 2 mM of buffer, 3 mM of buffer, 4 mM of buffer, 5 mM of buffer, 6 mM of buffer, 7 mM of buffer, 8 mM of buffer, 9 mM of buffer, 10 mM of buffer, 11 mM of buffer, 12 mM of buffer, 13 mM of buffer, 14 mM of buffer, 15 mM of buffer, 16 mM of buffer, 17 mM of buffer, 18 mM of buffer, 19 mM of buffer, or 20 mM of buffer.

[0080] In a preferred embodiment of the invention, the buffer is a tris(hydroxymethyl)aminomethane (Tris) buffer.

[0081] More preferably, the formulation comprises 1 mM Tris buffer, 2 mM Tris buffer, 3 mM Tris buffer, 4 mM Tris buffer, 5 mM Tris buffer, 6 mM Tris buffer, 7 mM Tris buffer, 8 mM Tris buffer, 9 mM Tris buffer, 10 mM Tris buffer, 11 mM Tris buffer, 12 mM Tris buffer, 13 mM Tris buffer, 14 mM Tris buffer, 15 mM Tris buffer, 16 mM Tris buffer, 17 mM Tris buffer, 18 mM Tris buffer, 19 mM Tris buffer, or 20 mM Tris buffer.

[0082] More preferably, the formulation contains 10 mM Tris buffer.

[0083] In a further embodiment of the invention, the tensioning agent is selected from mannitol, sucrose, trehalose, glycerol, propylene glycol, sodium chloride, and arginine hydrochloride.

[0084] Tensor agents are excipients selected to adjust the tension of a formulation. Tensile typically relates to the osmotic pressure of a solution, usually relative to the osmotic pressure of human serum. Formulations can be hypotonic, isotonic, or hypertonic. The concentration of a tensor agent depends on the desired tension and the molecular weight of the specific active agent chosen. For example, 25 mg / mL glycerol has a similar tension to an aqueous solution of 95 mg / mL sucrose. Other excipients can affect the tension of a formulation, and the concentration of a tensor agent varies depending on the desired result and the molecular weight of the active agent used.

[0085] In a further embodiment of the invention, the formulation comprises a tensile agent of 5 mg / mL to 150 mg / mL.

[0086] Preferably, the formulation contains a tensile agent at a concentration of 10 mg / mL to 120 mg / mL.

[0087] More preferably, the formulation contains a tensile agent at a concentration of 20 mg / mL to 100 mg / mL.

[0088] More preferably, the formulation contains a tensile agent at a concentration of 30 mg / mL to 80 mg / mL.

[0089] More preferably, the formulation contains a tensile agent at a concentration of 40 mg / mL to 60 mg / mL.

[0090] More preferably, the formulation contains a tensile agent at a concentration of 45 mg / mL to 55 mg / mL.

[0091] In a preferred embodiment of the present invention, the tensioning agent is mannitol.

[0092] More preferably, the formulation contains 10 mg / mL to 90 mg / mL of mannitol.

[0093] More preferably, the formulation contains 20 mg / mL to 80 mg / mL of mannitol.

[0094] More preferably, the formulation contains 30 mg / mL to 70 mg / mL of mannitol.

[0095] More preferably, the formulation contains 40 mg / mL to 60 mg / mL of mannitol.

[0096] More preferably, the formulation contains 45 mg / mL to 55 mg / mL of mannitol.

[0097] More preferably, the formulation contains 50 mg / mL of mannitol.

[0098] In a further embodiment of the invention, the antioxidant is selected from free radical scavengers, chelating agents, or chain terminators.

[0099] In a further embodiment of the invention, the formulation comprises 0.05-10.0 mg / mL of an antioxidant.

[0100] Preferably, the formulation contains 0.1-5.0 mg / mL of antioxidant.

[0101] More preferably, the formulation contains 0.2-1.0 mg / mL of antioxidant.

[0102] Optionally, preferably, the formulation comprises 0.05 mg / mL of antioxidant, 0.075 mg / mL of antioxidant, 0.1 mg / mL of antioxidant, 0.2 mg / mL of antioxidant, 0.3 mg / mL of antioxidant, 0.4 mg / mL of antioxidant, 0.5 mg / mL of antioxidant, 0.6 mg / mL of antioxidant, 0.7 mg / mL of antioxidant, 0.8 mg / mL of antioxidant, 0.9 mg / mL of antioxidant, 1.0 mg / mL of antioxidant, 1.1 mg / mL of antioxidant, 1.2 mg / mL of antioxidant, 1.3 mg / mL of antioxidant, 1.4 mg / mL of antioxidant, 1.5 mg / mL of antioxidant, 1.6 mg / mL of antioxidant, 1.7 mg / mL of antioxidant. Antioxidant at concentrations of 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, or 10.0 mg / mL.

[0103] In a preferred embodiment of the present invention, the antioxidant is a free radical scavenger.

[0104] More preferably, the antioxidant is selected from EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, histidine, methionine, ethanol, and N-acetylcysteine.

[0105] Further preferably, the antioxidant is EDTA.

[0106] More preferably, the formulation contains 0.05-10.0 mg / mL of EDTA.

[0107] More preferably, the formulation contains 0.1-5.0 mg / mL of EDTA.

[0108] More preferably, the formulation contains 0.2-1.0 mg / mL of EDTA.

[0109] Optionally, and preferably, the formulation comprises 0.05 mg / mL of EDTA, 0.075 mg / mL of EDTA, 0.1 mg / mL of EDTA, 0.2 mg / mL of EDTA, 0.3 mg / mL of EDTA, 0.4 mg / mL of EDTA, 0.5 mg / mL of EDTA, 0.6 mg / mL of EDTA, 0.7 mg / mL of EDTA, 0.8 mg / mL of EDTA, 0.9 mg / mL of EDTA, 1.0 mg / mL of EDTA, and 1.1 mg / mL of E. DTA, 1.2 mg / mL EDTA, 1.3 mg / mL EDTA, 1.4 mg / mL EDTA, 1.5 mg / mL EDTA, 1.6 mg / mL EDTA, 1.7 mg / mL EDTA, 1.8 mg / mL EDTA, 1.9 mg / mL EDTA, 2.0 mg / mL EDTA, 2.5 mg / mL EDTA, 3.0 mg / mL EDTA, 3.5 mg / mL EDTA, 4.0 mg / mL EDTA, 4.5 mg / mL EDTA in 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, or 10.0 mg / mL.

[0110] More preferably, the formulation contains 0.5 mg / mL of EDTA.

[0111] Optionally, preferably, the antioxidant is citric acid.

[0112] Further preferably, the formulation contains 1-20 mM of citric acid.

[0113] More preferably, the formulation contains 5-15 mM of citric acid.

[0114] Further preferably, the formulation contains 8-12 mM of citric acid.

[0115] Optionally, preferably, the formulation comprises 1 mM citric acid, 1.5 mM citric acid, 2 mM citric acid, 2.5 mM citric acid, 3 mM citric acid, 3.5 mM citric acid, 4 mM citric acid, 4.5 mM citric acid, 5 mM citric acid, 5.5 mM citric acid, 6 mM citric acid, 6.5 mM citric acid, 7 mM citric acid, 7.5 mM citric acid, 8 mM citric acid, 8.5 mM citric acid, 9 mM citric acid, 9.5 mM citric acid, 10 mM citric acid, 10 Citric acid at concentrations of 0.5 mM, 11 mM, 11.5 mM, 12 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, 15 mM, 15.5 mM, 16 mM, 16.5 mM, 17 mM, 17.5 mM, 18 mM, 18.5 mM, 19 mM, 19.5 mM, or 20 mM.

[0116] More preferably, the formulation contains 10 mM citric acid.

[0117] In an alternative embodiment of the present invention, the antioxidant is ascorbic acid.

[0118] In a further alternative embodiment of the invention, the antioxidant is butylated hydroxytoluene (BHT).

[0119] In a further alternative embodiment of the invention, the antioxidant is butylated hydroxyanisole (BHA).

[0120] In a further alternative embodiment of the invention, the antioxidant is sodium sulfite.

[0121] In a further alternative embodiment of the invention, the antioxidant is p-amino acid benzoic acid.

[0122] In a further alternative embodiment of the invention, the antioxidant is glutathione.

[0123] In a further alternative embodiment of the invention, the antioxidant is propyl gallate.

[0124] In a further alternative embodiment of the invention, the antioxidant is cysteine.

[0125] In a further alternative embodiment of the invention, the antioxidant is histidine.

[0126] In a further alternative embodiment of the invention, the antioxidant is methionine.

[0127] In a further alternative embodiment of the invention, the antioxidant is ethanol.

[0128] In a further alternative embodiment of the invention, the antioxidant is N-acetylcysteine.

[0129] In a preferred embodiment of the present invention, the pH of the formulation is 8.0-8.6.

[0130] More preferably, the pH of the formulation is 8.0-8.3.

[0131] In a preferred embodiment of the present invention, the pharmaceutical preparation comprises:

[0132] (i) Compounds of the following formula with concentrations ranging from 1 mg / mL to 100 mg / mL:

[0133] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Glu-Phe-Val--Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly

[0134] Where Xaa2 is Aib;

[0135] The 20-position of Lys was chemically modified by conjugating the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18CO2H; and

[0136] The C-terminal amino acid is amidated (SEQ ID NO: 1) (Compound 1)

[0137] Or its pharmaceutically acceptable salt;

[0138] (ii) 10 mM Tris buffer;

[0139] (iii) Mannitol at 46 mg / mL;

[0140] (iv) 0.5 mg / mL EDTA,

[0141] The pH of the formulation is 8.0-8.3.

[0142] In a further embodiment of the invention, a method for treating and / or preventing type 2 diabetes, obesity, non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH) is provided, wherein the method comprises administering to a patient a therapeutically effective amount of a pharmaceutical preparation as described herein.

[0143] In a further embodiment of the invention, a pharmaceutical preparation as described herein is provided for the treatment and / or prevention of type 2 diabetes, obesity, NAFLD, and / or NASH.

[0144] In a further embodiment of the invention, the use of the pharmaceutical formulation as described herein in the preparation of a medicament for the treatment of type 2 diabetes, obesity, NAFLD, and / or NASH is provided.

[0145] As used herein, the term "pharmaceutical formulation" refers to a solution having at least one active pharmaceutical ingredient (API) capable of exerting a biological effect in humans and at least one inactive ingredient (e.g., a buffer, excipient, surfactant, etc.), wherein, when combined with the API, the inactive ingredient is suitable for therapeutic administration to humans. The pharmaceutical formulations disclosed herein are stable formulations in which the degree of degradation, modification, aggregation, loss of biological activity, etc., of the therapeutic compound is acceptablely controlled and does not unacceptably increase over time.

[0146] In the context of this invention, API is compound 1 or a pharmaceutically acceptable salt thereof, compound 2 or a pharmaceutically acceptable salt thereof, compound 3 or a pharmaceutically acceptable salt thereof, or compound 4 or a pharmaceutically acceptable salt thereof. Compounds 1, 2, 3, and 4, their pharmaceutically acceptable salts, and methods of their preparation are described in U.S. Patent No. 9,938,335.

[0147] As used herein, the term "pharmaceuticalally acceptable excipient" refers to any ingredient that does not have therapeutic activity and has acceptable toxicity, such as buffers, solvents, tensioning agents, stabilizers, antioxidants, surfactants, or polymers used to formulate pharmaceutical products. They are generally safe for human use according to standards set by the U.S. Food and Drug Administration.

[0148] As used herein, the term "buffer" refers to a solution that is resistant to pH changes. Buffers can include weak acids and their salts, or weak bases and their salts, which help maintain pH stability. Examples of buffers used in pharmaceutical formulations include bicarbonate buffers, carbonate buffers, citrate buffers, histidine buffers, phosphate buffers, tartrate buffers, tris(hydroxymethyl)aminomethane (or 2-amino-2-hydroxymethyl-prop-1,3-diol [(HOCH2)3CNH2]) buffers, and combinations thereof. Some of these buffers are suitable for pharmaceutical formulations administered subcutaneously. The buffer used in a pharmaceutical formulation is selected according to the desired pH of the formulation. For example, the pharmaceutical formulation of the present invention has a pH of 7.8 to 9.0. Suitable buffers for achieving this pH include bicarbonate buffers, carbonate buffers, phosphate buffers, tris(hydroxymethyl)aminomethane (or 2-amino-2-hydroxymethyl-propane-1,3-diol [(HOCH2)3CNH2]) buffers, and sodium hydroxide (NaOH) buffers. Phosphate buffers and Tris buffers are preferred for use in injectable formulations. The pH of the formulation can be adjusted using physiologically appropriate acids and bases, if necessary, to achieve the desired pH. (For example, pH adjustment may be necessary when the concentration of API in the formulation increases or decreases).

[0149] Tris(hydroxymethyl)aminomethane or tris(hydroxymethyl)aminomethane buffers can be called “TRIS,” “Tris,” “Tris base,” “Tris buffer,” “Trisamine,” “THAM,” and other names. Furthermore, many buffers and / or buffer systems include Tris. Examples include Tris-buffered saline (“TBS”), Tris-hydrochloride buffer (“Tris-HCl”), Tris base (pH 10.6), Tris / borate / ethylenediaminetetraacetic acid (“EDTA”) buffer (“TBE”), and Tris / acetate / EDTA buffer (“TAE”). Tris bases are often used with Tris-HCl to prepare Tris buffers at the desired pH.

[0150] As used herein, the term "tense agent" refers to a pharmaceutically acceptable excipient used to adjust the tensile properties of a formulation. Tensile properties generally relate to the osmotic pressure of a solution, typically relative to the osmotic pressure of human serum. Formulations can be hypotonic, isotonic, or hypertonic. Suitable tensile agents include, but are not limited to, salts, amino acids, and sugars. Preferred tensile agents for use in the pharmaceutical formulations of this invention include mannitol, sucrose, trehalose, propylene glycol, glycerol, sodium chloride, and arginine hydrochloride.

[0151] The term "antioxidant" refers to a pharmaceutically acceptable excipient that prevents the oxidation of APIs. Antioxidants suitable for pharmaceutical formulations of this invention include chelating agents (EDTA, citric acid), reactive oxygen species scavengers (ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate), and chain terminators (histidine, cysteine, methionine, ethanol, and N-acetylcysteine).

[0152] The alternative buffers, tensioners and antioxidants applicable to the pharmaceutical formulations of this invention are described in Remington: The Science and Practice of Pharmacy, 23rd edition (Editor-Adeboye Adejare).

[0153] The pharmaceutical formulations described herein may include other suitable pharmaceutically acceptable excipients, such as solubilizers, emulsifiers, surfactants, preservatives, colorants, viscosity modifiers, and stabilizers.

[0154] As may be used herein, the term “about” or “approximately” when used to refer to a particular enumerated numerical value or range of values ​​means that the value can vary from the enumerated values ​​by no more than 10% (e.g., + / - 10%). For example, the expression “about 100” as used herein includes 90 and 110 and all values ​​in between (e.g., 91, 92, 93, 94, etc.).

[0155] As used interchangeably herein, “treatment” is intended to refer to all processes in which there may be complete elimination, mitigation or delay, reduction in severity or frequency (e.g., flares or attacks), interruption or cessation of progression of the disease and / or its symptoms, but not necessarily complete elimination of all disease symptoms. Treatment includes administering the pharmaceutical preparations of this disclosure to treat a person who will benefit from at least one of the processes listed above, including: (a) inhibiting further progression of disease symptoms and effects, i.e., preventing their development; (b) alleviating the disease, i.e. causing elimination or remission of the disease, disease symptoms, or complications thereof; and (c) preventing or reducing the frequency of disease attacks or flares. Depending on the specific implementation, the pharmaceutical preparations provided herein may be used to treat at least one of type II diabetes, obesity, NAFLD, and NASH.

[0156] As may be used interchangeably herein, the terms “patient,” “subject,” and “individual” refer to a person. Unless otherwise stated, an individual is also characterized by having symptoms of a disease from which they will benefit from the administration of the pharmaceutical preparations disclosed herein, being at risk of developing symptoms of a disease, or experiencing symptoms of a disease.

[0157] As may be used interchangeably herein, the “effective amount” or “therapeutic effective amount” of the pharmaceutical formulations of this disclosure refers to the amount (in terms of dosage, frequency of administration, and time period of a particular method of administration) required to achieve the desired therapeutic outcome. The effective amount of the pharmaceutical formulations of this disclosure can vary depending on factors such as an individual’s disease state, age, sex, and weight, as well as the ability of the pharmaceutical formulations of this disclosure to elicit the desired response in an individual. An effective amount is also the amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the pharmaceutical formulations of this disclosure.

[0158] The pharmaceutical formulation of the present invention can be administered to patients via enteral administration. As understood in the medical field, enteral administration refers to the injection of a dose into the body via a sterile syringe or some other drug delivery system, including an autoinjector or infusion pump. Exemplary drug delivery systems for use with the pharmaceutical formulations disclosed herein are described in the following references, the disclosures of which are expressly incorporated herein by reference in their entirety: U.S. Patent Publication No. 2014 / 0054883, entitled “Infusion Pump Assembly,” filed March 7, 2013, by Lanigan et al.; U.S. Patent No. 7,291,132, entitled “Medication Dispensing Apparatus with Triple Screw Threads for Mechanical Advantage,” filed February 3, 2006, by Deruntz et al.; U.S. Patent No. 7,517,334, entitled “Medication Dispensing Apparatus with Spring-Driven Locking Feature Enabledby Administration of Final Dose,” filed September 18, 2006, by Jacobs et al.; and U.S. Patent No. 8,734,394, entitled “Automatic Injection Device with Delay Mechanism Including Dual Functioning Biasing Member,” filed August 24, 2012, by Adams et al. Non-gut routes include administration of IM, SO, and IP. Brief description of the attached diagram

[0160] Figure 1 Example: The concentration of compound 1 in solution when the pH changes from about 5.0 to about 7.0.

[0161] Figure 2aExamples include the total aggregates of a solution formulation of compound 1 measured by size exclusion chromatography (SEC) in a formulation matrix containing 10 mM phosphate buffer, wherein NaCl or glycerol is used as a tensile agent.

[0162] Figure 2b Example: Total aggregates of a compound 1 solution formulation measured by SEC in a formulation matrix containing 10 mM Tris buffer, where NaCl or glycerol is used as a tensile agent.

[0163] Figure 3a Example: When compound 1 at 2 mg / mL is prepared under various pH conditions and incorporated into protofibrils, the risk of protofibril formation as a function of pH for compound 1 is considered.

[0164] Figure 3b Example: When compound 1 at 12 mg / mL is formulated under various pH conditions and incorporated into fibrils, the risk of fibril formation of compound 1 as a function of pH is considered.

[0165] Figure 4 The RP-HPLC chromatogram illustrates the effect of thermal stress when a 2 mg / mL solution of Compound 1 is stored at 40 °C for up to 4 weeks.

[0166] Figure 5a The RP-HPLC chromatogram of a 2 mg / mL compound 1 drug product prepared with 0.5 mg / mL EDTA is an example of the effects of transition metals and H2O2.

[0167] Figure 5b The RP-HPLC chromatogram of a 2 mg / mL compound 1 drug product formulated without EDTA is an example of the effects of transition metals and H2O2.

[0168] Figure 6a Examples include the total aggregates in a formulation of compound 1 stored at 5°C, measured by size exclusion chromatography (SEC) at 0, 1, and 3 months.

[0169] Figure 6b Examples include total aggregates in a formulation of compound 1 stored at 25°C, measured by size exclusion chromatography (SEC) at 0, 1, and 3 months.

[0170] Figure 6c Examples include the total aggregates in a formulation of compound 1 stored at 30°C, measured by size exclusion chromatography (SEC) at 0, 1, and 3 months. Example

[0171] Preparation of compounds 1, 2, 3 and 4

[0172] Compound 1

[0173] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG

[0174] Where Xaa2 is Aib;

[0175] Through ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 18 -CO2H is chemically modified at the 20-position of K by ε-amino conjugation with the K side chain; and

[0176] The C-terminal amino acid is amidated to form a C-terminal primary amide (SEQ ID NO: 1).

[0177]

[0178] The diagram above depicts the structure of compound 1 using standard single-letter amino acid codes, except for residues Aib2 and K20, whose structures have been expanded.

[0179] Compound 1 was prepared as described in Example 2 of U.S. Patent No. 9,938,335. Alternative synthetic methods are described in U.S. Provisional Patent Application Serial No. 63 / 038,363.

[0180] Compound 2

[0181] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG

[0182] Where Xaa2 is Aib;

[0183] Through ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO-(CH2) 18 -CO2H is chemically modified at the 20-position of K by ε-amino conjugation with the K side chain; and

[0184] The C-terminal amino acid is amidated to form a C-terminal primary amide (SEQ ID NO: 2).

[0185]

[0186] The diagram above depicts the structure of compound 2 using standard single-letter amino acid codes, except for residues Aib2 and K20, whose structures have been expanded.

[0187] Compound 2 was prepared as described in Example 4 of U.S. Patent No. 9,938,335.

[0188] Compound 3

[0189] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLEGGPSSG

[0190] Where Xaa2 is Aib;

[0191] Through ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) 16 -CO2H is chemically modified at the 20-position of K by ε-amino conjugation with the K side chain; and

[0192] The C-terminal amino acid is amidated to form a C-terminal primary amide (SEQ ID NO: 3).

[0193]

[0194] The above figure depicts the structure of compound 3 using standard single-letter amino acid codes, except for residues Aib2 and K20, whose structures have been expanded.

[0195] Compound 3 was prepared as described in Example 1 of U.S. Patent No. 9,938,335.

[0196] Compound 4

[0197] HXaa2QGTFTSDYSKYLDEKKAKEFVEWLLSGGPSSG

[0198] Where Xaa2 is Aib;

[0199] Through ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO-(CH2) 16 -CO2H is chemically modified at the 20-position of K by conjugation with the ε-amino group of the K side chain; and

[0200] The C-terminal amino acid is amidated to form a C-terminal primary amide (SEQ ID NO: 4).

[0201]

[0202] The diagram above depicts the structure of compound 4 using standard single-letter amino acid codes, except for residues Aib2 and K20, whose structures have been expanded.

[0203] Compound 4 was prepared as described in Example 3 of U.S. Patent No. 9,938,335.

[0204] Synthesis of Compound 1

[0205] Compound 1 is being evaluated in a clinical trial in human patients for the treatment of type 2 diabetes. The drug is expected to be administered orally. The solubility of compound 1 under different pH conditions is being evaluated.

[0206] Material

[0207] The drug substance and excipients used in this study are detailed in Table 1. All other laboratory reagents were used as is.

[0208] Table 1: Materials used for solubility assessment

[0209]

[0210]

[0211] method

[0212] The solubility of compound 1 was assessed at 25°C in pH ranges from 5.0 to 7.0. All solutions contained 10 mM Tris (1.21 g / L) and 0.05% EDTA (0.5 g / L). The pH of the solutions was determined by titration with 1 N hydrochloric acid or a concentrated Tris base stock solution.

[0213] The concentration of compound 1 was measured at 280 nm using a UV-Vis spectrophotometer (SoloVPE). UV-Vis spectrophotometers are commonly used to quantify proteins or peptides in solution. The characteristic UV absorption spectrum near 280 nm primarily originates from aromatic amino acids, such as tryptophan (Trp, W) and tyrosine (Tyr, Y). When the molar extinction coefficient of a protein or peptide is known, the amount of protein or peptide is accurately quantified based on UV absorbance using Beer-Lambert's law, thereby inferring that the molecule does not contain non-protein components that absorb UV, such as bound nucleotide cofactors, heme, or iron-sulfur centers. Compound 1 has tyrosine amino acid residues at positions 10 and 13 and a tryptophan amino acid residue at position 25, and has a concentration of 1.86 mL·mg. -1 ·cm -1 The extinction coefficient (e.g., calculated using the Pace method). This method is applicable to measuring peptide concentrations under different pH conditions for compound 1.

[0214] result

[0215] The solubility data for compound 1 are shown in Table 2, and examples are provided in... Figure 1 middle.

[0216] Table 2: Solubility data of compound 1 at pH 5.0–7.0 (approximately).

[0217]

[0218] The solubility of compound 1 increased significantly as the pH increased from 5.3 to 6.8. Due to the limited supply of compound 1 at the time of this study, this study was not conducted at pH above 6.8. Extrapolation of the data suggests that the solubility of compound 1 can be even higher at pH 7.0 and above. The data indicate that compound 1 should be formulated at pH 7.0 or higher to ensure sufficient solubility of compound 1 during the manufacturing process of the pharmaceutical product and / or in the final dosage form.

[0219] Feasibility study of solution formulation of compound 1

[0220] A study was conducted to assess the feasibility of preparing compound 1 in solution. Solubility data for compound 1 indicate that it should be prepared at pH 7.0 or higher.

[0221] Material

[0222] The compound 1API and excipients used in this study are detailed in Table 3. All other laboratory reagents were used as is.

[0223] Table 3: Materials used in the feasibility study of compound 1

[0224]

[0225] method

[0226] The formulation of compound 1 is shown in Table 4.

[0227] Table 4: Drug Product Formulation of Compound 1

[0228]

[0229] The solution was filtered through a 0.22-mm PVDF filter. In a laminar flow hood, the solution was filled into glass vials. The vials were capped and stored at 5°C, 25°C, and 30°C. Samples were extracted and submitted for the following tests: (a) initial test; (b) two weeks; and (c) one month. The formation of covalent aggregates was measured by size exclusion chromatography (SEC).

[0230] result

[0231] Figure 2a and 2bData on aggregate formation at 25°C are shown. Rapid aggregate formation was observed for all formulations. Aggregation kinetics varied depending on pH, buffers, and tensile agents. The amount of aggregate present after one month at 25°C suggests that none of the formulations is feasible as an injectable product with a 24-month shelf life and desired duration of use. Further investigation is needed to understand the degradation mechanism of compound 1.

[0232] The fibrillation tendency and appropriate pH conditions of the solution formulation of compound 1

[0233] Compound 1 shares some sequence similarities with natural glucagon and is considered to have a potential tendency to form fibrils. Fibril formation can lead to loss of function of the native protein and the acquisition of potential toxic functions, such as inducing immunogenic responses. The tendency of compound 1 to form fibrils will be assessed to determine whether it contributes to the degradation of the compound. The risk of fibril formation by compound 1 as a function of solution pH will be assessed.

[0234] Material

[0235] Prepare solutions of compound 1 at concentrations of 2 mg / mL and 12 mg / mL, and adjust the pH of the solutions to 7.5, 7.8, 8.0, 8.3, and 8.5, respectively. The composition of the preparations is shown in Table 5. All other laboratory reagents were used as is.

[0236] Table 5: Composition of the solution formulation of compound 1

[0237] Element supplier batch number concentration <![CDATA[Compound 1 a > Corden / Eli Lilly BO1704P007 2 mg / mL or 12 mg / mL Tris Eli Lilly C469901 1.21 mg / mL (10 mM Tris buffer) EDTA disodium dihydrate SAFC CDBB4550V 0.5 mg / mL (0.05%) Mannitol Eli Lilly C470786 46 mg / mL (4.6%) <![CDATA[Hydrochloric acid, 1N b > Fisher 164777 Appropriate amount <![CDATA[Sodium hydroxide, 1N b > Fisher 171239 Appropriate amount pure water Hospira 74-602-4B-01 n / a - solvent

[0238] a Adjust the amount of compound 1 to indicate the amount reported in the analytical certificate by HPLC.

[0239] b Amount sufficient to adjust pH

[0240] Preparation of protofibril seeds of compound 1

[0241] Compound 1 fibrils were generated by incubating 500 μL of 2 mg / mL and 12 mg / mL solutions at pH 7.5 in a 37°C incubator with constant end-to-end rotation for 72 hours. After 72 hours, the solution became turbid. Then, 200 μL of the turbid solution was sonicated in a sonic bath for multiple 2-minute cycles until the solution became clear to generate fibril seeds. The 2 mg / mL sample was sonicated for two 2-minute cycles, while the 12 mg / mL sample was sonicated for six 2-minute cycles.

[0242] The formation of fibrils by compound 1 was observed using a thioflavin T (ThT) fluorescence assay.

[0243] Prepare solution formulations of Compound 1 with or without fibril seeds. For inoculated samples, add 5 μL of 2-mg / mL or 12-mg / mL seeds to 150 μL of the corresponding drug product. For uninoculated samples, add 5 μL of water to 150 μL of Compound 1 drug product. A negative control with seeded buffer was also run.

[0244] The formation of fibrils in compound 1 was observed using thioflavone T (ThT) assays (as described in Schlein, M, The AAPS Journal 2017, 19, (2), 397-408). Briefly, 20 μL of each sample was added to each of the three wells of a black transparent-bottomed 384-well plate. ThT was added to each well to a final ThT concentration of 4 μM / well. The plate was sealed with an optical adhesive film and loaded into a SpectraMax i3x (Molecular Devices) plate reader, incubated at 37°C each time. Readings were performed every 15 minutes using an excitation wavelength of 450 nm and an emission wavelength of 480 nm for 36 hours, with 3-second oscillations between readings.

[0245] Results: Fibrous formation of compound 1 was measured by ThT fluorescence assay.

[0246] Fibrous structures are large, self-assembled proteins or peptides with specific biophysical properties. Most notably, individual peptide backbones transform into β-sheet-rich conformations. This can potentially introduce undesirable physical, chemical, and therapeutic risks. Experimentally, fibrillation can be visually observed as increased turbidity, precipitation, or gelation.

[0247] In this study, fluorescence spectroscopy was used to assess the risk of protofibril formation of compound 1 as a function of solution pH, with thioflavin T (ThT) as the binding dye. ThT is an effective fluorescent marker of protofibrils. Once selectively bound to protofibril deposits, the fluorescence signal exhibited a significant increase in fluorescence intensity. Compound 1 was formulated under various pH conditions (7.5, 7.8, 8.0, 8.3, and 8.6) and incorporated with pre-prepared protofibril seeds to accelerate protofibril formation kinetics. Figure 3a and 3b An increase in ThT fluorescence signal can be clearly seen in formulations with pH 8.0 or lower. Fibrillation of compound 1 is clearly particularly sensitive to pH. Below pH 7.8, fibrillation is rapid at both concentrations (2 mg / mL and 12 mg / mL). When the pH increases above 8.0, the fluorescence signal remains flat, indicating the absence of amyloid fibrillation.

[0248] Experimental data from ThT fluorescence assays illustrate the risk of fibrillation formation in Compound 1 at pH ≤ 8.0. At pH > 8.0, fibrillation formation can be successfully prevented even in the presence of inoculated fibrils. From the perspective of mitigating the risk of fibrillation formation, suitable pH conditions for the solution formulation of Compound 1 are above 7.8, preferably above 8.0. Furthermore, this study reveals that stable pH control is crucial during the product's shelf life. Suitable buffers with sufficient buffering strength, such as tris, can be used.

[0249] Degradation study of compound 1 solution formulation

[0250] The amino acid sequence of compound 1 contains residues that potentially make the peptide susceptible to chemical and / or physical degradation. For example, compound 1 has glutamine (Gln, Q) and glycine (Gly, G) at positions 3 and 4, respectively, which may be prone to deamidation. Deamidation is primarily affected by temperature and pH. Compound 1 also has histidine (His, H) and tryptophan (Trp, W) at positions 1 and 25, respectively, which may be potential oxidation hotspots. The risk of oxidation of compound 1 in solution is assessed.

[0251] Material

[0252] Prepare a 2 mg / mL solution of compound 1 at pH 8.0. The composition of the study formulation is shown in Table 6. All other laboratory reagents were used as is.

[0253] Table 6: Composition of the solution formulation of compound 1

[0254]

[0255] a Compound 1API (lot number: BO1704P007) was obtained from Corden Pharma.

[0256] b Adjust the amount of compound 1 to indicate the amount reported in the analytical certificate by HPLC.

[0257] Degradation stress of compound 1

[0258] Compound 1 solution formulations are subjected to various conditions, as summarized in Table 7.

[0259] Table 7: Composition of the solution formulation of compound 1

[0260]

[0261] Results and Conclusions

[0262] (a) Effect of thermal stress

[0263] Compound 1 was prepared as a solution and stored at 40°C for up to 4 weeks. Compositions of batches 1 and 3 contained 0.5 mg / mL EDTA (Table 6), while compositions of batches 2 and 4 did not contain EDTA. EDTA is known to be an effective free radical scavenger. If the oxidation of Trp is ROS-initiated, it is presumed that the presence of 0.5 mg / mL EDTA could inhibit the observed chemical degradation. Samples were taken at appropriate times and subsequently analyzed using RP-HPLC.

[0264] RP-HPLC chromatogram shows Figure 4 In batches 2 and 4, a new peak with a retention time of approximately 4 minutes was observed. Based on historical data, this new peak was attributed to the oxidation of Trp. Similarly, another new peak was identified at a retention time of approximately 7 minutes, corresponding to double Trp oxidation. However, these new peaks were not present at the same retention times in the chromatograms of batches 1 and 3.

[0265] (b) Effects of transition metals and H2O2

[0266] When compound 1 formulation is doped with 2 ppm Fe 3+ At 1 ppm H₂O₂, the presence of 0.5 mg / mL EDTA inhibits Trp oxidation, meaning that no new peaks are observed at retention times of approximately 4 and 7 minutes in the RP-HPLC chromatogram. Figure 5a In the absence of EDTA, 2 ppm of Fe was added. 3+ Or a sample with 1 ppm H2O2 produces Trp oxides ( Figure 5b Doped with Cu 2+ or Ni 2+ The formulation of compound 1 did not show the same degree of degradation.

[0267] (c) Mitigation measures proposed to reduce potential oxidation in solution

[0268] Besides enzymatic oxidation, the oxidation of proteins in solution is typically initiated by free radicals, or reactive oxygen species (ROS). ROS can exist as a result of chemical sterilization processes, through impurity formation, or exposure to light. For example, when hydrogen peroxide (H₂O₂) is used for sterilization, residual H₂O₂ levels can remain adsorbed on the container walls and can initiate oxidation reactions. When sterilization is performed using g-radiation, ROS are generated through radiation-induced chemical processes. Transition metal ions such as iron (Fe₂O₃) can also contribute to ROS formation. 3+ ), copper (Cu) 2+ ) or nickel (Ni 2+Proteins can be another source of ROS and are commonly found as impurities in pharmaceutical formulations, in APIs / drug substances, or excipients. They can also leach from equipment used for storing and processing protein products, such as stainless steel containers. Elastomer components on the equipment may also contain metals due to their curing process. If a protein solution is exposed to light, UV light can be absorbed by aromatic amino acids, leading to ROS generation.

[0269] Although all amino acid side chains are prone to oxidation, free radicals tend to preferentially attack a few amino acid residues, most notably methionine (Met, M), cysteine ​​(Cys, C), histidine (His, H), or tryptophan (Trp, W). In the amino acid sequence of compound 1, the presence of His and Trp amino acids at positions 1 and 25, respectively, makes the peptide potentially prone to oxidation.

[0270] Degradation studies suggest that compound 1 may be prone to oxidation. Including antioxidants, particularly free radical scavengers (e.g., EDTA), is a reasonable strategy to mitigate oxidation in solution.

[0271] Stability study of compound 1 solution formulation

[0272] This study evaluated the stability of Compound 1 as a solution drug product in twelve (12) formulations in prefilled syringes under nominal, accelerated, and stress conditions. Formulation stability was assessed by varying excipient type, pH, and the addition of fibrils. The stability of Compound 1 was assessed using mannitol, sucrose, and propylene glycol as excipients while varying pH (8.0, 8.3, and 8.6). All formulations used in this study are listed in Table 8. Samples were stored at 5 ± 3 °C and 25 ± 2 °C / 60 ± 5% RH for up to 6 months, and at 30 ± 2 °C / 65 ± 5% RH for up to 2 months. The completed analytical program is shown in Table 9 below. Analytical tests performed at each stability time point were described according to physical appearance, RP-HPLC, SEC, and AEX. Test methods were performed at each stability time point.

[0273] Table 8: Feasibility Study of Compound 1 Solution Formulation

[0274]

[0275] 1 The amount of compound 1 was adjusted based on the amount reported in the analytical certificate as determined by HPLC. The purity of the material was reported as 84%. Therefore, to achieve a final concentration of 12 mg / mL, a concentration of 14.29 mg / mL was prepared.

[0276] 2 Add fibrin seeds to the preparation.

[0277] Table 9: Feasibility Study of the Test Plan

[0278]

[0279] Material

[0280] The materials used in this study are as follows:

[0281] i) Test sample of compound 1, batch number NB7956p7A-L, concentration of 12 mg / mL;

[0282] ii) Compound 1, enterprise reference standard, batch number RS1237, concentration 0.89 mg / mL

[0283] iii) Compound 1, protocellulose seeds, batch number C241836-2018-0176-B1, concentration 12 mg / mL

[0284] iv) Glass Prefillable Syringe Platforms, BD Neopak, C / N 47433010, Lot No. 4357108

[0285] v) Syringe Plungers, West Pharma, C / N 11402007, Batch No. D000077885

[0286] vi) EDTA disodium salt, dihydrate, Sigma, C / N E1644, lot number SLBV1798

[0287] vii) Mannitol, JTBaker, C / N 2553-01, Batch No. 212595

[0288] viii) Propylene glycol, Fisher Chemical, C / N P355-1, batch number 180248

[0289] ix) Sucrose, Sigma, C / N S3929, Lot No. SLBV6651

[0290] x) Tris base, Sigma, C / N T6791, lot number SLBQ2306V

[0291] (xi)Aeris Peptide XB-C18, 2.6μm, 4.6×250mm, C / N 00G-4505-E0, Lot Nos. H18-303286, H19 051410, H19-079474, H19-084509, H19-131060 and H19-131061

[0292] xii) BioPro IEX QF, 5μm, 4.6×100mm, C / N QF00505-1046WP, Lot No. 14153

[0293] TOSOH TSKgel G2000SWXL, 5μm, 7.8×300mm, C / N 08540, lot numbers 008C-00776D and A02353-05A

[0294] equipment

[0295] The equipment used in this study is as follows:

[0296] i) Agilent 1100 / 1200 HPLC system

[0297] ii) Eisai mechanical observation lamp model MIH-DX

[0298] iii) Fisher light meter model 06-662-63

[0299] iv) HIAC optical particle counting system, model 9703

[0300] v)Metter Toledo SevenMulti pH meter

[0301] vi) ProteinSimple microfluidic imaging microscope, DPA 5200 with Bot 1

[0302] method

[0303] a) Based on physical appearance description

[0304] Based on the physical appearance description, tests were conducted at all time points using a protocol titled "Physical Appearance Testing for Client 055 Bioproduct Drug Substance and Liquid Drug Product".

[0305] b) RP-HPLC

[0306] The purity and protein content of the samples were determined by RP-HPLC at all time points using a protocol entitled “Identity and Purity Determination of Compound 1 by RP-HPLC”.

[0307] c) Size exclusion chromatography

[0308] The sample aggregates at all time points were determined using a protocol entitled “Determination of Compound 1 Purity by Size Exclusion Chromatography”.

[0309] d) Anion exchange chromatography

[0310] The charge heterogeneity curves of the samples at all time points were determined using a protocol entitled “Determination of Compound 1 Charge Heterogeneity by Anion Exchange Chromatography”.

[0311] result

[0312] a) Based on physical appearance description

[0313] The physical appearance results are shown in Table 10. At all time points and under all conditions, all prepared samples were microemulsified, pale yellow liquid solutions. For each formulation, no particulate matter was observed at the initial, half-month, or one-month time points. At the two-month time point, almost no particulate matter was observed in formulations 4, 5, and 12 at 25±2℃ / 60±5%RH, and almost no particulate matter was observed in formulations 2, 5, and 6 at 30±2℃ / 65±5%RH. At two months, under the corresponding stability conditions, no particulate matter was observed in the remaining formulations. At the three-month and six-month time points, under both stability conditions, almost no particulate matter was observed in all formulations.

[0314] Table 10: Overview of Physical Appearance

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324] b) Reversed-phase high-performance liquid chromatography

[0325] The purity results obtained by RP-HPLC analysis are shown in Table 11. In this group of formulations, the initial average main peak purity percentage ranged from 97.3% to 97.5%, and the average percentage of total related substances (TRS) ranged from 2.5% to 2.7%. In the formulation group under the 6M / 5±3℃ time point and conditions, the average main peak purity percentage ranged from 96.4% to 97.2%, and the average percentage of TRS ranged from 2.8% to 3.6%. In the formulation group under the 6M / 25±2℃ / 60±5%RH time point and conditions, the average main peak purity percentage ranged from 89.4% to 93.1%, and the average percentage of TRS ranged from 6.9% to 10.6%. In the formulation group under the 2M / 30±2℃ / 65±5%RH time point and conditions, the average main peak purity percentage ranged from 92.0% to 94.3%, and the average percentage of TRS ranged from 5.7% to 8.0%. For each formulation, a similar pattern of decreasing peak percentage and increasing TRS percentage was observed under accelerated stabilization conditions. A marginal decrease in peak purity was observed at up to 6 months at 5 ± 3 °C compared to the initial values. The decrease in peak purity percentage was pH-dependent, with higher pH values ​​exhibiting the largest change.

[0326] Results of protein content assessment by RP-HPLC at all time points and under all conditions (Table 11). Each of the twelve (12) individual formulations was prepared at a single concentration level of approximately 12 mg / mL. Throughout the study, protein content ranged from 9.8 to 13.7 mg / mL. Therefore, percentage labeling requirements ranged from 84% to 111% when comparing protein concentrations with initial staging time points throughout the study. Decreased protein concentrations were observed under accelerated six-month time point conditions. More specifically, the decrease in content was pH-dependent, with higher pH values ​​exhibiting the greatest variation.

[0327] Table 11: Overview of RP-HPLC Results

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] (c) Size exclusion chromatography

[0336] The purity analysis results by SEC are shown in Table 12. In this group of formulations, the initial monomer percentage ranged from 98.8% to 99.2%, the aggregate percentage for all formulations was 0.3%, and the fragment percentage ranged from 0.5% to 0.9%. In the formulation group under the 6M / 5±3℃ time point and conditions, the monomer percentage ranged from 98.7% to 99.0%, the aggregate percentage ranged from 0.4% to 0.5%, and the fragment percentage ranged from 0.6% to 0.9%. In the formulation group under the 6M / 25±2℃ / 60±5%RH time point and conditions, the monomer percentage ranged from 98.2% to 98.7%, the aggregate percentage ranged from 0.7% to 1.0%, and the fragment percentage ranged from 0.6% to 1.0%. In the formulation group under the 2M / 30±2℃ / 65±5%RH time point and conditions, the monomer percentage ranged from 98.4% to 99.0%, the aggregate percentage ranged from 0.5% to 0.8%, and the fragment percentage ranged from 0.5% to 1.0%. For each formulation, a similar pattern of decreasing monomer percentages and increasing aggregate and fragment percentages was observed under the stability conditions involved. While marginal changes may be attributed to methodological variability, sucrose-based formulations exhibited the largest variations in monomer percentages, while mannitol-based formulations showed the smallest. Furthermore, for each excipient type, higher pH values ​​exhibited the largest variations, except for propylene glycol. Additionally, when comparing the same formulation components with and without fibrillary seeds, those with fibrillary seeds generally exhibited higher purity, particularly under accelerated conditions.

[0337] In summary, the data showed minimal changes in aggregates and fragments after 6 months of storage under accelerated conditions.

[0338] Table 12: Overview of SEC Results

[0339]

[0340]

[0341]

[0342]

[0343]

[0344] (d) Anion exchange chromatography

[0345] The results of charge heterogeneity determined by AEX are shown in Table 13. In this formulation group, the initial percentage of the main peak ranged from 98.1% to 98.7%, the percentage of basic variants ranged from 0.6% to 1.1%, and the percentage of acidic variants ranged from 0.6% to 0.8%. In the formulation group under the conditions of 6M / 5±3℃, the percentage of the main peak ranged from 93.3% to 97.1%, the percentage of basic variants ranged from 0.5% to 0.9%, and the percentage of acidic variants ranged from 2.3% to 6.0%. In the formulation group under the conditions of 6M / 25±2℃ / 60±5%RH, the percentage of the main peak ranged from 86.5% to 91.6%, the percentage of basic variants ranged from 0.9% to 1.9%, and the percentage of acidic variants ranged from 7.3% to 11.8%. In the formulation groups at 2M / 30±2℃ / 65±5%RH time points and under the specified conditions, the percentage of the main peak ranged from 92.4% to 93.3%, the percentage of the basic variant ranged from 1.0% to 1.4%, and the percentage of the acidic variant ranged from 5.3% to 6.5%. For each formulation, a similar pattern of decreasing main peak percentage and increasing percentage of acidic and basic variants was observed under the stability conditions involved.

[0346] Table 13: Overview of AEX Results

[0347]

[0348]

[0349]

[0350]

[0351] Effect of antioxidants on the stability of compound 1

[0352] introduction

[0353] Degradation studies suggest that compound 1 may be prone to oxidation. The presence of EDTA (a commonly used antioxidant) effectively mitigates oxidation in solution. Other excipients may also be considered to reduce oxidation. In this study, EDTA, citrate, and methionine were evaluated as antioxidants in the solution formulation of compound 1. Samples without any antioxidants were included as controls.

[0354] Materials and methods

[0355] The compound 1API and excipients used in this study are detailed in Table 14. All other laboratory reagents were used as is.

[0356] Table 14: Research Materials

[0357]

[0358] a Adjust the amount of compound 1 to account for the amount reported in the analytical certificate by HPLC.

[0359] b Amount sufficient to adjust pH

[0360] Formulations containing compound 1 under study are shown in Table 15.

[0361] Table 15: Composition of Compound I Formulation

[0362]

[0363] The solution was filtered through a 0.22 mM PVDF filter. In a laminar flow hood, the solution was filled into glass vials. The vials were capped and stored at 5°C, 25°C, and 30°C. Four formulations containing Compound 1 were prepared to evaluate the stabilizing efficacy of the antioxidants (i.e., EDTA, citrate, and methionine). Control samples without any antioxidants were also included (Table 15, batch numbers 25-1, 25-2, 25-3, and 25-4). Subsequently, a fifth sample containing higher concentrations of methionine (100 mM and 10 mM) was prepared to evaluate the effect of methionine concentration (Table 15, batch number 26). Samples were prepared and stored at 5°C, 25°C, and 30°C for up to 3 months. Stability indicator assays were performed at appropriate times to assess physical and chemical stability.

[0364] Appearance of Compound I formulation

[0365] Visual inspection of the appearance of Compound 1 formulations after three months revealed significant differences between formulations (Table 15). At 5°C, the solutions were colorless. However, at 25°C and 30°C, the control and methionine-containing samples were slightly yellow, while the samples containing EDTA and citrate remained colorless. Color change is generally an indicator of chemical degradation. At 5°C, the chemical degradation rate was likely slow enough, regardless of the stabilizer, that all solutions remained colorless. At elevated temperatures, divergent degradation kinetics reflected the effects of antioxidants.

[0366] Table 16: Appearance of Compound 1 formulation after three (3) months

[0367]

[0368] The effect of antioxidants on the chemical stability of compound 1 was determined by RP-HPLC.

[0369] The data in Table 17 show that the chemical degradation of compound 1 was not significant under refrigeration conditions. Significant differences in stabilizing efficacy were observed at 25°C and 30°C. In the absence of any antioxidant, the control sample showed rapid chemical degradation. Methionine (a commonly used antioxidant in monoclonal antibody formulations) exhibited lower stabilizing efficacy (10 mM or 100 mM) compared to EDTA and citrate. Chemical degradation was inhibited by either EDTA or citrate, with EDTA being slightly more effective than citrate.

[0370] Table 17: Total impurities of Compound 1 as determined by RP-HPLC

[0371]

[0372]

[0373] Data from the above experiments indicate that Trp oxidation is one of the main degradation pathways. Table 18 shows that possible Trp oxidation is almost entirely inhibited by EDTA or citrate, with less effect obtained from methionine.

[0374] Table 18: Compound 1 impurities involved in Trp oxidation identified by RP-HPLC

[0375]

[0376] Effect of antioxidants on the physical stability of compound 1

[0377] Covalent aggregate formation was assessed by size exclusion chromatography (SEC). Total aggregates measured by SEC over three months for vials stored at 5°C were shown... Figure 6a In the text, the display shows that for vials stored at 25°C... Figure 6b In, and for vials stored at 30°C, it shows Figure 6c All formulations performed well at 5°C. EDTA and citrate showed significant stability at 25°C and 30°C.

[0378] In addition to SEC, subvisible particulate matter from three months' worth of samples was tested using High-Intensity Optical Anchoring (HIAC) and Motion Flow Imaging (MFI) methods. Experimental data are shown in Table 19. No obvious trends were observed. Particle counts obtained via HIAC were all within the specifications for measurements ≥10 μm and ≥25 μm.

[0379] Table 19: Total aggregates of Compound 1 obtained through SEC

[0380]

[0381] sequence list <110> Eli Lilly <120> Therapeutic peptide preparations <130> X22102 <150> US 63 / 129,157 <151> 2020-12-22 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2)..(2) <223> The 2-digit Xaa is Aib <220> <221> MOD_RES <222> (20)..(20) <223> The 20-position of Lys was chemically modified by conjugating the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18CO2H. <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 1 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Glu Gly Gly Pro Ser 20 25 30 Ser Gly <210> 2 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2)..(2) <223> The 2-digit Xaa is Aib <220> <221> MOD_RES <222> (20)..(20) <223> The 20-position of Lys was chemically modified by conjugating the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2)18CO2H. <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Ser Gly Gly Pro Ser 20 25 30 Ser Gly <210> 3 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2)..(2) <223> The 2-digit Xaa is Aib <220> <221> MOD_RES <222> (20)..(20) <223> The 20-position of Lys was chemically modified by conjugating the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)16CO2H. <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 3 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Glu Gly Gly Pro Ser 20 25 30 Ser Gly <210> 4 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2)..(2) <223> The 2-digit Xaa is Aib <220> <221> MOD_RES <222> (20)..(20) <223> The 20-position of Lys was chemically modified by conjugating the ε-amino group of the Lys side chain with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2)16CO2H. <220> <221> MOD_RES <222> (34)..(34) <223> Gly at position 34 is amidated <400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Ser Gly Gly Pro Ser 20 25 30 Ser Gly <210> 5 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (2)..(2) <223> The 2-digit Xaa is Aib <220> <221> MOD_RES <222> (20)..(20) <223> The 20-position Lys was chemically modified by conjugating the ε-amino group of the Lys side chain to a C14-C24 fatty acid via a linker, wherein the linker was ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)t, where t was 1. <220> <221> MISC_FEATURE <222> (28)..(28) <223> 28-bit Xaa is either Glu or Ser <220> <221> MOD_RES <222> (34)..(34) <223> 34-bit Gly is optionally amidated <400> 5 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Lys Ala Lys Glu Phe Val Glu Trp Leu Leu Xaa Gly Gly Pro Ser 20 25 30 Ser Gly

Claims

1. Pharmaceutical preparations, comprising: (i) Compounds of the following formula from 1 mg / mL to 100 mg / mL in Xaa2 is Aib; Xaa28 is either Glu or Ser; By via 20-bit Lys and C 14 -C 24 The linker between fatty acids allows the ε-amino group of the Lys side chain to bind with C. 14 -C 24 Fatty acid conjugation was used to chemically modify the 20-position Lys, with the linker being ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu). t , where t is 1 or 2; and The C-terminal amino acid may or may not be amidated; (ii) Buffers selected from phosphate buffers and tris(hydroxymethyl)aminomethane (Tris) buffers, ranging from 1 mM to 20 mM; (iii) A tonic agent selected from mannitol, sucrose, trehalose, propylene glycol, glycerol, sodium chloride, and arginine hydrochloride at concentrations ranging from 5 mg / mL to 150 mg / mL; and (iii) an antioxidant selected from 0.05-10.0 mg / mL EDTA and 1-20 mM citric acid. The pH of the formulation is 7.8-9.

0.

2. The pharmaceutical preparation according to claim 1, wherein the compound is selected from: (a) Compounds of the following formula: Where Xaa2 is Aib; By making the Lys sidechain -Amino group with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 CO2H conjugation is used to chemically modify the 20-position Lys; and The C-terminal amino acid is amidated; (b) Compounds of the following formula: Where Xaa2 is Aib; By making the Lys sidechain -Amino group with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2) 18 CO2H conjugation is used to chemically modify the 20-position Lys; and The C-terminal amino acid is amidated; (c) Compounds of the following formula: Where Xaa2 is Aib; By making the Lys sidechain -Amino group with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 16 CO2H conjugation is used to chemically modify the 20-position Lys; and The C-terminal amino acid is amidated; (d) Compounds of the following formula: Where Xaa2 is Aib; By making the Lys sidechain -Amino group with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)2-CO-(CH2) 16 CO2H conjugation is used to chemically modify the 20-position Lys; and The C-terminal amino acid is amidated.

3. The pharmaceutical preparation according to claim 1, wherein the compound has the following formula: Where Xaa2 is Aib; By making the Lys sidechain -Amino group with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 CO2H conjugation is used to chemically modify the 20-position Lys; and The C-terminal amino acid is amidated.

4. The pharmaceutical formulation according to any one of claims 1-3, wherein the buffer is a tris(hydroxymethyl)aminomethane buffer.

5. The pharmaceutical formulation according to claim 4, wherein the formulation comprises 10 mM Tris buffer.

6. The pharmaceutical preparation according to any one of claims 1-3, wherein the antioxidant is EDTA.

7. The pharmaceutical preparation according to any one of claims 1-3, wherein the antioxidant is citric acid.

8. The pharmaceutical formulation according to claim 7, wherein the formulation comprises 5 mM-15 mM citric acid.

9. The pharmaceutical formulation according to claim 7, wherein the formulation comprises 8 mM-12 mM citric acid.

10. The pharmaceutical formulation according to claim 7, wherein the formulation comprises 10 mM citric acid.

11. The pharmaceutical preparation according to any one of claims 1-3, wherein the pH of the preparation is 8.0-8.

6.

12. The pharmaceutical preparation according to claim 11, wherein the pH of the preparation is 8.0-8.

3.

13. The pharmaceutical preparation according to claim 1, comprising: (i) Compounds of the following formula with concentrations ranging from 1 mg / mL to 100 mg / mL: Where Xaa2 is Aib; By making the Lys sidechain -Amino group with ([2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2) 18 CO2H conjugation is used to chemically modify the 20-position Lys; and The C-terminal amino acid is amidated; (ii) 10 mM Tris buffer; (iii) Mannitol at 46 mg / mL; (iv) 0.5 mg / mL EDTA, The pH of the formulation is 8.0-8.

3.

14. The pharmaceutical preparation according to any one of claims 1-3 and 13, for the treatment and / or prevention of type 2 diabetes.

15. The pharmaceutical preparation according to any one of claims 1-3 and 13, for the treatment and / or prevention of obesity.

16. The pharmaceutical preparation according to any one of claims 1-3 and 13, for the treatment and / or prevention of non-alcoholic fatty liver disease (NAFLD).

17. The pharmaceutical preparation according to any one of claims 1-3 and 13, for the treatment and / or prevention of non-alcoholic steatohepatitis (NASH).

18. Use of the pharmaceutical preparation according to any one of claims 1-13 in the preparation of a medicament for treating type 2 diabetes.

19. Use of the pharmaceutical preparation according to any one of claims 1-13 in the preparation of a medicament for treating obesity.

20. Use of the pharmaceutical preparation according to any one of claims 1-13 in the preparation of a medicament for treating NAFLD.

21. Use of the pharmaceutical preparation according to any one of claims 1-13 in the preparation of a medicament for treating NASH.

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