Liquid formulations of glucagon analogs

Abstract

The present invention relates to formulations of glucagon analogues or pharmaceutically acceptable salts and / or derivatives thereof; and their medical use, e.g. in the treatment of hypoglycemia. In particular, the present invention relates to stable aqueous liquid formulations of glucagon analogues comprising a combination of excipients that make them suitable for long-term storage as liquids and capable of being used in single dose (SD) or multiple dose (MD) formulations.

Description

Technical Field

[0001] This invention relates to formulations of glucagon analogs and their medical use, for example, in the treatment of hypoglycemia. In particular, this invention relates to stable aqueous liquid formulations of glucagon analogs. Background Technology

[0002] Human proglucagon is a 158-amino acid precursor polypeptide that is selectively processed in tissues to form a number of structure-related proglucagon-derived peptides, including glucagon (Glu), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM). These molecules are involved in a wide range of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, and regulation of food intake.

[0003] Natural glucagon is a 29-amino acid peptide corresponding to amino acids 53 to 81 of proglucagon. Glucagon helps maintain blood glucose levels by binding to glucagon receptors on hepatocytes, causing the liver to release glucose stored as glycogen through glycogenolysis. As these stores are depleted, glucagon also stimulates the liver to synthesize additional glucose through gluconeogenesis. This glucose is released into the bloodstream, preventing hypoglycemia. This has led to the development of therapeutic formulations of glucagon or glucagon analogs designed to be used in "rescue" situations of acute hypoglycemia in diabetic subjects, such as those who have received high doses of insulin. However, due to the relatively low physical and chemical stability of natural glucagon itself, most commercially available glucagon products are currently offered in the form of freeze-dried solid dosage forms (which need to be reconstituted in a suitable liquid medium immediately before use). Because individuals with hypoglycemia may exhibit symptoms of dizziness and / or confusion, or in some cases even become unconscious or semi-conscious, this means they may be unable to perform or complete the necessary initial fluid remodeling, thus complicating the subsequent injection of glucagon. Therefore, within the limited available time before excessive glucagon buildup occurs, this remodeling and injection may have to be performed by another person without experience processing the product.

[0004] This has led to efforts in the field to develop formulations of glucagon analogs that, in addition to exhibiting satisfactory high activity at the glucagon receptor, are sufficiently soluble in liquid formulations (particularly at physiological pH, where natural glucagon is substantially insoluble at neutral pH) and are physically and chemically stable, enabling immediate use without reconstitution. These analogs (i) are advantageously available in ready-to-use liquid pharmaceutical formulations suitable for immediate injection, and (ii) can be stored (including by the relevant subject or patient under environmental conditions) for a sufficiently long period prior to use.

[0005] WO 2011 / 117417 (Novo Nordisk A / S) discloses a modified glucagon peptide containing a mutation at the 25th amino acid of glucagon 1 to 29, which resists aggregation in aqueous solution.

[0006] WO 2016 / 133863 (Eli Lilly and Company) describes a powder composition for nasal administration of glucagon, which contains cyclodextrin and phospholipid surfactant in a phase characterized by the midpoint of XRPD.

[0007] WO 2019 / 110838 (Adocia) relates to formulations of glucagon, copolyamino acids with carboxyl salt fillers and hydrophobic groups.

[0008] WO 2008 / 084237 (Arecor Limited) describes an aqueous protein composition in which the pKa of the displacement buffer used is at least one unit greater than the pH at which the protein is stable.

[0009] WO 2014 / 016300 (Zealand Pharma A / S) discloses glucagon analogs suitable for use in liquid formulations, wherein the analogs have improved solubility and / or stability compared to natural human glucagon. One of the glucagon analogs described in WO 2014 / 016300 is called dasiglucagon and is in clinical trials as an off-the-shelf treatment for severe hypoglycemia, for the treatment of type 1 diabetes, and for the treatment of congenital hyperinsulinemia. The pharmacokinetic and pharmacodynamic characteristics of dasiglitin and its interaction with other drugs were disclosed in Diabetes Care, 2017 (https: / / doi.org / 10.2337 / dc17-1402). A comparison.

[0010] WO 2017 / 053822 (Xeris Pharmaceuticals, Inc.) discloses a liquid formulation of a natural glucagon peptide using dimethyl sulfoxide (DMSO) as an aprotic polar solvent system. Ionizing stabilizers may also be added to improve solubility. WO 2014 / 124151 (Xeris Pharmaceuticals, Inc.) discloses a liquid formulation of glucagon peptide in DMSO, which additionally contains a cryoprotectant, such as trehalose, to protect the formulation from freeze-thaw cycles. However, the use of DMSO in the solvent system of these formulations has several significant drawbacks, as it results in relatively high viscosity, making injection difficult, and leading to undesirable side effects such as skin reactions, dry skin, headache, dizziness, drowsiness, nausea, vomiting, diarrhea, constipation, respiratory problems, vision problems, blood problems, and allergic reactions. DMSO also causes a garlic-like odor, breath, and body odor. Furthermore, DMSO dissolves plastics and rubbers, which can be disadvantageous when applied via delivery devices such as pens, syringes, or pump systems. In some cases, DMSO is used in combination with 15% to 20% of a co-solvent (e.g., propylene glycol or ethanol) to avoid the relatively high freezing temperatures of formulations containing 80% to 100% DMSO. However, if a formulation using DMSO and a co-solvent contains more than 5% water, the chemical stability of glucagon in the formulation will be reduced.

[0011] Therefore, improving formulations of glucagon analogues, particularly stable liquid formulations that can be stored for extended periods without excessive levels of physical and chemical degradation of the active monomeric form of the peptide and that overcome some of the drawbacks associated with existing formulations of glucagon or glucagon analogues, remains a problem in the art. Invention Overview

[0013] In a broad sense, the present invention is based on studies reported in the embodiments, which have explored and identified excipients suitable for use in aqueous liquid formulations of glucagon analogs, which are suitable for long-term liquid storage and can be used in single-dose (SD) or multi-dose (MD) formulations.

[0014] In these studies, the inventors discovered that buffers in formulations containing glucagon analogs play a crucial role in the stability of aqueous formulations containing glucagon analogs, particularly dasiglitin. Experiments disclosed herein demonstrate that TIS, Bis-TRIS, ACES, and MES buffers and / or acetate, citrate, or succinate buffers provide chemical stability to glucagon analog formulations at weakly acidic to neutral pH levels, as determined by the detection and quantification of degradation products. Comparative analysis of degradation profiles shows that these buffers provide enhanced stability relative to other tested buffer systems (e.g., histidine or phosphate), even when the formulation otherwise has the same pH, i.e., the stability provided by the buffer is at least partially independent of its pH-regulating properties. Importantly, in some aspects of the invention where the formulation also contains a preservative such as m-cresol, the liquid formulation exhibits long-term stability and is therefore suitable for multiple administrations. This opens up the possibility of delivering glucagon analogue formulations in the form of drug delivery devices, and particularly single- or multi-dose delivery devices, such as pre-filled syringes, syringe devices, injection pens, adjustable-dose autoinjectors, disposable autoinjectors, wearable syringes, or infusion pumps, thereby providing patients with ready-to-use formulations in simpler, safer, and more patient-friendly devices.

[0015] Therefore, in a first aspect, the present invention provides a stable aqueous liquid pharmaceutical preparation comprising a glucagon analog or a pharmaceutically acceptable salt and / or derivative thereof, wherein the glucagon analog is:

[0016] Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH;

[0017] The formulation contains:

[0018] (a) Glucagon analogs or their pharmaceutically acceptable salts and / or derivatives, present at a concentration of about 0.5 mg / mL to about 10 mg / mL;

[0019] (b) TRIS, Bis-TRIS, ACES or MES, which are present as buffers at a concentration of about 25 mM to about 75 mM;

[0020] (c) Sodium chloride, which acts as a tension modifier and is present at a concentration of about 50 mM to about 600 mM; and

[0021] (d) pH from about 5.6 to about 7.0.

[0022] In another aspect, the present invention provides a stable aqueous liquid pharmaceutical formulation comprising a glucagon analog or a pharmaceutically acceptable salt and / or derivative thereof, wherein the glucagon analog is:

[0023] Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH;

[0024] The formulation contains:

[0025] (a) Glucagon analogues, present at concentrations of about 0.5 mg / mL to about 10 mg / mL;

[0026] (b) TRIS, Bis-TRIS, ACES or MES, which are present as a buffer at a concentration of about 25 mM to about 75 mM; and / or citrate, acetate or succinate, which are present as a buffer at a concentration of about 1 mM to about 30 mM.

[0027] (c) Sodium chloride, which acts as a tension modifier and is present at a concentration of about 50 mM to about 600 mM; and

[0028] (d) pH from about 5.6 to about 7.0.

[0029] In some embodiments of a formulation comprising a glucagon analog or its pharmaceutically acceptable salt and / or derivative, the formulation comprises a glucagon analog, its pharmaceutically acceptable salt or derivative thereof present at a concentration of about 0.1 mg / mL to about 1.0 mg / mL, such as about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL or about 1.0 mg / mL.

[0030] In some aspects, the inventors have discovered that TRIS, Bis-TRIS, ACES, or MES buffers can directly stabilize glucagon analogs independently of the pH of the formulation provided by the buffer. This improvement in direct chemical stability can be determined relative to the stability of the glucagon analog in the corresponding formulation in phosphate buffers and / or histidine buffers under the same test conditions.

[0031] In another aspect, the present invention provides a method for producing the stable aqueous liquid pharmaceutical composition of the present invention.

[0032] In another aspect, the present invention provides a delivery device comprising a stable aqueous liquid formulation containing a glucagon analog of the present invention. For example, the delivery device includes a pre-filled syringe, a syringe device, an injection pen, an adjustable-dose autoinjector, a disposable autoinjector, a wearable syringe, or an infusion pump.

[0033] In another aspect, the present invention provides an article or medicine box comprising a container holding a stable aqueous liquid pharmaceutical composition of the present invention.

[0034] In another aspect, the present invention provides a stable aqueous liquid formulation of the glucagon analogue of the present invention for therapeutic purposes.

[0035] In another aspect, the present invention provides a stable aqueous liquid formulation of the glucagon analog of the present invention for treating diseases or conditions selected from hypoglycemia (including but not limited to severe hypoglycemia, acute hypoglycemia, and chronic hypoglycemia), type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, coronary heart disease, atherosclerosis, hypertension, dyslipidemia, hepatic steatosis, beta-blocker poisoning, insulinoma, or von Gilke's disease. In particular, this formulation can be used to treat various forms of hypoglycemia, such as hypoglycemia selected from diabetic hypoglycemia, acute insulin-induced hypoglycemia, hyperinsulinemia-induced hypoglycemia, nondiabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, congenital hyperinsulinemia-induced hypoglycemia, alcohol-induced hypoglycemia, hypoglycemia induced by gastric bypass surgery (including but not limited to postprandial hypoglycemia after Roux-en-Y gastric bypass surgery), hypoglycemia after weight loss, or hypoglycemia occurring during pregnancy. The method comprises administering a therapeutically effective amount of the glucagon analogue of the present invention to a patient in need of treatment.

[0036] In some implementations, severe hypoglycemia is characterized by very low blood glucose levels. In some implementations, treatment for acute hypoglycemia includes treating acute episodes of severe hypoglycemia, including but not limited to episodes of severe hypoglycemia characterized by very low blood glucose levels.

[0037] In another aspect, the present invention provides a method for treating patients suffering from diseases or conditions selected from: hypoglycemia (including but not limited to severe hypoglycemia, acute hypoglycemia, and chronic hypoglycemia), type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, coronary heart disease, atherosclerosis, hypertension, dyslipidemia, hepatic steatosis, beta-blocker poisoning, insulinoma, or von Gilke's disease. In particular, this formulation can be used to treat various forms of hypoglycemia, such as hypoglycemia selected from: diabetic hypoglycemia, acute insulin-induced hypoglycemia, hyperinsulinemia-induced hypoglycemia, nondiabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, congenital hyperinsulinemia-induced hypoglycemia, alcohol-induced hypoglycemia, hypoglycemia induced by gastric bypass surgery, hypoglycemia after weight loss (including but not limited to postprandial hypoglycemia after Roux-en-Y gastric bypass surgery), or hypoglycemia occurring during pregnancy, the method comprising administering a therapeutically effective amount of a stable aqueous liquid formulation of the glucagon analog of the present invention to a patient in need of treatment.

[0038] In another aspect, the present invention provides the use of a stable aqueous liquid formulation of the glucagon analog of the present invention in the preparation of a medicament for treating diseases or conditions selected from: hypoglycemia (including but not limited to severe hypoglycemia, acute hypoglycemia, and chronic hypoglycemia), type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, coronary heart disease, atherosclerosis, hypertension, dyslipidemia, hepatic steatosis, beta-blocker poisoning, insulinoma, or von Gilke's disease. Specifically, this formulation can be used to treat various forms of hypoglycemia, including hypoglycemia selected from: diabetic hypoglycemia, acute insulin-induced hypoglycemia, hyperinsulinemia-induced hypoglycemia, nondiabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, congenital hyperinsulinemia-induced hypoglycemia, alcohol-induced hypoglycemia, hypoglycemia induced by gastric bypass surgery (including but not limited to postprandial hypoglycemia after Roux-en-Y gastric bypass surgery), hypoglycemia after weight loss, or hypoglycemia occurring during pregnancy. The method comprises administering a therapeutically effective amount of the glucagon analogue of the present invention to a patient in need of treatment.

[0039] In some embodiments, patients treated with the formulation according to the invention are not in a state of starvation, do not suffer from adrenal insufficiency, and / or do not suffer from chronic hypoglycemia. In some embodiments, patients treated with the formulation according to the invention do not experience decreased liver glycogen. In some embodiments, patients treated with the formulation according to this disclosure do not have tumors in their adrenal glands, including but not limited to pheochromocytoma. In some embodiments, patients treated with the formulation according to the invention do not have tumors in their pancreas, including but not limited to insulinoma.

[0040] In some implementations, patients treated with the formulation according to this disclosure are six years of age or older.

[0041] In another aspect, the present invention provides a method for producing a stable aqueous liquid pharmaceutical formulation comprising a glucagon analog or a pharmaceutically acceptable salt and / or derivative thereof, wherein the glucagon analog is:

[0042] Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH;

[0043] The method comprises formulating the following to produce a stable aqueous liquid pharmaceutical preparation: (a) a glucagon analog or pharmaceutically acceptable salt and / or derivative thereof, present at a concentration of about 0.5 mg / mL to about 10 mg / mL; (b) TRIS, Bis-TRIS, ACES, or MES, present as a buffer at a concentration of about 25 mM to about 75 mM; (c) sodium chloride, present as a tonic modifier at a concentration of about 50 mM to about 600 mM; (d) a pH of about 5.6 to about 7.0; and optionally (e) m-cresol, at a concentration of about 1.0 mg / mL to about 5.0 mg / mL. In some preferred embodiments, the method provides a stable aqueous liquid pharmaceutical preparation capable of being stored stably at 2 to 8°C for at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

[0044] In another aspect, the present invention provides a method for producing a stable aqueous liquid pharmaceutical formulation comprising a glucagon analog or a pharmaceutically acceptable salt and / or derivative thereof, wherein the glucagon analog is:

[0045] Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH;

[0046] The method comprises formulating the following to produce a stable aqueous liquid pharmaceutical preparation: (a) a glucagon analog or pharmaceutically acceptable salt and / or derivative thereof, present at a concentration of about 0.5 mg / mL to about 10 mg / mL; (b) TRIS, Bis-TRIS, ACES, or MES, present as a buffer at a concentration of about 25 mM to about 75 mM; (c) sodium chloride, present as a tonic modifier at a concentration of about 50 mM to about 600 mM; (d) a pH of about 5.6 to about 7.0; and optionally (e) m-cresol, at a concentration of about 1.0 mg / mL to about 5.0 mg / mL. In some preferred embodiments, the method provides a stable aqueous liquid pharmaceutical preparation capable of being stored stably at 2 to 8°C for at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

[0047] In some embodiments, glucagon analogs or their pharmaceutically acceptable salts and / or derivatives are present at concentrations of about 0.1 mg / mL to about 1.0 mg / mL, such as about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1.0 mg / mL.

[0048] In another aspect, the present invention provides the use of a stable aqueous liquid pharmaceutical formulation comprising a glucagon analog or a pharmaceutically acceptable salt and / or derivative thereof, wherein the glucagon analog is:

[0049] Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH;

[0050] It is used to provide an aqueous liquid pharmaceutical formulation that is stable for 24 months when stored at 2 to 8°C, wherein the formulation comprises the following to produce a stable aqueous liquid pharmaceutical formulation:

[0051] (a) Glucagon analogues, present at concentrations of about 0.5 mg / mL to about 10 mg / mL;

[0052] (b) TRIS, Bis-TRIS, ACES or MES, which are present as buffers at a concentration of about 25 mM to about 75 mM;

[0053] (c) Sodium chloride, which acts as a tension modifier and is present at a concentration of about 50 mM to about 600 mM;

[0054] (d) pH from about 5.6 to about 7.0; and optional

[0055] (e) m-cresol, at a concentration of about 1.0 mg / mL to about 5.0 mg / mL.

[0056] In some embodiments, glucagon analogs or their pharmaceutically acceptable salts and / or derivatives are present at concentrations of about 0.1 mg / mL to about 1.0 mg / mL, such as about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1.0 mg / mL.

[0057] In other aspects, the present invention relates to aqueous liquid pharmaceutical formulations comprising glucagon analogs, which are substantially composed of or consist of glucagon analogs and other components of the formulation as defined herein. Further aspects and embodiments of the invention will be apparent to those skilled in the art in light of this disclosure.

[0058] As used herein, “and / or” should be considered to specifically disclose each of the two indicated features or components, with or without the other. For example, “A and / or B” is considered to specifically disclose each of the following: (i) A, (ii) B, and (iii) A and B, as if each were stated separately herein.

[0059] Unless otherwise indicated by the context, the description and limitations of the features listed above are not limited to any specific aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described. Invention Details

[0061] definition

[0062] Unless otherwise stated, the following definitions are provided for the specific terms used in the written description above.

[0063] Throughout the specification and claims, conventional single-letter and three-letter codes for natural amino acids are used. All amino acid residues in the peptides of this invention preferably have an L-configuration; however, D-configuration amino acids may also be present. Aib represents α-aminoisobutyric acid.

[0064] glucagon analogues

[0065] The term "natural glucagon" refers to natural human glucagon having the following sequence: H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (SEQ ID NO: 1).

[0066] The preferred compounds used in the formulations of this invention have at least glucagon agonist activity. This can be evaluated in in vitro assays, for example, as described in WO 2014 / 016300, wherein the generation of cellular cyclic AMP (cAMP) is used to monitor human glucagon receptor activity in the presence of a glucagon analog.

[0067] The sequences disclosed herein are such that a "Hy-" moiety is incorporated at the amino terminus (N-terminus) and a "-OH" moiety is incorporated at the carboxyl terminus (C-terminus). In this case, and unless otherwise indicated, the "Hy-" moiety at the N-terminus of the sequence under discussion represents a hydrogen atom, and the "-OH" moiety at the C-terminus represents a hydroxyl group.

[0068] Compared to natural glucagon, glucagon analogs present in the formulations of this invention have one or more amino acid substitutions, deletions, inversions, or additions. This definition also includes the synonyms glucagon mimics and / or glucagon agonists. Furthermore, the analogs of this invention may additionally have one or more chemical modifications to their amino acid side groups, α-carbon atoms, terminal amino groups, or terminal carboxylic acid groups. Chemical modifications include, but are not limited to: adding chemical moieties, forming new bonds, and removing chemical moieties. Modifications at the amino acid side groups include, but are not limited to: acylation of the ε-amino group of lysine, N-alkylation of arginine, histidine, or lysine, alkylation of the carboxylic acid group of glutamic acid or aspartic acid, and deamidation of glutamine or asparagine. Modifications of the terminal amino group include, but are not limited to: deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxyl group include, but are not limited to: amides, lower alkylamides, dialkylamides, and lower alkyl ester modifications. Preferred lower alkyl groups herein are C1-C4 alkyl groups. Furthermore, one or more side groups or terminal groups can be protected by protecting groups known to peptide chemists of common technique. The α-carbon of an amino acid can be monomethylated or dimethylated.

[0069] In some aspects, the liquid formulations of the present invention use glucagon analogs or pharmaceutically acceptable salts or derivatives thereof represented by the following formula:

[0070] Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH (SEQ ID NO: 2), Compound 1.

[0071] In the three-letter amino acid code, this glucagon analog (dasi glucagon) is represented by the following formula:

[0072] H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr-OH

[0073] As reported in WO 2014 / 016300, compound 1 (SEQ ID NO: 2) exhibits in vitro agonist activity at the human glucagon receptor, wherein EC 50 The values ​​are 0.0095 nM and 0.030 nM, respectively.

[0074] It should be understood that the peptides (pharmaceutical substances) of the present invention may also be provided in the form of salts or other derivatives. As defined, salts include pharmaceutically acceptable salts.

[0075] Other derivatives of the glucagon analogues of the present invention include those containing metal ions (e.g., Mn). 2+ and Zn 2+ The prodrug can be a coordination complex, ester (e.g., an in vivo hydrolyzable ester), free acid or base, hydrate, prodrug, or lipid. Using techniques known in the art, an ester can be formed between a hydroxyl or carboxylic acid group present in the compound and a suitable carboxylic acid or alcohol reaction partner. The derivative, as a prodrug of the compound, can be converted in vivo or in vitro to one of the parent compounds. Generally, at least one biological activity of the compound is reduced in the prodrug form and can be activated by conversion of the prodrug to release the compound or its metabolites. Some examples of prodrugs include the use of a protecting group that can be removed in situ to release the active compound or to inhibit the clearance of the drug in vivo.

[0076] The glucagon analog used in the formulation of this invention can be synthesized using solid-phase peptide synthesis techniques known in the art. For example, this can be performed using Tentagel S PHB-Thr(tBu) resin (1.13 g, 0.24 mmol / g), COMU as a coupling agent, DMF as a solvent, and the Fmoc-chemicals as described above on a CEM Liberty Peptide Synthesizer. The sequence uses pseudoproline Fmoc-Phe-Thr(Ψ,Me,Me pro)-OH (at positions 6 / 7) and Fmoc-Glu-Ser(Ψ,Me,Me pro)-OH (at positions 15 / 16). Following synthesis, the glucagon analogue was cleaved from the resin and purified on a Gemini-NX column (5 cm, C18, 10 μm) at a flow rate of 35 mL / min for a mixture of buffer A (0.1% aqueous TFA) and buffer B (an aqueous solution containing 0.1% TFA and 90% MeCN). The combined fractions were lyophilized and redissolved in water prior to further purification. The purity of the product was determined by analytical HPLC, and the monoisotope mass was determined by MS.

[0077] glucagon analogues

[0078] As described herein, the stable aqueous liquid pharmaceutical formulations according to the invention comprise a glucagon analog, a buffer, a tonic modifier, and are formulated to have a pH that is physiologically compatible with the patient to whom the formulation is administered. In some embodiments, the formulations of the invention also comprise a preservative, for example, to enable the production of multi-dose formulations. In the following examples, the inventors determined that the choice of buffer used to formulate the compositions of the invention plays a decisive role in their long-term stability, and that many commonly used buffers, including phosphate buffers, histidine buffers, maleic acid, and Milli-Q water (MQW), do not provide sufficient stability in aqueous liquid formulations, for example, for storage at 25°C for 13, 26, or 52 weeks. However, other types of buffers, particularly TRIS, Bis-TRIS, ACES, or MES buffers, can be used to formulate stable aqueous liquid formulations of glucagon analogs. The experiments described herein have also found that the stability provided by these buffers directly stabilizes the glucagon analog, i.e., in a manner independent of the pH of the formulation provided by the buffer. This effect can be determined under the same test conditions relative to phosphate buffer and / or histidine buffer.

[0079] In IUPAC nomenclature, TRIS buffer refers to (tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol).

[0080] In the IUPAC nomenclature, Bis-TRIS buffer refers to (bis-trimethylane or 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol).

[0081] In the IUPAC nomenclature, ACES buffers refer to (N-(2-acetamido)-2-aminoethanesulfonic acid or 2-(carbamoylmethylamino)ethanesulfonic acid).

[0082] In the IUPAC nomenclature, MES buffer refers to (2-(N-morpholino)ethanesulfonic acid or as 2-morpholino-4-ylethanesulfonic acid.

[0083] Other buffers that can be used as alternatives to or supplements to these buffers include citrates, acetates, or succinates, for example, in the form of sodium citrate, sodium acetate, or sodium succinate. Preferably, these buffers are present at a concentration of about 1 mM to about 30 mM, more preferably about 10 mM to about 30 mM, and even more preferably at a concentration of about 15 mM. The actual concentration used can be determined by whether the buffer is used alone or in combination.

[0084] The experimental work leading to this invention demonstrates that stable formulations of glucagon analogs avoid the inclusion of polar aprotic solvents in liquid formulations, such as dimethyl sulfoxide (DMSO) as used in WO 2017 / 053822 and WO 2014 / 124151, which can introduce adverse effects on the formulation in use. Therefore, preferably, the formulations of this invention contain substantially no aprotic polar solvents, and more preferably, the formulations contain substantially no dimethyl sulfoxide (DMSO). In some particularly preferred embodiments, water is the only solvent used to prepare the aqueous liquid formulation.

[0085] The work described herein also demonstrates that, contrary to experience with formulations based on the use of DMSO (optionally in combination with a cosolvent), formulations in which water is present can be produced without substantially affecting the chemical or physical stability of the glucagon analogue in the formulation. Some particularly useful embodiments of the liquid pharmaceutical formulations of the present invention are aqueous formulations, i.e., formulations containing water. Such formulations may be in the form of aqueous solutions or aqueous suspensions. Some preferred embodiments of the aqueous pharmaceutical formulations of the present invention are aqueous solutions. In the context of the present invention, the term "aqueous formulation" will generally refer to a formulation containing at least 50% (50% w / w) water by weight, more preferably at least 75% w / w water, more preferably at least 80% w / w water, more preferably at least 85% w / w water, more preferably at least 90% w / w water, and most preferably at least 95% w / w water. Similarly, the term "aqueous solution" will generally refer to a solution containing at least 50% w / w water, and the term "aqueous suspension" refers to a suspension containing at least 50% w / w water. In some embodiments, the aqueous formulations of the present invention substantially do not contain aprotic polar solvents, such as dimethyl sulfoxide (DMSO). In this sense, it essentially means that the aqueous formulation contains less than 5% (v / v) of aprotic solvent by volume, more preferably less than 2% (v / v) of aprotic solvent by volume, and even more preferably less than 1% (v / v) of aprotic solvent by volume. According to some embodiments, water is the only solvent used to prepare the aqueous liquid formulation.

[0086] In some aspects of the invention, stable aqueous liquid formulations of glucagon analogs, their pharmaceutically acceptable salts and / or derivatives comprise:

[0087] (a) Glucagon analogues, present at concentrations of about 0.5 mg / mL to about 10 mg / mL;

[0088] (b) TRIS, Bis-TRIS, ACES or MES, which are present as buffers at a concentration of about 25 mM to about 75 mM;

[0089] (c) Sodium chloride, which acts as a tension modifier and is present at a concentration of about 50 mM to about 600 mM; and

[0090] (d) pH from about 5.6 to about 7.0.

[0091] In some embodiments of formulations comprising glucagon analogs or their pharmaceutically acceptable salts and / or derivatives, the formulation comprises a glucagon analog, its pharmaceutically acceptable salt, or a derivative thereof present at a concentration of about 0.1 mg / mL to about 1.0 mg / mL, such as about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1.0 mg / mL.

[0092] The term "medicatable carrier" includes any standard pharmaceutical carrier or diluent, such as those used in compositions or formulations suitable for oral, pulmonary, rectal, nasal, topical, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, transdermal, or vaginal administration. Medicatable carriers for therapeutic purposes are well known in the pharmaceutical industry and are described in, for example... Remington's Pharmaceutical Sciences According to Mack Publishing Co. (ARGennaroedit. 1985), liquid formulations typically use unbuffered or buffered aqueous solutions as a carrier. For example, sterile saline or phosphate-buffered saline (PBS) at slightly acidic, slightly alkaline, or physiological pH can be used. Relevant pH buffers include tris(hydroxymethyl)aminomethane (TRIS), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (Bis-TRIS), 2-(carbamoylmethylamino)ethanesulfonic acid (ACES), maleic acid, 2-morpholino-4-ylethanesulfonic acid (MES), and mixtures thereof. The term also covers any reagent listed in the United States Pharmacopeia for use in animals or humans.

[0093] In the context of this invention, the term "medicinal salt" refers to a salt that is harmless to the patient or subject to be treated therein. Such salts are typically acid addition salts or basic salts. Acid addition salts include salts of organic acids and salts of inorganic acids. Some non-limiting examples of suitable acid addition salts include hydrochlorides, phosphates, formates, acetates, trifluoroacetates, and citrates. Some examples of basic salts include salts in which the cation is selected from: alkali metal ions, such as sodium and potassium ions; alkaline earth metal ions, such as calcium ions; and substituted ammonium ions, such as NR(R')3. + ammonium ions of the type, where R and R' independently represent optionally substituted C. 1-6 Alkyl groups, optionally substituted C 2-6Alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. Other examples of pharmaceutically usable salts are described in Remington's Pharmaceutical Sciences, 17th edition, Ed. Alfonso R. Gennaro (ed.), Mack Publishing Company, Easton, PA, USA, 1985 and the latest edition, and in the Encyclopaedia of Pharmaceutical Technology.

[0094] As used in the context of this invention, the term "treatment" (and its other grammatical variations) refers to a method for obtaining a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization of disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also refer to a prolonged survival compared to expected survival without treatment. "Treatment" is an intervention aimed at preventing the occurrence of a condition or altering its pathological condition. Therefore, "treatment" refers to both therapeutic treatment and preventative or preventative measures. As used in the context of preventative or preventative measures, pharmaceutical preparations do not necessarily require complete prevention of the disease or condition. Those requiring treatment include those already suffering from the condition, as well as those for which the occurrence of the condition is to be prevented. "Treatment" also implies the suppression or reduction of the improvement of a pathological condition or symptom (e.g., weight gain or hypoglycemia) compared to no treatment, and does not necessarily imply the complete cessation of the relevant condition.

[0095] A “stable” formulation is one in which the peptides therein substantially retain their physical and / or chemical stability and / or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability and its biological activity after storage. Storage period is typically selected based on the expected shelf life of the formulation. The formulations of the present invention are provided as stable liquid formulations, such as stable aqueous liquid formulations. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). In this invention, a "stable" formulation includes one in which, after being stored at 2 to 8°C for at least 18 months, at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% of the glucagon analogue remains undegraded. Stability can also be tested under accelerated conditions, which typically use increased storage temperatures to assess stability over shortened time periods. For example, storage at 25°C (sometimes referred to as under accelerated conditions) can be used to assess stability over periods of 13, 26, 39, or 52 weeks. In comparison, the use of other buffers, such as phosphate buffers or histidine buffers, results in stability levels typically below 80% after the corresponding storage time.

[0096] Stability can be measured at a selected temperature for a selected period of time, for example, by using an elevated temperature to reduce the time span of the test formulation. Generally, storage at a temperature of 2 to 8°C indicates storage under normal refrigeration conditions. In some embodiments, the formulation is stable under such conditions for at least 6 months, more preferably at least 12 months, more preferably at least 18 months, and more preferably at least 24 months. Stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including evaluating aggregate formation (e.g., using size exclusion chromatography, UV light scattering, dynamic light scattering, circular dichroism, by measuring turbidity and / or by visual inspection); assessing charge heterogeneity by using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; N-terminal or C-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced antibodies with intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing antibody bioactivity or antigen-binding function; etc. Instability may involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine ​​residues, N-terminal extension, C-terminal processing, differential glycosylation, adduct formation, etc.

[0097] If a peptide does not show any signs (or very few signs) of aggregation, precipitation, and / or denaturation when examined visually for color and / or clarity, or when measured by UV light scattering, dynamic light scattering, circular dichroism, or size exclusion chromatography, then the peptide “maintains its physical stability” in the pharmaceutical formulation and is considered to still retain its biological activity.

[0098] If a peptide is considered to retain its biological activity at a given time due to its chemical stability, then the peptide is "chemically stable" in a pharmaceutical formulation, as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the peptide. Chemical alterations may involve isomerization, oxidation, and size modification (e.g., trimming), which can be evaluated using, for example, HPLC or size exclusion chromatography, SDS-PAGE, and / or mass spectrometry. Other types of chemical alterations include charge changes (e.g., due to deamidation), which can be evaluated, for example, by HPLC or ion exchange chromatography or icIEF.

[0099] As disclosed herein, stable aqueous liquid formulations of glucagon analogs or pharmaceutically acceptable salts or derivatives of the present invention typically comprise:

[0100] (a) Glucagon analogues or their pharmaceutically acceptable salts or derivatives, present at a concentration of about 0.5 mg / mL to about 10 mg / mL;

[0101] (b) TRIS, Bis-TRIS, ACES or MES, which are present as buffers at a concentration of about 25 mM to about 75 mM;

[0102] (c) Sodium chloride, which acts as a tension modifier and is present at a concentration of about 50 mM to about 200 mM; and

[0103] (d) pH from about 5.6 to about 7.0.

[0104] In some embodiments of formulations comprising glucagon analogs or their pharmaceutically acceptable salts and / or derivatives, the formulation comprises a glucagon analog, its pharmaceutically acceptable salt, or a derivative thereof present at a concentration of about 0.1 mg / mL to about 1.0 mg / mL, such as about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1.0 mg / mL.

[0105] For example, in some implementations, glucagon analogs are present at a concentration of about 1.0 mg / mL or about 4.0 mg / mL.

[0106] Preferably, in the stable aqueous liquid formulation of the present invention, TRIS, Bis-TRIS or ACES are present as buffers at a concentration of about 50 mM.

[0107] Preferably, in the stable aqueous liquid formulation of the present invention, sodium chloride is present as a tension modifier at a concentration of about 50 mM to about 600 mM, about 50 mM to about 500 mM, about 150 mM to about 200 mM, or about 60 mM to about 120 mM.

[0108] Preferably, in the stable aqueous liquid formulation of the present invention, the formulation has a pH of about 5.8 to 6.7, or about 6.0 to 7.0, and more preferably about 6.0 or about 6.5. In some embodiments disclosed herein, the single-dose (SD) formulation has a pH of about 6.5, while the formulation intended for multiple-dose use has a pH of about 6.0 or about 6.5.

[0109] Preferably, in the stable aqueous liquid formulation of the present invention, the formulation does not contain an ionization-stabilizing excipient selected from hydrochloric acid, nitric acid, sulfuric acid, or combinations thereof.

[0110] Preferably, in the stable aqueous liquid formulation of the present invention, the formulation is a ready-to-use formulation.

[0111] Preferably, in the stable aqueous liquid formulation of the present invention, the formulation is stable at 2 to 8°C for at least 6 months, at least 12 months, at least 18 months, or at least 24 months. Preferably, in the stable aqueous liquid formulation of the present invention, the glucagon analog in the formulation retains at least about 90% of its biological activity after being stored at 2 to 8°C for 18 months. Although storage at 2 to 8°C reflects storage under refrigerated conditions from production to final use, the liquid formulation of the present invention preferably exhibits long-term storage stability under environmental conditions, such as during the time between sale to the end user and application.

[0112] Preferably, the stable aqueous liquid formulation of the present invention is sterile.

[0113] Preferably, in the stable aqueous liquid formulation of the present invention, the formulation is administered to the subject by injection, for example, by subcutaneous injection.

[0114] Preferably, in the stable aqueous liquid formulation of the present invention, the buffer is TRIS or Bis-TRIS.

[0115] In some aspects, the stable aqueous liquid formulation of the present invention may further comprise a preservative. Preferably, the preservative is m-cresol, optionally at a concentration of about 1.0 mg / mL to about 5.0 mg / mL.

[0116] In one aspect, the present invention provides a stable aqueous liquid formulation wherein a glucagon analog is present at a concentration of about 0.75 mg / mL to about 1.25 mg / mL, a TRIS is present at a concentration of about 40 mM to about 60 mM, sodium chloride as a tonicotinic agent is present at a concentration of about 150 mM to about 200 mM, and the formulation has a pH of about 6.0 to about 7.0. In this aspect, preferably, the stable aqueous liquid formulation comprises a glucagon analog present at a concentration of about 1.0 mg / mL, a TRIS present at a concentration of about 50 mM, sodium chloride as a tonicotinic agent present at a concentration of about 175 mM, and the formulation has a pH of about 6.5. The formulation is generally intended as a single-dose formulation.

[0117] In another aspect, the present invention provides a stable aqueous liquid formulation wherein a glucagon analog is present at a concentration of about 3.0 to 5.0 mg / mL, a TRIS concentration of about 40 mM to about 60 mM, sodium chloride as a tonicotinic agent at a concentration of about 60 mM to about 120 mM, and m-cresol as a preservative at a concentration of about 3.0 mg / mL to about 4.0 mg / mL, and the formulation has a pH of about 6.0 to about 7.0. In this aspect, preferably, a glucagon analog is present at a concentration of about 4 mg / mL, a TRIS concentration of about 50 mM, sodium chloride as a tonicotinic agent at a concentration of about 90 mM, and m-cresol as a preservative at a concentration of about 3.0 to about 4.0 mg / mL, and the formulation has a pH of about 6.5. In this aspect, the formulation is generally intended as a multi-dose formulation and therefore includes a preservative. An example of a multi-dose formulation has a pH of about 6.5, 4.0 mg / mL of dasiglitin, TRIS present at a concentration of about 50 mM, sodium chloride present as a tension modifier at a concentration of about 90 mM, and m-cresol present as a preservative at a concentration of about 3.15 mg / mL.

[0118] In another aspect, the present invention provides a stable aqueous liquid formulation in which a glucagon analog or pharmaceutically acceptable salt and / or derivative thereof is present at a concentration of about 3.0 to 5.0 mg / mL, TRIS, Bis-TRIS, ACES or MES are present as a buffer at a concentration of about 25 mM to about 75 mM, and / or citrate, acetate or succinate are present as a buffer at a concentration of about 1 mM to about 30 mM, sodium chloride is present as a tension modifier at a concentration of about 50 mM to about 150 mM, m-cresol is present as a preservative at a concentration of about 3.0 mg / mL to about 4.0 mg / mL, and the formulation has a pH of about 6.0 to 7.0. In this respect, preferably, a glucagon analog or its pharmaceutically acceptable salt and / or derivative is present at a concentration of about 4 mg / mL, a TRIS, Bis-TRIS, ACES, or MES buffer is present at a concentration of about 50 mM, or a citrate buffer, acetate buffer, and / or succinate buffer is present at a concentration of about 15 mM, sodium chloride is present as a tension modifier at a concentration of about 90 mM, m-cresol is present as a preservative at a concentration of about 3.0 to about 4.0 mg / mL, and the formulation has a pH of about 6.5.

[0119] As used in this article, the term "stable formulation" refers to a formulation having enhanced physical stability, enhanced chemical stability, or enhanced physical and chemical stability.

[0120] As used herein, the term "physical stability" refers to a measure of the tendency of a peptide (e.g., the compounds of this invention) to form soluble or insoluble aggregates as a result of exposing a peptide (e.g., the compounds of this invention) to stress and / or interacting with unstable interfaces and surfaces (e.g., hydrophobic surfaces and interfaces). The physical stability of an aqueous peptide formulation can be evaluated by visual inspection and / or turbidity measurement after the formulation, filled into a suitable container (e.g., a box or vial), has been exposed to mechanical / physical stress (e.g., stirring) at different temperatures for varying periods of time. When a formulation exhibits visible turbidity, it can be classified as physically unstable in terms of peptide aggregation. Alternatively, the turbidity of the formulation can be evaluated by simple turbidity measurements known to those skilled in the art.

[0121] The physical stability of aqueous peptide formulations can also be evaluated using reagents that act as spectroscopic probes representing the conformational state of the peptide. The probes are preferably small molecules that preferentially bind to non-native conformational isomers of the peptide. An example of such a small molecule spectroscopic probe is thioflavin T, a fluorescent dye widely used for the detection of amyloid fibrils. In the presence of fibrils and possibly other peptide conformations, thioflavin T exhibits a new excitation maxima at approximately 450 nm and enhanced emission at approximately 482 nm when bound to the fibril-like form of the peptide. Unbound thioflavin T is substantially non-fluorescent at the wavelengths in question.

[0122] As used herein, the term "chemical stability" refers to peptide stability with respect to a covalent / chiral chemical change in the peptide structure that leads to the formation of chemically degraded products, which have potentially lower biopotency and / or potentially increased immunogenicity compared to the native peptide structure. A variety of chemically degraded products can form depending on the type and detailed properties of the native peptide and the environment in which it is exposed. In practice, as is known to those skilled in the art, the elimination of chemical degradation in peptide formulations is generally not entirely avoidable, and increased amounts of chemically degraded products are frequently observed during the storage and use of such formulations. Many peptides are susceptible to degradation processes in which the side-chain amide groups of glutamine or asparagine residues are hydrolyzed to form free carboxylic acids. Other degradation pathways involve the formation of high molecular weight conversion products, in which two or more peptide molecules become covalently bonded to each other through transamidation and / or disulfide interactions, resulting in the formation of covalently bonded oligomers and polymeric degradation products (see, for example, Stability of Protein Pharmaceuticals(Ahern, T.J. and Manning MC, Plenum Press, New York, 1992). Furthermore, hydrolysis of the peptide backbone leading to peptide fragments is well-known. Oxidation (e.g., oxidation of methionine residues) is another form of chemical degradation of peptides. The chemical stability of peptide formulations can be evaluated by measuring the amount of chemical degradation products at multiple time points after exposure to different environmental conditions (e.g., degradation product formation can generally be promoted by increasing temperature). The amount of each individual degradation product can be determined by separating the degradation products to generate a “degradation profile.” Separation is typically performed using various chromatographic techniques (e.g., SEC-HPLC and / or RP-HPLC) based on molecular size and / or charge. Oligomers can be covalent or non-covalent. Covalent oligomers are two or more molecules linked by covalent chemical bonds, and therefore the bonds are generally irreversible. Conversely, in non-covalent oligomers, one or more molecules are linked together by non-covalent chemical bonds (e.g., ionic interactions, hydrophobic interactions, etc.). While this can be reversible in some cases, the interactions are often so strong that they are practically irreversible, and can render active pharmaceutical substances pharmaceutically unusable, such as covalent oligomers.

[0123] At low pH, the chemical instability of glucagon itself is primarily attributed to the isomerization and cleavage of aspartic acid residues, the deamidation of glutamine residues, and the oxidation of methionine. Generally, the deamidation of Asn and Gln occurs at high pH via a cyclic imide ring intermediate, exhibiting a significant rate at physiological pH of approximately 7.4. This cyclic imide ring intermediate can open to form L-Asp and L-isoAsp or L-Glu and L-isoGlu, respectively. The cyclic imide ring intermediate can also lead to the formation of small amounts of the corresponding D-isomers, indicating a slow racemic reaction of the cyclic imide.

[0124] At pH values ​​below physiological pH, the rate of deamidation of Asn and Gln decreases, but as pH decreases, the rate of cyclic imide formation from Asp and Glu, and consequently the rate of isomerization, increases. Cyclic imide formation is highest between pH 4 and pH 6. The formation of cyclic imide intermediates can also lead to peptide sequence cleavage.

[0125] As stated above, "stable formulation" can therefore refer to a formulation with improved physical stability, or improved chemical stability, or improved physical and chemical stability. Generally, a formulation should be stable during use and storage (under recommended use and storage conditions), at least until the specified expiration date.

[0126] In some embodiments of the liquid pharmaceutical formulations of the present invention, the formulation is stable under conditions of at least 2 weeks of use and at least 6 months of storage. In other embodiments, the formulation is stable under conditions of at least 2 weeks of use and at least 1 year of storage. In still other embodiments, the formulation is stable under conditions of at least 2 weeks of use and at least 2 years of storage. In still other embodiments, the formulation is stable under conditions of at least 4 weeks of use and at least 2 years of storage, or even under conditions of at least 4 weeks of use and more than 3 years of storage. Some particularly useful embodiments of such pharmaceutical formulations of the present invention are stable under conditions of at least 6 weeks of use and at least 3 years of storage. In this respect, for the purposes of this paragraph, the term "use" means removing the pharmaceutical formulation from storage and subjecting it to different environmental conditions (light, darkness, temperature, agitation, etc.) for the purpose of using the formulation for therapeutic purposes, while for the purposes of this paragraph, the term "storage" means storing the formulation in a refrigerator or freezer under non-stirred conditions at a temperature not exceeding about 5°C. Those skilled in the art will understand the typical range of use and storage conditions that these pharmaceutical formulations can withstand.

[0127] As described herein, the stable aqueous liquid formulation of the glucagon analogue of the present invention comprises a buffer and a tonic modifier that provide pH. According to conventional pharmaceutical practice, the formulation of the present invention is sterile and / or free of reducing agents. The liquid formulation of the present invention is an aqueous liquid formulation.

[0128] As used herein, the term "buffer" refers to a pharmaceutically acceptable reagent that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and are available in the literature. In some preferred embodiments, the buffer is selected from TIS buffers, Bis-TRIS buffers, ACES buffers, or MES buffers and / or citrate buffers, acetate buffers, or succinate buffers, because these buffers provide a stable formulation in which the glucagon analog dissolves without becoming viscous, cloudy, or causing the peptide drug to precipitate. In some preferred embodiments, the buffer is TIS or Bis-TRIS. Typically, the buffer will be present at a concentration of about 25 mM to about 75 mM. In some embodiments, the buffer will be present at a concentration of about 50 mM. In some embodiments, the TIS, Bis-TRIS, ACES, or MES buffer provides direct chemical stability to the glucagon analog independently of the pH of the formulation. In other embodiments, as determined under the same test conditions relative to phosphate buffers and / or histidine buffers, TIS, Bis-TRIS, ACES, or MES buffers provide improved direct chemical stability. In other embodiments, particularly those related to multi-dose formulations containing preservatives such as metallocresol, the buffer further includes the use of citrate, acetate, or succinate at a concentration of about 1 mM to about 30 mM. In some embodiments, formulations in which the TIS, BIS-TRIS, ACES, or MES buffers evaluated under the same test conditions are replaced with phosphate buffers and / or histidine buffers of the same concentration and pH, provide improved chemical stability. For example, the formulation may have a degradation profile after being stored at 25°C for 52 weeks, after which, after being stored at 25°C for 52 weeks, the formulation contains one or more of the following: less than 5% Pyro-Glu 4-29, less than 7% Trp / Tyr oxidation, less than 4% kynurenine, less than 5% F-4-29+F5-29 and / or less than 2% F3.29, wherein all percentages are determined by HPLC.

[0129] As used herein, the term "tonometer" refers to a pharmaceutically acceptable tonic agent used to adjust the tension of a formulation. The formulations of the present invention are preferably isotonic, i.e., they have substantially the same osmotic pressure as human serum. The tonic agent used in the formulation is preferably sodium chloride. The concentration of the tonic agent will depend on the concentrations of other components of the formulation, especially if the formulation is intended to be isotonic. Generally, sodium chloride will be used as a tonic agent at a concentration of about 50 mM to 600 mM. In some embodiments, the concentration of sodium chloride present as a tonic agent in the formulations of the present invention can be about 10 mM to about 150 mM, for example, about 20 mM to about 130 mM, about 50 mM to about 125 mM, about 75 mM to about 120 mM, about 90 mM to about 115 mM, or about 110 mM. In some embodiments, the concentration of sodium chloride present as a tonic agent in the formulations of the present invention can be about 150 mM to about 200 mM, optionally about 175 mM. In a preferred embodiment, sodium chloride may be present as a tension modifier in such a formulation comprising a preservative at a concentration of about 50 mM to about 150 mM, and most preferably at a concentration of about 90 mM, about 75 mM, or about 125 mM.

[0130] Typically, the components and amounts of the liquid formulation of the present invention are selected to provide a formulation having a pH of about 5.6 or a pH of about 6.0 to about 7.0. In a preferred embodiment, the formulation may have a pH of about 6.2 to about 6.8. More preferably, the formulation may have a pH of about 6.3 to about 6.7. More preferably, the formulation may have a pH of about 6.4 to about 6.6. Alternatively or supplementally, for both single-dose and multi-dose formulations, the formulation may have a pH of about 6.5.

[0131] In some embodiments, the formulations of the present invention further comprise a preservative. In some preferred embodiments, the preservative is m-cresol, present at a concentration of about 1.0 mg / mL to about 5.0 mg / mL, more preferably at a concentration of about 2.0 mg / mL to about 4.0 mg / mL, and most preferably at a concentration of about 3.0 mg / mL to about 4.0 mg / mL. The amount of preservative depends on the need to effectively kill a range of bacterial types, such as required for regulatory approval.

[0132] The formulations of the present invention have degradation profiles determined by chromatographic separation (e.g., by size-exclusion chromatography (SEC) and / or reverse-phase chromatography (RPC)). In particular, a further finding of the experiments described herein is that, after storage at 25°C for 52 weeks, the degradation product profiles of buffers producing stable formulations according to the present invention are very similar, while the degradation product profiles of buffers not producing stable formulations are different. In the table below, the percentages of degradation products were measured using HPLC.

[0133] Pyro-Glu (4 to 29) involves the reaction of the main-chain nitrogen of Gln at position 4 with a side-chain amide to form an internal lactam ring. Subsequently, the amide bond between Ser at position 3 and the lactamed Glu moiety is hydrolyzed to produce a degradation product in which the first three amino acids have been cleaved and a Pyro-Glu moiety is present at the N-terminus. Oxidation of Tyr / Trp results in the incorporation of hydroxyl groups into the aromatic rings of Tyr and Trp. Kynurenine is a known oxidation product of Trp residues. The addition of succinate involves the addition of dasigrease to the double bond in the maleate buffer. FX 1 -X 2 It contains amino acid X 1 To X 2 A fragment.

[0134]

[0135] In summary, compared with phosphate, histidine, and maleic acid, TIS, Bis-TRIS, MES, and ACES protect daczyme from hydrolysis and oxidation, and there is no significant interaction between TIS, Bis-TRIS, MES, and daczyme. A small amount (0.9% after 52 weeks at 25°C) of daczyme condensed with ACES (+165 Da) is formed within 52 weeks.

[0136] Therefore, in some aspects, as determined by HPLC, the formulation of the present invention will have less than 5% Pyro-Glu 4-29 after being stored at 25°C for 52 weeks, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1%, and most preferably less than 0.5%.

[0137] Therefore, in some cases, as determined by HPLC, the formulations of the present invention will have less than 7% Trp / Tyr oxidation after being stored at 25°C for 52 weeks, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1%, and most preferably less than 0.5%.

[0138] Therefore, in some cases, as determined by HPLC, the formulation of the present invention will have less than 4% kynurenine after being stored at 25°C for 52 weeks; more preferably less than 3%, more preferably less than 2%, more preferably less than 1%, and most preferably less than 0.5%.

[0139] Therefore, in some cases, as determined by HPLC, the formulation of the present invention will have less than 5% F-4-29+F5-29 after being stored at 25°C for 52 weeks, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1%, and most preferably less than 0.5%.

[0140] Therefore, in some cases, as determined by HPLC, the formulations of the present invention will have less than 2% F3.29 after being stored at 25°C for 52 weeks, more preferably less than 1% and most preferably less than 0.5%.

[0141] In one aspect, the stable aqueous liquid formulation comprises a glucagon analog at a concentration of about 0.75 mg / mL to about 1.25 mg / mL, a TRIS at a concentration of about 40 mM to about 60 mM, sodium chloride as a tonic modulator at a concentration of about 150 mM to about 200 mM, and the formulation has a pH of about 6.2 to about 6.8.

[0142] In another aspect, the stable aqueous liquid formulation comprises a glucagon analog at a concentration of about 3.0 to 5.0 mg / mL, a TRIS at a concentration of about 40 mM to about 60 mM, sodium chloride as a tension modifier at a concentration of about 60 mM to about 120 mM, m-cresol as a preservative at a concentration of about 3.0 mg / mL to about 4.0 mg / mL, and the formulation has a pH of about 6.0.

[0143] In some embodiments, stable liquid formulations of glucagon analogs are ready-to-use formulations. As used herein, the term "ready-to-use" means a formulation that does not require preparation or dilution with a prescribed amount of diluent (e.g., water for injection or other suitable diluent) prior to use via the specified route of administration.

[0144] Medical conditions

[0145] The glucagon analog pharmaceutical formulations of the present invention can be used to treat or prevent a variety of conditions or disorders. Optionally, the formulations may be used in combination with one or more other therapeutically active substances. Therefore, related therapeutic uses include: treating or preventing hypoglycemia (including, but not limited to, acute hypoglycemia, chronic hypoglycemia, or severe hypoglycemia), type 2 diabetes (including disease progression of type 2 diabetes), impaired glucose tolerance, type 1 diabetes, obesity (including diseases or conditions associated with overweight or obesity), coronary artery disease, atherosclerosis, hypertension, dyslipidemia, hepatic steatosis, beta-blocker toxicity, insulinoma, and von Gilke's disease; preventing overweight; reducing weight; reducing food intake; increasing energy expenditure; delaying the progression from impaired glucose tolerance (IGT) to type 2 diabetes; delaying the progression from type 2 diabetes to insulin-dependent diabetes; regulating appetite or inducing satiety (including treating bulimia and binge-eating); and preventing weight regain after successful weight loss. As a general principle, the pharmaceutical formulations of the present invention can be used to control blood glucose levels.

[0146] The various forms of hypoglycemia that can be treated or prevented according to the present invention include diabetic hypoglycemia, acute insulin-induced hypoglycemia, hyperinsulinic hypoglycemia, severe hypoglycemia, nondiabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, congenital hyperinsulinic hypoglycemia, alcohol-induced hypoglycemia, hypoglycemia induced by gastric bypass surgery (including but not limited to postprandial hypoglycemia after Roux-en-Y gastric bypass surgery), hypoglycemia after weight loss, or hypoglycemia occurring during pregnancy.

[0147] In another embodiment, the liquid formulation comprising the glucagon analogue of the present invention is included in a delivery device. In some embodiments, the delivery device is a pre-filled syringe, syringe device, injection pen, adjustable-dose autoinjector, disposable autoinjector, wearable syringe, or infusion pump.

[0148] The formulations of the present invention can be administered in various dosage forms, such as solutions, suspensions, or emulsions, and can be used to formulate drug delivery systems for controlled release, sustained release, extended release, delayed release, or slow release. More specifically, but not exclusively, the pharmaceutical formulations of the present invention can be used in conjunction with parenteral controlled release and sustained release systems known to those skilled in the art. In this regard, reference may be made generally to Handbook of Pharmaceutical Controlled Release (Wise, DL, ed., Marcel Dekker, New York, 2000) and Drugs and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, EJ, ed., Marcel Dekker, New York, 2000).

[0149] Parenteral administration of the liquid pharmaceutical formulation of the present invention can be performed, for example, by subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe (e.g., a pen-like syringe and / or a pre-filled syringe). Parenteral administration of the liquid pharmaceutical formulation of the present invention can also be performed, for example, by subcutaneous, intramuscular, intraperitoneal, or intravenous injection using an autoinjector (e.g., an autoinjector containing a syringe). The autoinjector can be for single or multiple use and can provide (and / or be configured to provide) one or more doses of the liquid pharmaceutical formulation. In some embodiments, parenteral administration is performed by injecting the formulation of the present disclosure into the patient's lower abdomen, buttocks, thigh, or upper arm (including, but not limited to, the outer upper arm). Alternatively, parenteral administration can be performed using an infusion pump, for example, in the form of a device or system carried by the subject or patient and comprising a reservoir containing the liquid formulation of the present invention and an infusion pump for delivering / administering the formulation to the subject or patient, or in the form of a correspondingly small device suitable for implantation into the subject or patient.

[0150] In some cases, a patient may be treated by administering a single dose of the formulation or by administering multiple doses. In some cases, a first dose of the formulation may be administered to the patient, followed by a second dose. In some cases, a first dose of the formulation may be administered to the patient, followed by a second dose, and then one or more additional doses of the formulation (e.g., a third dose, which may also be a fourth dose or additional dose). Example

[0151] The following embodiments are provided to illustrate preferred aspects of the invention and are not intended to limit the scope of the invention.

[0152] Example 1

[0153] Materials and methods

[0154] Synthesis of Darcy glucagon

[0155] Darcy glucagon is synthesized by Bachem AG. Darcy glucagon formulations are prepared and analyzed by Zealand Pharma, A / S, Denmark. Alternatively, glucagon analogs may be synthesized as described in WO 2014 / 016300 (Zealand Pharma A / S), the contents of which are incorporated herein by reference in their entirety.

[0156] Chemical stability test

[0157] Dash glucagon was dissolved in MilliQ water to achieve a stock concentration of 25 mg / mL. The formulation was prepared by aliquoting the peptide stock solution (937 μL) into tubes, followed by adding the buffer component stock solution, and then adding MilliQ water to 90% of the volume. The pH was measured and adjusted as needed, then MilliQ water was added to obtain the final volume. All formulations were then filtered using a Minisart high-flow 0.2 μm filter. The filtered solution was bubbled with nitrogen and aliquoted into vials, each containing 1.0 mL. The vials were inverted (crimped) and placed in a stabilization chamber.

[0158] The formulation was tested under accelerated conditions at 5°C and 25°C at weeks 0, 9, 13, 26, 34, 39, and 52. The purity of the formulation was determined by HPLC, and the degradation product profile was determined by LC-MS analysis.

[0159] The compositions of the single-dose (SD) formulations of this invention are shown in Tables 1 and 5. All formulations contain mg / mL daczyme glucagon and are formulated at pH 6.5. Formulations 1 to 3 investigated the effect of different amounts of sodium chloride in the formulations, while formulations 4 to 10 investigated the effect of the buffers used in the formulation preparation. Formulation 11 is a control formulation in Milli-Q water, and formulation 12 was designed as a multi-dose formulation and is included in this study by comparison with the single-dose formulations. The composition of formulation 12 (MD formulation) is pH 6.5, 4.0 mg / mL glucagon analog, 50 mM TRIS, 90 mM NaCl, and 3.15 mg / mL m-cresol.

[0160] The compositions of the multi-dose formulations of the present invention are shown in Tables 2 to 4 and Table 6. All formulations comprise m-cresol and 4 mg / mL dasigreatin and are prepared at pH 6.0 or pH 6.5. Tables 2a, 3a, and 4a report the results of HPLC determinations. Tables 2b, 3b, and 4b report the results of SEC determinations.

[0161] The formulations in Table 2 investigated the effects of different buffers (Tris, Bis-TRIS, MES, sodium citrate, sodium acetate, and sodium succinate) on formulation stability at pH 6.0 and pH 6.5 and at two concentrations of sodium chloride (90 mM and 125 mM) as isotonic regulators.

[0162] The formulations listed in Tables 3, 4, and 6 are similar to those listed in Table 2, and the effects of combinations of buffers on formulation stability were investigated at pH 6.0 and pH 6.5 and with a concentration of sodium chloride (75 mM) as an isotonic regulator.

[0163] HPLC

[0164] The purity of dacey glucagon samples was determined by HPLC. Samples were analyzed using a Dionex Ultimate 3000 HPLC system. Chromeleon was used as the analytical software. Dacey glucagon was separated from degradation products using a Kinetex C8 column (150 mm × 4.6 mm, 2.6 μm). The mobile phase consisted of A: 0.45% TFA (v / v) in MilliQ water (MQW) and B: 0.45% TFA (v / v) in MeCN / MQW (90:10). The gradient was 35% to 38% B over 40 minutes at a flow rate of 0.5 mL / min. The column oven temperature was set to 30 °C, the detector wavelength to 220 nm, and 2 μL was injected.

[0165] SEC

[0166] Size exclusion chromatography (SEC) experiments were performed on a Dionex Ultimate 3000 HPLC system (Thermo Fisher) using isocranial elution at a flow rate of 0.5 mL / min to determine the presence of oligomers in the formulation. The mobile phase consisted of 0.1% TFA and 45% acetonitrile in MQW. The detection wavelength was 215 nm. The injected volume was 2 μg of peptide. The column used for SEC analysis was a TSKgel SuperSW2000 (TOSOH Corporation), 4 μm, 30 × 4.6 mm, with a column temperature of 25 °C. The run time was 12 minutes.

[0167] result

[0168] After 52 weeks of accelerated storage at 25°C, the following results were obtained regarding the purity % of the remaining dasiglitin in the formulation.

[0169] Stability studies showed that, relative to other buffers and the control (Milli-Q water), all TRIS, Bis-TRIS, ACES, and MES buffers produced acceptable levels of stability after 52 weeks of storage at 25°C, with 80% or more of d'aceglucosamine remaining intact despite accelerated stress conditions used in the experiments, taking into account the accelerated storage conditions used in the experiments.

[0170] For multi-dose formulations containing m-cresol, TRIS, Bis-TRIS, ACES, or MES, as well as citrate buffers, acetate buffers, or succinate buffers, good levels of stability are provided for the formulation, as assessed by HPLC and SEC.

[0171] Table 5

[0172]

[0173] Table 5b

[0174]

[0175]

[0176] Given the challenges reported in the prior art regarding the stability of glucagon formulations containing water, these observations are surprising.

[0177] The results were also unexpected, because although TIS and Bis-TRIS have similar chemical structures, ACES and MES have different structures.

[0178] The experiment also showed that the buffers commonly used in liquid peptide formulations, namely phosphate, histidine, and maleic acid, cannot provide formulations that can maintain the integrity of dasiglitin for long-term storage (75%, 68%, and 57%, respectively).

[0179] The experiment also showed that an appropriate amount of NaCl can be included as a tension modifier without affecting the stability provided by having four buffers.

[0180] Data showed that the glucagon loss trend was similar between single-dose (SD) formulations (0.31% weekly, formulation 1) and multiple-dose (MS) formulations (0.32% weekly, formulation 12).

[0181] Experiments also showed that preservatives (m-cresol) can be included in formulations while maintaining storage stability, allowing for the development of multi-dose formulations and formulations used in conjunction with delivery devices.

[0182] Example 2 - Darcyline improved postprandial hypoglycemia after Roux-en-Y gastric bypass surgery

[0183] Postprandial hypoglycemia is a common and debilitating complication following Roux-en-Y gastric bypass (RYGB), for which no effective treatment exists. In a proof-of-concept study, we investigated the effects of daczyme glucagon (a novel, stable glucagon analog) on ​​postprandial hypoglycemia following RYGB. The primary objective of this study was to examine the effects of two doses of daczyme glucagon on postprandial minimum blood glucose concentration (PG) and the time taken to reach hypoglycemia (<3.9 mmol / L) in individuals who underwent RYGB with confirmed postprandial hypoglycemia.

[0184] Materials and methods

[0185] Ten individuals who underwent RYGB surgery (2 men, 8 women; BMI 34.6 (range 21.8 to 39.0) kg / m²) with confirmed symptomatic postprandial hypoglycemia (PG < 3.5 mmol / L as verified by 6 days of continuous glucose monitoring) were included. 2 Age 46 (29 to 67 years); HbA1c 1c 32 (29 to 42) mmol / mol; time since surgery was 9.3 (6 to 11.5) years; weight loss since surgery was 53.9 (25.1 to 92.0) kg. A double-blind, randomized, crossover study was completed, consisting of three separate treatment days, each including a standardized liquid-mixed dietary test (25 kJ / kg body weight; 50% carbohydrates, 35% fat, and 15% protein). After the postprandial PG peak (approximately 10 minutes before the expected time point for PG to return to fasting levels using a subject-specific linear regression model), placebo, 80 or 200 μg of dasigreatin (D) was administered. 80μg and D 200μg Subcutaneous injection of [the drug / method] was administered. Blood samples were taken and hypoglycemic symptoms were assessed at fixed time intervals using the Edinburgh Hypoglycaemia Symptom Scale. Data were analyzed using a linear mixture model and a Tukey-corrected model for multiple comparisons.

[0186] result

[0187] Compared to placebo, after drug administration, D80μg and D 200μg Both treatments significantly improved the lowest PG (placebo: 3.0 ± 0.2 mmol / L; D) level. 80μg 3.9 ± 0.3 mmol / L; D 200μg : 4.5 ± 0.2 mmol / L; p = 0.002 and p = 0.0002) and PG increment AUC (iAUC) 70至240分钟 (Placebo: 752 ± 19 minutes × mmol / L; D) 80μg : 917±22 minutes × mmol / L; D 200μg The results showed that the effective doses were 992 ± 28 minutes × mmol / L; p < 0.0001 and p < 0.0001. Furthermore, compared to placebo (< 3.9 mmol / L), both doses reduced the time spent in hypoglycemia (placebo: 62.0 ± 8 minutes; p < 0.0001 and p < 0.0001). 80μg : 27.5±12 minutes; D 200μg The time to blood glucose level was 14.0 ± 9 minutes; p = 0.05 and p = 0.003. Five participants experienced grade 2 hypoglycemia (<3.1 mmol / L) after placebo administration, while those who received placebo experienced hypoglycemia after placebo administration experienced hypoglycemia after placebo administration. 200μg None of the participants subsequently experienced this condition (time spent in grade 2 hypoglycemia (n=5): placebo: 31.0 ± 5 minutes; D 80μg 7±5 minutes; D 200μg (0.0 ± 0 minutes; p > 0.05 and p = 0.01). Hypoglycemic symptoms did not change significantly between the three study days.

[0188] in conclusion

[0189] A single dose of dasiglitin effectively improves postprandial hypoglycemia and represents a promising new treatment option for managing postprandial hypoglycemia after RYGB.

[0190] References

[0191] All references mentioned in this article are explicitly included in their entirety through citation.

[0192] WO 2014 / 016300(Zealand Pharma A / S)

[0193] et al, Diabetes Care, 2017(https: / / doi.org / 10.2337 / dc17-1402).

[0194] WO 2017 / 053822 (Xeris Pharmaceuticals, Inc.)

[0195] WO 2014 / 124151 (Xeris Pharmaceuticals, Inc.)

[0196] WO 2016 / 133863(Eli Lilly and Company)

[0197] WO 2019 / 110838(Adocia)

[0198] WO 2008 / 084237(Arecor Limited)

[0199] Table 1

[0200]

[0201] Formulation 12 (MD formulation) is pH 6.5, 4.0 mg / mL dasiglitin, 50 mM TRIS, 90 mM NaCl and 3.15 mg / mL m-cresol.

[0202] Table 2

[0203]

[0204] Table 2a

[0205]

[0206]

[0207] *Precipitation at 5℃

[0208] Table 2b

[0209]

[0210]

[0211] *Precipitation at 5℃

[0212] Table 2c

[0213]

[0214]

[0215] Table 2d

[0216]

[0217]

[0218] Table 3

[0219]

[0220] Table 3a

[0221]

[0222]

[0223] *Precipitation at 5℃

[0224] Table 3b

[0225]

[0226]

[0227] *Precipitation at 5℃

[0228] Table 3c

[0229]

[0230]

[0231] Table 3d

[0232]

[0233]

[0234] Table 4

[0235]

[0236] Table 4a

[0237]

[0238]

[0239] *Precipitation at 5℃

[0240] Table 4b

[0241]

[0242] *Precipitation at 5℃

[0243] Compared with the control formulation, the DLS and absorbance of all acetate-containing formulations were acceptable.

[0244] Table 4c

[0245]

[0246]

[0247] Table 4d

[0248]

[0249] Table 6

[0250]

[0251] Table 6a

[0252]

[0253] Table 6b

[0254]

[0255] sequence list <110> ZEALAND PHARMA A / S <120> Liquid formulation of glucagon analogues <130> 007371412 <140> 20163408.6 <141> 2020-03-16 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_FEATURE <222> (16)..(16) <223> Xaa = Aib <400> 1 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Ala Arg Ala Glu Glu Phe Val Lys Trp Leu Glu Ser Thr 20 25

Claims

1. A stable aqueous liquid pharmaceutical formulation comprising a glucagon analogue or a pharmaceutically acceptable salt thereof, said glucagon analogue being: Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH wherein the formulation comprises: (a) the glucagon analogue present at a concentration of 0.5 mg / mL to 10 mg / mL; (b) TRIS present as a buffering agent at a concentration of 25 mM to 75 mM; (c) sodium chloride present as a tonicity adjusting agent and at a concentration of 50 mM to 600 mM; and (d) a pH of 5.6 to 7.

0.

2. The stable aqueous liquid pharmaceutical formulation of claim 1, wherein the TRIS buffer directly chemically stabilizes the glucagon analogue independent of the pH of the formulation provided by the buffer.

3. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the formulation provides improved chemical stability relative to a formulation in which the TRIS buffer is replaced with a phosphate buffer and / or a histidine buffer at the same concentration and pH evaluated under the same testing conditions.

4. The stable aqueous liquid pharmaceutical formulation of the preceding claim 1 or claim 2, wherein the formulation has a degradation profile after 52 weeks of storage at 25 °C after which the formulation comprises one or more of the following after 52 weeks of storage at 25 °C: less than 5% Pyro-Glu 4-29, less than 7% Trp / Tyr oxidation, less than 4% kynurenine, less than 5% F-4-29+F5-29, and / or less than 2% F3.29, wherein all percentages are determined by HPLC.

5. The stable aqueous liquid pharmaceutical formulation of the preceding claim 1 or claim 2, wherein the formulation has a degradation profile in which succinic acid addition of maleic acid does not occur to the glucagon analogue.

6. The stable aqueous liquid pharmaceutical formulation of the preceding claim 1 or claim 2, wherein the formulation comprises less than 5% of an aprotic polar solvent by volume.

7. The stable aqueous liquid pharmaceutical formulation of the preceding claim 1 or claim 2, wherein the formulation comprises less than 5% of dimethyl sulfoxide (DMSO) by volume.

8. The stable aqueous liquid pharmaceutical formulation of the preceding claim 1 or claim 2, wherein water is the only solvent used to prepare the aqueous liquid pharmaceutical formulation.

9. The stable aqueous liquid pharmaceutical formulation of the preceding claim 1 or claim 2, wherein the glucagon analogue or a pharmaceutically acceptable salt thereof is present at a concentration of 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 1.0 mg / mL, or 4.0 mg / mL.

10. The stable aqueous liquid pharmaceutical formulation of the preceding claim 1 or claim 2, wherein the TRIS buffer is present as a buffering agent at a concentration of 50 mM. ​ ​ ​ ​ ​ ​ 11. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the sodium chloride is present as a tonicity adjusting agent at a concentration of 150 mM to 200 mM or at a concentration of 50 mM to 150 mM.

12. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the formulation has a pH of 5.8 to 6.

7.

13. The stable aqueous liquid pharmaceutical formulation of claim 12, wherein the formulation has a pH of 6.

5.

14. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the formulation does not comprise an ionizing stabilizing excipient selected from the group consisting of hydrochloric acid, nitric acid, sulfuric acid, or a combination thereof.

15. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the formulation is a ready-to-use formulation.

16. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the formulation is stable at 2 to 8 °C for at least 6 months.

17. The stable aqueous liquid pharmaceutical formulation of claim 16, wherein the formulation is stable at 2 to 8 °C for at least 12 months.

18. The stable aqueous liquid pharmaceutical formulation of claim 16, wherein the formulation is stable at 2 to 8 °C for at least 18 months.

19. The stable aqueous liquid pharmaceutical formulation of claim 16, wherein the formulation is stable at 2 to 8 °C for at least 24 months.

20. The stable aqueous liquid pharmaceutical formulation of claim 16, wherein the glucagon analog in the formulation retains at least 90% of its biological activity after 18 months of storage at 2 to 8 °C.

21. The stable aqueous liquid pharmaceutical formulation of claim 16, wherein the glucagon analog in the formulation retains at least 80% of its biological activity after 18 months of storage at 2 to 8 °C.

22. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the formulation is sterile and / or free of a reducing agent.

23. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the formulation is formulated for administration to a subject by injection.

24. The stable aqueous liquid pharmaceutical formulation of claim 23, wherein the injection is a subcutaneous injection.

25. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, further comprising a preservative.

26. The stable aqueous liquid pharmaceutical formulation of claim 25, wherein the preservative is meta-cresol.

27. The stable aqueous liquid pharmaceutical formulation of claim 26, wherein the preservative is meta-cresol at a concentration of 1.0 mg / mL to 5.0 mg / mL.

28. The stable aqueous liquid pharmaceutical formulation of claim 1 or claim 2, wherein the glucagon analogue or pharmaceutically acceptable salt thereof is present at a concentration of 0.75 mg / ml to 1.25 mg / mL, the TRIS buffer is present at a concentration of 40 mM to 60 mM, sodium chloride is present as a tonicity adjusting agent at a concentration of 150 mM to 200 mM, and the formulation has a pH of 6.0 to 6.

8.

29. The stable aqueous liquid pharmaceutical formulation of claim 28, wherein the glucagon analogue or pharmaceutically acceptable salt thereof is present at a concentration of 1.0 mg / mL, TRIS is present at a concentration of 50 mM, sodium chloride is present as a tonicity adjusting agent at a concentration of 175 mM, and the formulation has a pH of 6.

5.

30. The stable aqueous liquid pharmaceutical formulation of claim 26, wherein the glucagon analogue or pharmaceutically acceptable salt thereof is present at a concentration of 3.0 to 5.0 mg / mL, the TRIS buffer is present at a concentration of 40 mM to 60 mM, sodium chloride is present as a tonicity adjusting agent at a concentration of 50 mM to 150 mM, m-cresol is present as a preservative at a concentration of 3.0 mg / mL to 4.0 mg / mL, and the formulation has a pH of 6.0 to 7.

0.

31. The stable aqueous liquid pharmaceutical formulation of claim 30, wherein the glucagon analogue or pharmaceutically acceptable salt thereof is present at a concentration of 4 mg / mL, TRIS is present at a concentration of 50 mM, sodium chloride is present as a tonicity adjusting agent at a concentration of 90 mM, m-cresol is present as a preservative at a concentration of 3.0 to 4.0 mg / mL, and the formulation has a pH of 6.

5.

32. The stable aqueous liquid pharmaceutical formulation of claim 26, wherein the glucagon analogue or pharmaceutically acceptable salt thereof is present at a concentration of 3.0 to 5.0 mg / mL, TRIS is present as a buffer at a concentration of 25 mM to 75 mM, sodium chloride is present as a tonicity adjusting agent at a concentration of 50 mM to 150 mM, m-cresol is present as a preservative at a concentration of 3.0 mg / mL to 4.0 mg / mL, and the formulation has a pH of 6.0 to 7.

0.

33. The stable aqueous liquid pharmaceutical formulation of claim 32, wherein the glucagon analogue or pharmaceutically acceptable salt thereof is present at a concentration of 4 mg / mL, TRIS buffer is present at a concentration of 50 mM, sodium chloride is present as a tonicity adjusting agent at a concentration of 90 mM, m-cresol is present as a preservative at a concentration of 3.0 to 4.0 mg / mL, and the formulation has a pH of 6.

5.

34. A delivery device comprising the stable aqueous liquid pharmaceutical formulation of any one of claims 1 to 33.

35. The delivery device of claim 34, wherein the delivery device is a syringe device.

36. The delivery device of claim 34, wherein the delivery device is a pre-filled syringe, an adjustable dose auto-injector, or an infusion pump.

37. The delivery device of claim 34, wherein the delivery device is an injection pen, a disposable auto-injector, or a wearable injector.

38. The stable aqueous liquid pharmaceutical formulation of claim 1 for use in therapy.

39. The stable aqueous liquid pharmaceutical formulation of claim 1 for use in the treatment of hypoglycemia.

40. The stable aqueous liquid pharmaceutical formulation for use in a method of treatment of claim 39, wherein the hypoglycemia includes, but is not limited to, severe hypoglycemia, acute hypoglycemia, chronic hypoglycemia.

41. The stable aqueous liquid pharmaceutical formulation for use in a method of treatment of claim 39, wherein the hypoglycemia is selected from the group consisting of: diabetic hypoglycemia, acute insulin-induced hypoglycemia, severe hypoglycemia, non-diabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, alcohol-induced hypoglycemia, gastric bypass-induced hypoglycemia, or hypoglycemia occurring during pregnancy.

42. The stable aqueous liquid pharmaceutical formulation for use in a method of treatment of claim 41, wherein the hypoglycemia is postprandial hypoglycemia following Roux-en-Y gastric bypass surgery.

43. The stable aqueous liquid pharmaceutical formulation for use in a method of treatment of any one of claims 38 to 42, for use in the treatment of a human patient.

44. Use of the formulation of any one of claims 1 to 33 in the manufacture of a medicament for the treatment of a patient.

45. Use of the formulation of any one of claims 1 to 33 in the manufacture of a medicament for the treatment of a disease or condition in a patient, wherein the disease or condition is hypoglycemia.

46. The use of claim 45, wherein the hypoglycemia includes, but is not limited to, severe hypoglycemia, acute hypoglycemia, chronic hypoglycemia.

47. The use of claim 45, wherein the hypoglycemia is selected from the group consisting of: diabetic hypoglycemia, acute insulin-induced hypoglycemia, severe hypoglycemia, non-diabetic hypoglycemia, reactive hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, alcohol-induced hypoglycemia, gastric bypass-induced hypoglycemia, or hypoglycemia occurring during pregnancy.

48. The use of claim 47, wherein the hypoglycemia is postprandial hypoglycemia following Roux-en-Y gastric bypass surgery.

49. The use of any one of claims 44 to 48, wherein the patient is a human.

50. A method for producing a stable aqueous liquid pharmaceutical formulation comprising a glucagon analogue or a pharmaceutically acceptable salt thereof, the glucagon analogue being: Hy-HSQGTFTSDYSKYLD-Aib-ARAEEFVKWLEST-OH. ​ The method comprises formulating the following to produce the stable aqueous liquid pharmaceutical formulation: (a) the glucagon analogue or a pharmaceutically acceptable salt thereof, present at a concentration of 0.5 mg / mL to 10 mg / mL; (b) TRIS, present as a buffering agent at a concentration of 25 mM to 75 mM; (c) sodium chloride, present as a tonicity adjusting agent and at a concentration of 50 mM to 600 mM; (d) a pH of 6.0 to 7.0; and optionally (e) m-cresol at a concentration of 1.0 mg / mL to 5.0 mg / mL.

51. A formulation produced by the method of claim 50, wherein the formulation is a stable aqueous liquid pharmaceutical formulation of any one of claims 1 to 33.

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