Oxyntomodulin peptide analogue preparations

Abstract

The present invention relates to GLP-1 and / or glucagon receptor agonists (e.g., oxyntomodulin peptide analogs), pharmaceutically acceptable salts thereof, formulations containing same, and their use for treating diabetes and / or obesity or related diseases or disorders.

Description

[Technical Field]

[0001] The present invention relates to GLP-1 and / or glucagon receptor agonists (e.g., oxyntomodulin peptide analogs), pharmaceutically acceptable salts thereof, formulations containing same, and their use for treating diabetes, obesity, and related diseases or disorders. [Background technology]

[0002] Type 2 diabetes mellitus (T2DM) is a progressive disease characterized by insulin resistance, loss of beta cell function, and progressive loss of beta cell insulin secretion function. Obesity is a major contributor to the development of insulin resistance in T2DM. A correlation between adiposity and insulin resistance has been reported in both adults and children.

[0003] Oxyntomodulin (OXM) is a 37-amino acid peptide hormone released from the L-cells of the small intestine along with an additional hormone, glucagon-like peptide 1 (GLP-1). OXM reduces body weight in obese patients as a result of increased satiety and increased energy expenditure (Wynne et al., Curr Opin Investig Drugs 2010;11:1151-1157). The satiety-inducing effect of OXM is thought to be mediated by activation of the GLP-1R antagonist exendin (Baggio et al., Gastroenterol 2004:127:546-558), (Sowden et al., Am J Physiol. Reg, Integr comp. Physiol. 2007;292:R962-R970). Other effects of OXM, such as improved beta cell function and increased energy expenditure, are attributed to the glucagon receptor pathway (Kosinski et al, Obesity 2012;20:1566-1571).

[0004] Oxyntomodulin (OXM) improves glucose tolerance and stimulates insulin secretion in mice (Maida et al., Endocrinol 2008;149:5670-5678). Oxyntomodulin peptide analogs, such as PEGylated derivatives, may be useful in the treatment of type 2 diabetes and related disorders. These PEGylated derivatives are long-acting analogs that bind to and activate both the glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GcgR). Such derivatives must be chemically and physically stable in liquid formulations for both quality / safety / delivery purposes and shelf life. Summary of the Invention

[0005] The present invention provides a stable liquid formulation of a GLP-1 and / or glucagon receptor agonist, comprising a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, and an aqueous buffer solution, wherein the formulation has a pH of about 3.5 to about 8.0, and the GLP-1 and / or glucagon receptor agonist is native oxyntomodulin, an oxyntomodulin peptide analog, or an active fragment thereof. In one embodiment, the GLP-1 and / or glucagon receptor agonist is a PEGylated oxyntomodulin peptide analog.

[0006] In one embodiment, the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or an active fragment thereof, or the GLP-1 and / or glucagon receptor agonist is represented by the structure of Formula 3, Formula 4, or Formula 5, Formula 3 is represented by the following structure:

[0007] [ka]

[0008] Formula 4 is represented by the following structure:

[0009] [ka]

[0010] During the ceremony, L is a linking group, OXM * is a naturally occurring OXM, an OXM peptide analog, or an active fragment thereof, PEG is a branched or linear polyethylene glycol polymer, Y is —OH, OR, NR2, or an amino acid, which is optionally substituted with L-PEG, which is optionally amidated; R is H or a C1-C4 alkyl group; Formula 5 is represented by the following structure:

[0011] [ka]

[0012] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala -Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Gly-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa 38 -Xaa 39 (SEQ ID NO: 1), Xaa 38 is Cys, Cys-PEG, or absent, Xaa 39 is Cys, Cys-PEG, or absent, The C-terminal amino acid is optionally amidated.

[0013] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala- Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa 38 -Xaa 39 (SEQ ID NO: 6), Xaa 38 is Cys, Cys-PEG, or absent, Xaa 39 is Cys, Cys-PEG, or absent, The C-terminal amino acid is optionally amidated.

[0014] The present invention further provides a stable PEGylated oxyntomodulin liquid formulation comprising a PEGylated oxyntomodulin peptide analog, or a pharmaceutically acceptable salt thereof, and an aqueous buffer solution, wherein the formulation is substantially free of destabilizing substances and has a pH value of about 3.5 to about 8.0, and the PEGylated oxyntomodulin peptide analog comprises any one of SEQ ID NOs: 1 to 9 or active fragments thereof, or is represented by the structure of Formula 3, Formula 4, or Formula 5.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising a GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient selected from the group consisting of methionine, sorbitol, and a buffer, wherein the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or an active fragment thereof, or is represented by a compound of Formula 3, 4, or 5. In one embodiment, the present invention provides a formulation comprising 70 mg / mL of a PEGylated oxyntomodulin peptide analog of Formula 3 in 10 mM acetate buffer at a pH value of about 4.5.

[0016] In another aspect, the present invention provides a method for treating patients with type 2 diabetes, obesity, or any related medical disorder, disease, or condition that can be treated or treated by GLP-1 and / or glucagon receptor agonists, comprising administering to the patient a pharmaceutically effective amount of a liquid dosage form of the GLP-1 and / or glucagon receptor agonist formulation of the present invention.Diseases and conditions include, but are not limited to, patients with glucose-related metabolic diseases or disorders, or obesity-related diseases and disorders.Diseases such as fatty liver disease or cardiovascular diseases and disorders may be alleviated or positively affected by the treatment regimen contemplated herein below.

[0017] Patients who are treated or can be treated with the formulations of the present invention are administered a stable liquid dosage form containing the active ingredient. Such liquid dosage forms may be administered at any suitable interval. In a preferred embodiment, such patients are treated once a week with a stable PEGylated oxyntomodulin formulation at any suitable dose or dosage range.

[0018] In another aspect, the present invention provides a method for lowering HbA1c in a patient in need thereof, comprising administering to the patient a pharmaceutically effective amount of a liquid dosage form of a GLP-1 and / or glucagon receptor agonist formulation of the present invention.

[0019] In one aspect, the present invention provides a method of treating a patient with type 2 diabetes, comprising administering to the patient by weekly subcutaneous administration a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist formulation of the present invention, wherein the effective weekly dose of the GLP-1 and / or glucagon receptor agonist ranges from about 10 to about 150 mg / week.

[0020] In another aspect, the present invention provides a pre-filled syringe having a stable GLP-1 and / or glucagon receptor agonist formulation of the present invention.

[0021] In yet another aspect, the present invention provides a multi-dose container comprising a stable liquid formulation of the present invention.

[0022] In yet another aspect, the present invention provides a method of treating a patient having a disease or condition selected from the group consisting of obesity, metabolic disease, type 1 or type 2 diabetes, and impaired glucose metabolism, impaired glucose tolerance, and impaired fasting glucose, comprising administering to the patient by subcutaneous administration a pharmaceutically effective dose of a GLP-1 and / or glucagon receptor agonist formulation of the present invention, wherein the effective weekly dose of the GLP-1 and / or glucagon receptor agonist is in the range of about 10 to about 150 mg / week.

[0023] The present invention further provides a method for treating, suppressing, inhibiting, or reducing the incidence of type II diabetes, obesity or obesity-related metabolic disorders, weight loss, glucose intolerance, hyperinsulinemia, insulin resistance, hypertension, fatty liver disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, Alzheimer's disease, and / or stroke in a patient in need thereof, comprising administering to the patient a GLP-1 and / or glucagon receptor agonist formulation of the present invention.

[0024] In yet another aspect, the present invention provides a pharmaceutically acceptable salt of a GLP-1 and / or glucagon receptor agonist, wherein the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or an active fragment thereof, or is represented by a compound of Formula 3, 4, or 5.

[0025] In one aspect, the invention provides an acetate salt of a GLP-1 and / or glucagon receptor agonist, wherein the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or an active fragment thereof, or is represented by a compound of Formula 3, 4, or 5. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3.

[0026] In one aspect, the present invention provides a hydrochloride salt of a GLP-1 and / or glucagon receptor agonist, wherein the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or an active fragment thereof, or is represented by a compound of Formula 3, 4, or 5. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3.

[0027] In another aspect, the present invention provides a GLP-1 and / or glucagon receptor agonist formulation comprising an acetate salt of a GLP-1 and / or glucagon receptor agonist described herein and an aqueous buffer, wherein the formulation has a pH value of about 3.5 to about 8.0 and one or more additional pharmaceutically acceptable excipients.

[0028] In yet another aspect, the invention provides a method of administering a formulation of the invention, the method comprising a first administration period of a PEGylated oxyntomodulin peptide analog at a first once-weekly (QW) dose followed by a second administration period at a second once-weekly (QW) dose prior to administration of a pharmaceutically effective amount of the QW stable liquid dosage formulation, wherein the second QW dose is greater than the first QW dose.

[0029] The present invention further provides a process for preparing an acetate salt of a PEGylated oxyntomodulin peptide analog, comprising: (a) dissolving an oxyntomodulin (OXM) peptide analog in a buffer to form an OXM peptide analog solution, wherein the buffer has a pH value of about 6.6 to about 7.0; (b) adding an OXM peptide analog solution to a stirred solution of PEG to produce a reaction mixture comprising a crude PEGylated oxyntomodulin peptide analog product, wherein the solution of PEG is formed by dissolving PEG in the same buffer solution as that in which the OXM peptide analog of step (a) is dissolved; (c) adjusting the reaction mixture to a pH of about 3.95 to about 4.05 using acetic acid; (d) purifying the crude PEGylated oxyntomodulin peptide analog product on an HPLC column with a gradient of acetonitrile and dilute aqueous acetic acid, followed by concentration and lyophilization to provide the acetate salt of the PEGylated oxyntomodulin peptide analog. [Brief explanation of the drawings]

[0030] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. The invention, however, both as to organization and method of operation, together with its objects, features, and advantages, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings.

[0031] [Figure 1] 1 shows a flow diagram for the preparation of acetate salts of Formula 3. [Figure 2] 1 shows the deconvoluted mass spectrum of Formula 3 obtained by LC / MS analysis with post-column neutralization by DEMA. [Figure 3] The sequence ions detected in the CID-MS / MS spectrum of the peptide intermediate ([M+5H]5+ m / z 902.0) are shown. [Figure 4] The sequence ions detected in the ECD-MS / MS spectrum of the peptide intermediate ([M+5H]5+ m / z 902.0) are shown. [Figure 5] LC-UV chromatograms from LC / MS analysis of pyrolytic digestion of Formula 3 are shown. [Figure 6]Shown are SEC-HPLC results obtained for F38 samples (formulated with 25 mM cysteine) stored at 5±3° C. for 4 weeks and at 40±2° C. for several weeks. [Figure 7] Shown are RP-HPLC results obtained for F38 samples stored at 5±3° C. for 4 weeks and at 40±2° C. for several weeks. [Figure 8] 3D surface plot of % back main peak impurities at different methionine concentrations and pH with 0 mM arginine and 0 mM sorbitol. [Figure 9] 3D surface plot of % back main peak impurities at different methionine concentrations and pH with 125 mM arginine and 0 mM sorbitol. [Figure 10] 3D surface plot of % total aggregate impurities at different methionine concentrations and pH with 0 mM arginine and 0 mM sorbitol. [Figure 11] 3D surface plot of % total aggregate impurities at different methionine concentrations and pH with 125 mM arginine and 0 mM sorbitol.

[0032] It will be understood that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides a stable GLP-1 and / or glucagon receptor agonist liquid formulation comprising a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, and an aqueous buffer solution, wherein the formulation has a pH value of about 3.5 to about 8.0.

[0034] The present invention further provides a stable GLP-1 and / or glucagon receptor agonist liquid formulation comprising a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, and an aqueous buffer, wherein the formulation has a pH value of about 3.5 to about 8.0, and the GLP-1 and / or glucagon receptor agonist is native oxyntomodulin, an oxyntomodulin peptide analog, or an active fragment thereof.

[0035] In one embodiment, the GLP-1 and / or glucagon receptor agonist is a PEGylated oxyntomodulin peptide analog.

[0036] In one embodiment, as used herein, "active" or "active" means having agonist activity at the GLP-1 receptor and / or the glucagon receptor.

[0037] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala -Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Gly-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa 38 -Xaa 39 (SEQ ID NO: 1), Xaa 38 is Cys, Cys-PEG, or absent, Xaa 39 is Cys, Cys-PEG, or absent, the C-terminal amino acid is optionally amidated; Xaa 38 and Xaa 39 If both are absent, Ala 37 The NH2 group of is PEGylated and has an optional linker.

[0038] As used herein, "Cys-PEG" refers to the attachment of one or more poly(ethylene glycol) (PEG) molecules to a cysteine ​​residue, either directly or via a linking group. The PEG and linking group are as described herein or known in the art.

[0039] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala -Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Gly-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa 38 -Xaa 39 (SEQ ID NO: 1), Xaa 38 is Cys, Cys-PEG, or absent, Xaa 39 is Cys, Cys-PEG, or absent, The C-terminal amino acid is optionally amidated.

[0040] In one embodiment, Xaa 38 is Cys-PEG, and Xaa 39 In one embodiment, Xaa is absent. 38 is Cys and Xaa 39 is Cys-PEG. 38 is Cys-PEG, and Xaa 39 is Cys. 38 is Cys-PEG, and Xaa 39 is Cys-PEG. 38 and Xaa 39 Both are absent, and Ala 37 is PEGylated. 37 The NH2 group of is optionally PEGylated via a linker.

[0041] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Gln-Glu -Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Gly-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Cys-Cys (SEQ ID NO: 2), the Cys residue at position 38 is optionally PEGylated; the Cys residue at position 39 is optionally PEGylated; The carboxyl group of Cys at position 39 is optionally amidated.

[0042] In one embodiment, a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog) is PEGylated at positions 38 and / or 39 of SEQ ID NO: 1 or SEQ ID NO: 2, with the PEG molecule covalently attached to the thiol group of the Cys residue either directly or via a linking group attached to the PEG molecule. In one embodiment, the PEG molecule is linear or branched. In one embodiment, the PEG molecule has a molecular weight of about 20-40 kDa, e.g., about 20 kDa, or about 30 kDa, or about 40 kDa.

[0043] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala -Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Gly-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Cys (20 kDa PEG)-Cys(20kD PEG) (SEQ ID NO: 3), The carboxyl group of Cys at position 39 is optionally amidated.

[0044] In one embodiment, the carboxyl group of the PEGylated Cys at position 39 is amidated.

[0045] In one embodiment, the GLP-1 and / or glucagon receptor agonist is a compound of formula 4.

[0046] [ka]

[0047] During the ceremony, L is a linking group, OXM * is a naturally occurring OXM, an OXM peptide analog, or an active fragment thereof, PEG is a branched or linear polyethylene glycol polymer, Y is —OH, OR, NR2, or an amino acid, which is optionally substituted with L-PEG, which is optionally amidated; R is a C1-C4 alkyl group.

[0048] In one embodiment, the amino acid is a cysteine ​​substituted with an L-PEG group, and the linking group L is attached to the S atom on the cysteine.

[0049] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Gln -Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Gly-Arg-Asn-Arg-Asn-Asn-Ile-Ala (SEQ ID NO: 5).

[0050] In one embodiment, Ala 37 is PEGylated. 37 The NH2 group of is optionally PEGylated via a linker.

[0051] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala- Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa 38 -Xaa 39 (SEQ ID NO: 6), Xaa 38 is Cys, Cys-PEG, or absent, Xaa 39 is Cys, Cys-PEG, or absent, the C-terminal amino acid is optionally amidated; When both Xaa38 and Xaa39 are absent, the NH2 group of Ala37 is PEGylated with an optional linker.

[0052] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala- Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa 38 -Xaa 39 (SEQ ID NO: 6), Xaa 38 is Cys, Cys-PEG, or absent, Xaa 39 is Cys, Cys-PEG, or absent, The C-terminal amino acid is optionally amidated.

[0053] In one embodiment, Xaa 38 is Cys-PEG, and Xaa39 In one embodiment, Xaa is absent. 38 is Cys and Xaa 39 is Cys-PEG. 38 is Cys-PEG, and Xaa 39 is Cys. 38 is Cys-PEG, and Xaa 39 is Cys-PEG. 38 and Xaa 39 Both are absent, and Ala 37 is PEGylated. 37 The NH2 group of is optionally PEGylated via a linker.

[0054] In one embodiment, the PEG is a branched, linear, or multi-arm polyethylene glycol polymer. In one embodiment, the PEG molecule in the oxyntomodulin peptide analog has a molecular weight of 20-40 kDa, e.g., about 20 kDa, or about 30 kDa, or about 40 kDa.

[0055] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln- Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Cys-Cys (SEQ ID NO: 7), the Cys residue at position 38 is optionally PEGylated; the Cys residue at position 39 is optionally PEGylated; The carboxyl group of Cys at position 39 is optionally amidated.

[0056] In one embodiment, a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog) is PEGylated at positions 38 and / or 39 of SEQ ID NO: 6 or 7, with the PEG molecule covalently attached to the thiol group of the Cys residue, either directly or via a linking group attached to the PEG molecule. In one embodiment, the PEG is a branched, linear, or multi-arm polyethylene glycol polymer. In one embodiment, the PEG molecule in the oxyntomodulin peptide analog has a molecular weight of 20-40 kDa, e.g., about 20 kDa, or about 30 kDa, or about 40 kDa.

[0057] In one embodiment, an oxyntomodulin peptide analog of SEQ ID NO: 6 or 7 is PEGylated at either Cys at position 38 or Cys at position 39, or both, with a 20 kDa PEG molecule covalently attached to the thiol group of the Cys residue at these positions. In one embodiment, an oxyntomodulin peptide analog is PEGylated at both Cys residues at positions 38 and 39 with a 20 kDa PEG molecule covalently attached to each of the thiol groups of the Cys residues at these positions. In another embodiment, the Cys residue at position 39 may be absent in SEQ ID NO: 6 or 7, leaving a single site of PEGylation at position 38.

[0058] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala -Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Cys(20kDa PEG)-Cys(20kDa PEG) (SEQ ID NO: 8), The carboxyl group of Cys at position 39 is optionally amidated.

[0059] In one embodiment, the carboxyl group of the PEGylated Cys at position 39 is amidated.

[0060] In one embodiment, the GLP-1 and / or glucagon receptor agonist has the amino acid sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala- Contains Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala (SEQ ID NO: 9).

[0061] In one embodiment, the NH2 group of Ala37 is PEGylated. In one embodiment, the NH2 group of Ala37 is PEGylated, optionally via a linker.

[0062] In one embodiment, a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog) described herein is PEGylated at either Cys at position 38 or Cys at position 39, or both, with a PEG molecule covalently attached to the thiol group of the Cys residue at these positions, e.g., a 20 kDa PEG, a 30 kDa PEG, or a 40 kDa PEG. In one embodiment, the oxyntomodulin peptide analog is PEGylated at both Cys residues at positions 38 and 39 with a PEG molecule covalently attached to each thiol group of the Cys residue at these positions, e.g., a 20 kDa PEG, a 30 kDa PEG, or a 40 kDa PEG molecule. In another embodiment, the Cys residue at position 39 can be absent, leaving a single site of PEGylation at position 38.

[0063] In one embodiment, the PEG in the GLP-1 and / or glucagon receptor agonists (e.g., PEGylated oxyntomodulin peptide analogs) described herein can be attached to the oxyntomodulin 38 and / or 39Cys group via a linking group known in the art. In one embodiment, PEG is attached to the oxyntomodulin 38 and / or 39Cys group using mPEG-20kDa maleimide (Formula 1) or mPEG-20kDa iodoacetamide (Formula 2), where n is 10 to 2500, or n is 350 to 600, or n is 425 to 475.

[0064] [ka]

[0065] Other known linkers or linking groups may also be utilized to covalently attach OXM or its analogs to PEG moieties. Alternatively, activated PEG molecules can be reacted directly with OXM analogs having at least one reactive cysteine ​​at positions 38 and / or 39 to form PEGylated OXM analogs utilized as active pharmaceutical ingredients (APIs) in the stable liquid formulations of the present invention.

[0066] In one embodiment, PEG is attached to the oxyntomodulin 38 and 39 Cys groups using both iodoacetamide of mPEG-20kDa, and the carboxyl group of the PEGylated Cys at position 39 is amidated.

[0067] In one embodiment, PEG is conjugated to the oxyntomodulin 38 and 39 Cys groups using either mPEG-20kDa maleimide or mPEG-20kDa iodoacetamide, and the carboxyl group of the PEGylated Cys at position 39 is amidated. For example, PEG is conjugated to the oxyntomodulin 38 Cys group using mPEG-20kDa maleimide and to the oxyntomodulin 39 Cys group using mPEG-20kDa iodoacetamide. In another embodiment, PEG is conjugated to the oxyntomodulin 38 Cys group using mPEG-20kDa iodoacetamide and to the oxyntomodulin 39 Cys group using mPEG-20kDa maleimide.

[0068] In one embodiment, PEG is attached to the oxyntomodulin 38 and 39 Cys groups of SEQ ID NO: 1 using both maleimides of mPEG-20kDa, and the carboxyl group of the PEGylated Cys at position 39 is amidated (Formula 3).

[0069] [ka]

[0070] Formula 3 includes standard letter amino acid code designations (i.e., His), except for certain chemical structures depicted as embedded in the formula or otherwise indicated. In one embodiment, the compound of Formula 3 is in the form of a pharmaceutically acceptable salt. In one embodiment, the compound of Formula 3 is in the form of an acetate salt.

[0071] In another embodiment, PEG is attached to the oxyntomodulin 38 and 39 Cys groups of SEQ ID NO: 6 using both maleimides of mPEG-20kDa, and the carboxyl group of the PEGylated Cys at position 39 is amidated (Formula 5).

[0072] [ka]

[0073] In one embodiment, the compound of formula 5 is in the form of a pharmaceutically acceptable salt. In another embodiment, the compound of formula 5 is in the form of an acetate salt.

[0074] Other oxyntomodulin variants / analogs and / or activators of both the GLP-1 receptor and the glucagon receptor with at least one cysteine ​​residue at position 38 can also be utilized in the present invention. Suitable analogs with dual activity are described in Santoprete et al. J. Pept. Sci. 2011;17:270-280. In particular, substitution of D-Ser or Aib or D-Ala at position 2 of the N-terminus provides DPP-IV-resistant analogs while retaining GLP-1R / GcgR dual agonist properties. Aib at position 2 is preferred (Aib is 2-aminoisobutyric acid) because it provides enhanced potency and selectivity at the GLP-1 receptor. Other embodiments include active OXM (HSQGTFTSDYSKYLDSRAQDFVQWLMNTKLRNNIA) (SEQ ID NO: 4) containing Cys residues at positions 38 or 38 and 39, with further modification(s) to the PEGylated derivatives described herein.

[0075] In one embodiment, the formulation of the present invention has a pH value of about 4.0 to about 7.0. In one embodiment, the pH value is about 4.5 to about 6.5. In one embodiment, the pH value is about 4.0 to about 5.5. In one embodiment, the pH is about 4.5.

[0076] In one embodiment, for preparation of the formulations of the invention, a suitable buffer, e.g., acetate buffer, is adjusted with the desired excipients (i.e., methionine (a stabilizer), sorbitol (a tonicity adjuster)) to a pH about 0.5 pH units below the target range, e.g., about 4.0 to 7.0 (alternatively, the pH is adjusted to the preferred target pH range after addition of the GLP-1 and / or glucagon receptor agonist). A GLP-1 and / or glucagon receptor agonist, such as an oxyntomodulin peptide analog described herein, is added to the formulation buffer, which is then adjusted with a weak acid or weak base to decrease or increase the pH to within the target range.

[0077] The inventors have discovered that lower pH values ​​within the range of about 4.0 to about 7.0 actually improve the stability of liquid formulations, with a pH of about 4.5 being preferred. Furthermore, the inventors have discovered that certain excipients routinely used in such parenteral formulations actually destabilize the API (active pharmaceutical ingredient, e.g., the GLP-1 and / or glucagon receptor agonist described herein) in the pre-buffer solution, causing the formation of impurities (e.g., cysteine) as determined by HPLC. The inventors have also discovered that certain agents, such as methionine and / or arginine, improve the stability of liquid formulations.

[0078] Unexpectedly, pre-buffer formulations containing 100 nM sodium chloride and citrate, histidine, or acetate buffers were not stable with respect to impurity formation when tested in aqueous buffer solutions (citrate, histidine, or acetate) without sodium chloride. This finding led to the selection of stable formulations that contain no sodium chloride or low concentrations (0-50 nM) of such salts. Stable formulations of GLP-1 / glucagon receptor agonists may contain sodium chloride as long as it does not lead to API degradation. Preferred formulations are sodium chloride-free or substantially free of sodium chloride or other tonicity adjusters, which would result in API degradation in aqueous buffers such as citrate, histidine, or acetate buffers. The percentage of impurities by HPLC analysis is preferably less than 6.0 percent area. This impurity profile also depends to some extent on the solution pH. Higher pH in the range of 5.3 to 6.6 resulted in higher percentages of total impurities in the sodium chloride-containing samples (6.1 to 7.3 or 7.0 to 9.3 for samples stored at room temperature for 6 days versus samples stored at 40 degrees Celsius for 6 days).

[0079] A less stabilizing amount of an isotonic agent, such as sodium chloride, is defined as ≥ approximately 10-100 mM sodium chloride. This concentration may vary depending on the specific excipients in the formulation and the pH of the solution. A destabilizing amount of cysteine ​​is defined as ≥ 25 mM cysteine.

[0080] Thus, in another embodiment, the formulations of the present invention are substantially free of destabilizing substances. In one embodiment, the destabilizing substance is cysteine. In one embodiment, the amount of cysteine ​​is less than 25 mM. In one embodiment, the amount of cysteine ​​is less than 10 mM. In one embodiment, the formulations are substantially free of sodium chloride. In one embodiment, the amount of sodium chloride is less than 100 mM. In one embodiment, the amount of sodium chloride is less than 25 mM.

[0081] As used herein, the term "substantially free" should be understood to mean a relatively small or no amount of any substance in the aqueous buffer formulation of the present invention that results in up to a percentage degradation of the active pharmaceutical ingredient of greater than about 5% based on high performance liquid chromatography (HPLC) measured under standard test conditions.

[0082] The present invention further encompasses formulations that include additional pharmaceutically acceptable (generally recognized as safe, "GRAS") excipients, such as surfactants or isotonicity agents or other pharmaceutically acceptable additives. These may include, for example, methionine, arginine, sorbitol, sucrose, non-ionic surfactants (poloxamers and polysorbates), or similar excipients. In one embodiment, the formulation is 70 mg / mL of the PEGylated oxyntomodulin peptide of formula 3 (OPK88003) in a 10 mM acetate buffer solution at pH 4.5, containing methionine (30-35 mM) and sorbitol to adjust isotonicity.

[0083] The present invention provides stable GLP-1 and / or glucagon receptor agonist liquid formulations comprising a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog), or a pharmaceutically acceptable salt thereof, and an aqueous buffer, the formulations being substantially free of destabilizing substances and having a pH range of about 3.5 to about 8.0; The GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or an active fragment thereof, or is represented by the structure of Formula 3, Formula 4, or Formula 5, Formula 3 is represented by the following structure:

[0084] [ka]

[0085] Formula 4 is represented by the following structure:

[0086] [ka]

[0087] During the ceremony, L is a linking group, OXM * is a naturally occurring OXM, an OXM peptide analog, or an active fragment thereof, PEG is a branched or linear polyethylene glycol polymer, Y is —OH, OR, NR2, or an amino acid, which is optionally substituted with L-PEG, which is optionally amidated; R is H or a C1-C4 alkyl group; Formula 5 is represented by the following structure:

[0088] [ka]

[0089] In one embodiment, the PEGylated oxyntomodulin peptide analog is represented by the structure of Formula 3, Formula 4, or Formula 5. In another embodiment, the PEGylated oxyntomodulin peptide analog is represented by the structure of Formula 3.

[0090] In one embodiment, Y in formula 4 is a cysteine ​​substituted with an L-PEG group, and the linking group L is attached to the cysteine ​​through the S atom on the cysteine.

[0091] In one embodiment, the GLP-1 and / or glucagon receptor agonist is in the form of a pharmaceutically acceptable salt. In one embodiment, the GLP-1 and / or glucagon receptor agonist is in the form of an acetate salt.

[0092] In one embodiment, a formulation of the invention contains no more than about 10.0% total impurities and less than about 5% of any single individual impurity as measured by RP-HPLC. In one embodiment, a formulation of the invention contains no more than about 10.0% total impurities and less than about 5% of any single individual impurity as measured by size exclusion chromatography.

[0093] In one embodiment, the formulation of the present invention is substantially free of destabilizing substances. In one embodiment, the destabilizing substance is cysteine. In one embodiment, the amount of cysteine ​​is less than 25 mM. In one embodiment, the amount of cysteine ​​is less than 10 mM. In one embodiment, the formulation is substantially free of sodium chloride. In one embodiment, the amount of sodium chloride is less than 100 mM. In one embodiment, the amount of sodium chloride is less than 25 mM.

[0094] In one embodiment, the formulation of the present invention comprises an isotonicity agent. In one embodiment, the isotonicity agent comprises sorbitol. Other isotonicity agents may include sucrose, mannitol, or other tonicity adjusting agents.

[0095] In one embodiment, the formulations of the present invention further comprise one or more preservatives at about 0.001% (w / v) to about 1% (w / v), based on the total volume of the liquid formulation. In one embodiment, the preservative is about 0.001% (w / v) to about 0.5% (w / v), or about 0.001% (w / v) to about 0.1% (w / v), or about 0.001% (w / v) to about 0.05% (w / v), or about 0.001% (w / v) to about 0.01% (w / v). In one embodiment, the preservative is m-cresol, phenol, and / or benzyl alcohol. In certain embodiments, the preservative is m-cresol. In another embodiment, the preservative is phenol or benzyl alcohol.

[0096] In one embodiment, in a formulation of the present invention, the GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog), or a pharmaceutically acceptable salt thereof, is present in a suitable vial at a concentration ranging from about 5 mg / mL to about 150 mg / mL. In one embodiment, in a formulation of the present invention, the GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, is present in a suitable vial at a concentration ranging from about 20 mg / mL to about 150 mg / mL. In one embodiment, in a formulation of the present invention, the GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, is present in a suitable vial at a concentration ranging from about 50 mg / mL to about 120 mg / mL. In one embodiment, in a formulation of the present invention, the GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, is present in a suitable vial at a concentration of about 70 mg / mL. In one embodiment, the suitable vial is a single-use vial.

[0097] In one embodiment, the buffer in the formulation of the present invention is selected from the group consisting of acetate buffer, citrate buffer, and histidine buffer. In certain embodiments, the buffer is acetate buffer. In one embodiment, the buffer is citrate buffer. In one embodiment, the buffer is histidine buffer.

[0098] In one embodiment, the pH range of the formulation of the present invention is about 4.0 to about 7.0, or about 4.0 to about 6.5, or about 4.0 to about 6.0, or about 4.0 to about 5.5, or about 4.0 to about 5.0, or about 4.5 to about 6.5, or about 4.5 to about 6.0, or about 4.5 to about 5.5. In another embodiment, the pH value of the formulation of the present invention is about 4.5.

[0099] In one embodiment, the formulation comprises 70 mg / mL of a PEGylated oxyntomodulin peptide analog of Formula 3 in 10 mM acetate buffer, the formulation having a pH value of about 4.5.

[0100] In one embodiment, the formulation of the present invention further comprises one or more additional pharmaceutically acceptable excipients known in the art, hi one embodiment, the additional pharmaceutically acceptable excipient is methionine, a methionine derivative, arginine, or an arginine derivative.

[0101] In one embodiment, the formulations of the present invention have an osmolality of 500 mOsm / kg or less. In one embodiment, the formulations of the present invention have a viscosity of about 10 to about 60 cP. In one embodiment, the formulations of the present invention have a syringeability of a breaking relaxation force of less than 10 N and a sliding equilibrium stress of less than 30 N. In one embodiment, the formulations of the present invention cause no or little injection site reaction, such as irritation.

[0102] In one embodiment, the formulations of the invention are stable for at least 5 freeze / thaw cycles, hi another embodiment, the formulations of the invention are stable for at least 48 hours at 30°C / 65% RH and for 12 months at 5°C.

[0103] The present invention further provides a pharmaceutical composition comprising a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient selected from the group consisting of methionine, sorbitol, and a buffer. In one embodiment, the GLP-1 and / or glucagon receptor agonist is a PEGylated oxyntomodulin peptide analog.

[0104] In one embodiment, the present invention provides a pharmaceutical composition comprising a PEGylated oxyntomodulin peptide analog selected from the group consisting of PEGylated oxyntomodulin peptide analogs of Formula 3, Formula 4, and Formula 5, or a PEGylated oxyntomodulin peptide analog comprising any one of SEQ ID NOs: 1-9 or active fragments thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient selected from the group consisting of methionine, sorbitol, and a buffer.

[0105] The present invention further provides a stable liquid formulation comprising a compound of Formula 3 in an aqueous buffer substantially free of sodium chloride, the formulation having a pH range of about 4.0 to about 7.0. The formulation optionally contains additional excipients selected from organic bases, isotonicity agents, and other excipients that do not destabilize or negatively impact the formulation. In addition to, or as a substitute for, methionine, arginine may also be used as a stabilizing amino acid. In one embodiment, the stable liquid formulation of the present invention has a pH range of about 4.0 to about 5.0. In another embodiment, the stable liquid formulation of the present invention has a pH of about 4.5.

[0106] In one embodiment, the stable liquid formulations of the present invention are administered to patients from a suitable container (e.g., vial, prefilled syringe, cartridge, etc.) at a suitable dose ranging from 20 mg to 80 mg twice weekly, once weekly, or at longer intervals. Autoinjectors are also contemplated. Dosages may vary depending on the patient's weight, the nature and severity of the disease state or condition, and the dose may be titrated from a lower initial dose to a higher dose. Single-dose vials generally contain a solution with 70 mg / mL of GLP-1 and / or glucagon receptor agonist (e.g., PEGylated oxyntomodulin peptide analog). The various volumes administered at this concentration determine the actual amount of GLP-1 and / or glucagon receptor agonist delivered to the patient. Multi-dose containers containing the liquid formulations of the present invention are also contemplated.

[0107] The present invention provides the use of the stable liquid formulation of the present invention in a patient in need thereof for the treatment of diabetes and / or obesity or a metabolic disorder. The present invention provides a stable liquid formulation of GLP-1 and / or glucagon receptor agonist that effectively reduces HbA1c in patients with type 2 diabetes.

[0108] The present invention encompasses the use of stable GLP-1 and / or glucagon receptor agonist liquid formulations that effectively reduce HbA1c, further reduce body weight, and / or alter fasting plasma glucose (FPG) in patients with type 2 diabetes.

[0109] The present invention further provides a method for treating type 2 diabetes, obesity, or any related medical disorder, disease, or condition that is or can be treated by a GLP-1 and / or glucagon receptor agonist in a patient in need thereof by administering a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist formulation of the present invention to the patient. Diseases and conditions include, but are not limited to, patients with glucose-related metabolic diseases or disorders or obesity-related diseases and disorders. Diseases such as fatty liver disease or cardiovascular diseases and disorders may be alleviated or positively affected by the treatment regimens contemplated herein below. In one embodiment, the disease or condition includes, but is not limited to, fatty liver disease or cardiovascular disease. In certain embodiments, the fatty liver disease is non-alcoholic fatty liver disease. In one embodiment, the liquid dosage form is administered twice a week, once a week, or at a longer dosing interval. In one embodiment, the liquid dosage form is administered once a week.

[0110] In another aspect, the present invention provides a method for treating a patient with type 2 diabetes, comprising administering to the patient a pharmaceutically effective amount of a liquid dosage form of a GLP-1 and / or glucagon receptor agonist formulation of the present invention. In one embodiment, the liquid dosage form is administered twice a week, once a week, or at longer dosing intervals. In one embodiment, the liquid dosage form is administered once a week. In one embodiment, in the method of the present invention, the formulation buffer is an acetate buffer. In one embodiment, the solution further comprises methionine. In one embodiment, the GLP-1 and / or glucagon receptor agonist is a PEGylated oxyntomodulin peptide analog. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In another embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 5. In one embodiment, the GLP-1 and / or glucagon receptor agonist is in the form of an acetate salt.

[0111] In one embodiment, in the methods of the present invention, the GLP-1 and / or glucagon receptor agonist is present in a suitable container or delivery device at a concentration ranging from about 5 mg / mL to about 150 mg / mL. In one embodiment, the concentration is from about 20 mg / mL to about 150 mg / mL. In one embodiment, the concentration is from about 20 mg / mL to about 120 mg / mL. In another embodiment, the concentration is from about 20 mg / mL to about 100 mg / mL. In one embodiment, the concentration is from about 30 mg / mL to about 100 mg / mL. In one embodiment, the concentration is from about 40 mg / mL to about 100 mg / mL. In another embodiment, the concentration is from about 40 mg / mL to about 80 mg / mL.

[0112] In one embodiment, the liquid dosage form for the methods of the present invention is substantially free of sodium chloride. In one embodiment, the liquid dosage form for the methods of the present invention is substantially free of cysteine. In one embodiment, the pH of the liquid dosage is about 4.5 to about 6.5. In one embodiment, the dosage administered to a patient is in the range of 20-150 mg / week, or 20-125 mg / week, or 20-100 mg / week, or 20-75 mg / week, or 20-50 mg / week, or 40-150 mg / week, or 50-150 mg / week, or 75-150 mg / week, or 100-150 mg / week. In another embodiment, the dosage administered to a patient is in the range of 0.5-1.5 mg / kg / week, or 0.5-1.0 mg / kg / week, or 1.0-1.5 mg / kg / week.

[0113] In yet another aspect, the present invention provides a method for lowering HbA1c in a patient in need thereof, comprising administering to the patient a pharmaceutically effective amount of a liquid dosage form of a GLP-1 and / or glucagon receptor agonist formulation of the present invention. In one embodiment, the liquid dosage form is administered twice a week, once a week, or at longer dosing intervals. In one embodiment, the liquid dosage form is administered once a week. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In one embodiment, the concentration of the GLP-1 and / or glucagon receptor agonist in a suitable container is about 70 mg / mL. In one embodiment, the suitable container is a single-use vial.

[0114] In one embodiment, the patient's HbA1c level is reduced after 4, 8, 12, 22, 24, 30, or 34 weeks. In another embodiment, the patient's HbA1c level is reduced by about 0.5% to 3% compared to baseline levels. In another embodiment, the patient's HbA1c level is reduced by about 1.3% compared to baseline levels. In another embodiment, the patient's mean absolute reduction in HbA1c levels is about 0.01% to 0.50%. In another embodiment, the patient's mean absolute reduction in HbA1c levels is about 0.09%.

[0115] In yet another aspect, the present invention provides a method for reducing body weight in a patient in need thereof, comprising administering to the patient a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist formulation of the present invention. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In one embodiment, the formulation is administered twice a week, once a week, or at longer intervals. In one embodiment, the formulation is administered once a week.

[0116] In one embodiment, the patient's weight is reduced after 4, 8, 12, 22, 24, 30, or 34 weeks. In another embodiment, the patient's weight is reduced by about 1.8 kg to 8.8 kg compared to baseline levels. In another embodiment, the patient's weight is reduced by about 4.4 kg compared to baseline levels. In another embodiment, the patient's weight is reduced by about 2% to 10% compared to baseline levels. In another embodiment, the patient's weight is reduced by about 5% compared to baseline levels.

[0117] In yet another aspect, the present invention provides a method for lowering blood triglycerides in a patient in need thereof, comprising administering to the patient a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist formulation of the present invention. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In one embodiment, the formulation is administered twice a week, once a week, or at longer intervals. In one embodiment, the formulation is administered once a week.

[0118] In one embodiment, the patient's blood triglycerides are lowered after 4, 8, 12, 22, 24, 30, or 34 weeks. In another embodiment, the patient's blood triglycerides are lowered by about 30 mg / dL to 40 mg / dL compared to baseline levels. In another embodiment, the patient's blood triglycerides are lowered by about 31.2 mg / dL compared to baseline levels.

[0119] The present invention further provides a method for treating a patient with type 2 diabetes, comprising administering to the patient a pharmaceutically effective amount of a GLP-1 and / or glucagon receptor agonist formulation of the present invention by subcutaneous administration once weekly, wherein the effective weekly dose per patient of the GLP-1 and / or glucagon receptor agonist ranges from about 10 to about 150 mg / week. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In one embodiment, the formulation is administered from a pre-filled syringe, cartridge, pen, auto-injector, or vial. In another embodiment, the pre-filled syringe, cartridge, auto-injector, pen, or vial contains a solution having 70 mg / mL of the GLP-1 and / or glucagon receptor agonist.

[0120] In another aspect, the present invention provides a pre-filled syringe containing a stable GLP-1 and / or glucagon receptor agonist formulation of the present invention. In one embodiment, the GLP-1 and / or glucagon receptor agonist in the pre-filled syringe of the present invention is represented by a compound of Formula 3. In one embodiment, the pre-filled syringe of the present invention contains a solution having a pH range of about 3.5 to about 8.0, or about 4.0 to about 7.0. In one embodiment, the formulation contains an acetate buffer. In one embodiment, the pre-filled syringe contains a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutically acceptable excipient contains methionine. In one embodiment, the formulation in the pre-filled syringe is substantially free of sodium chloride. In one embodiment, the liquid dosage form for the method of the present invention is substantially free of cysteine. In one embodiment, the amount of sodium chloride is less than 100 mM. In one embodiment, the amount of cysteine ​​is less than 25 mM.

[0121] The present invention further provides a multi-dose container containing the stable liquid formulation of the present invention.

[0122] In another aspect, the present invention provides a method for treating a patient having a disease or condition selected from the group consisting of obesity, metabolic disease, type 1 or type 2 diabetes, and impaired glucose metabolism, impaired glucose tolerance, and impaired fasting glucose, comprising administering to the patient a pharmaceutically effective amount of a liquid dosage form of a GLP-1 and / or glucagon receptor agonist formulation of the present invention. In one embodiment, the formulation is administered twice a week, once a week, or at longer dosing intervals. In another embodiment, the formulation is administered once a week.

[0123] The present invention further provides a method for treating a patient having a disease or condition selected from the group consisting of obesity, metabolic disease, type 1 or type 2 diabetes, and impaired glucose metabolism, impaired glucose tolerance, and impaired fasting glucose, comprising subcutaneously administering to the patient a pharmaceutically effective dose of a GLP-1 and / or glucagon receptor agonist liquid formulation of the present invention, wherein the effective dose of the GLP-1 and / or glucagon receptor agonist per patient is in the range of about 10 to about 150 mg / week. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3:

[0124] The GLP-1 and / or glucagon receptor agonist described herein and the preparations containing it are useful for treating type 2 diabetes, obesity, or any related medical disorders, diseases or conditions.Diseases and conditions include but are not limited to patients with glucose-related metabolic diseases or disorders or obesity-related diseases and disorders.Diseases such as fatty liver disease or cardiovascular diseases and disorders can be alleviated or positively affected by the treatment regimen proposed herein below.

[0125] The present invention further provides a method of treating, suppressing, inhibiting, or reducing the incidence of type 2 diabetes, obesity or obesity-related metabolic disorders, weight loss, glucose intolerance, hyperinsulinemia, insulin resistance, hypertension, fatty liver disease (e.g., non-alcoholic steatohepatitis, or NASH), diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, Alzheimer's disease, and / or stroke in a patient in need thereof, comprising administering to a patient in need thereof a GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof.

[0126] In one embodiment, the present invention provides a method of treating type 2 diabetes, comprising administering to a subject in need thereof a GLPGLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof.

[0127] In one embodiment, the present invention provides a method for treating, suppressing, inhibiting, or reducing the incidence of obesity and / or obesity-related metabolic disorders, such as hypertension, diabetes, elevated blood pressure, stroke, or heart disease, comprising administering to a patient in need thereof a GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof. In one embodiment, the obesity-related metabolic disorder is hypertension. In another embodiment, the disorder is type 2 diabetes. In another embodiment, the disorder is elevated blood pressure. In another embodiment, the disorder is stroke. In another embodiment, the disorder is heart disease.

[0128] In another embodiment, the present invention relates to a method of promoting, increasing, or facilitating weight loss in a subject, comprising administering to a patient in need thereof a GLPGLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof.

[0129] Type 2 diabetes is characterized by insulin resistance and, at certain stages of disease pathogenesis, a relative deficiency in insulin secretion. Most individuals with type 2 diabetes present with abdominal (visceral) obesity and fatty liver, which are closely associated with the presence of insulin resistance. Patients' livers become insulin resistant, resulting in uncontrolled glycogenolysis, resulting in increased and unphysiological glucose delivery to the bloodstream. Cholesterol and VLDL particle production in the liver are also unregulated. Furthermore, hypertension, dyslipidemia (high triglycerides and low HDL cholesterol levels, postprandial hyperlipidemia), and elevated PPAI-1 levels are often present in these individuals. This clustering of abnormalities is referred to as the "insulin resistance syndrome," or "metabolic syndrome," or obesity-related disorders. These abnormalities place patients with type 2 diabetes at increased risk for developing macrovascular complications, such as myocardial infarction and stroke.

[0130] In another aspect of the present invention, the GLP-1 and / or glucagon receptor agonist formulations of the present invention, or GLPGLP-1 and / or glucagon receptor agonists, or pharmaceutically acceptable salts thereof described herein, are useful for treating, suppressing, or inhibiting diseases or conditions associated with type 2 diabetes, or diseases or conditions associated with type 2 diabetes. In one embodiment, the present invention provides a method for treating, suppressing, or inhibiting fatty liver pathology, comprising administering to a subject in need thereof a GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, as described herein. In one embodiment, the fatty liver disease is nonalcoholic steatohepatitis (NASH).

[0131] In one embodiment, the present invention provides a method for treating glucose intolerance, comprising administering to a subject in need thereof a GLP-1 and / or glucagon receptor agonist formulation of the present invention described herein, or a GLPGLP-1 and / or glucagon receptor agonist or a pharmaceutically acceptable salt thereof. Glucose intolerance is a pre-diabetic condition in which blood glucose is higher than normal, but not high enough to justify a diagnosis of diabetes.

[0132] In one embodiment, the present invention provides a method for treating, suppressing, or inhibiting hyperinsulinemia, comprising administering to a subject in need thereof a GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof. Hyperinsulinemia is a symptom of an underlying problem that causes the pancreas to secrete excessive amounts of insulin. The most common cause of hyperinsulinemia is insulin resistance, a condition in which the body resists the effects of insulin and the pancreas attempts to compensate by producing more insulin. Hyperinsulinemia is associated with type 2 diabetes.

[0133] In one embodiment, the present invention provides a method for treating atherosclerosis and related diseases, such as cardiovascular disorders, cerebrovascular disorders, peripheral vascular disorders, or intestinal vascular disorders, in a subject in need thereof, comprising administering to a subject in need thereof a GLPGLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof. Atherosclerosis refers to a slow, complex disease that can begin with damage to the innermost layer of an artery. Causes of damage to the arterial wall may include (a) elevated blood cholesterol levels, (b) high blood pressure, (c) tobacco smoking, or (d) diabetes. Similarly, in another embodiment, the method of the present invention may be useful for treating subjects with a family history of early cardiovascular disease, which increases the risk of atherosclerosis.

[0134] In one embodiment, the present invention provides a method for treating Alzheimer's disease, comprising administering to a subject in need thereof a GLPGLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof. Alzheimer's disease (AD) is characterized by cognitive impairment and behavioral changes that affect memory and learning ability, daily life functions, and quality of life. Hyperinsulinemia and insulin resistance, known pathophysiological characteristics of T2DM, have also been demonstrated to significantly affect cognitive impairment. GLP-1 is thought to affect neurological and cognitive function, as well as its regulatory effects on glucose metabolism.

[0135] In one embodiment, the present invention provides a method for treating diabetic neuropathy. Diabetic neuropathy is a complication of diabetes that typically progresses early, before a clinical diagnosis of diabetes is made. The earliest clinical evidence of neuropathy is the appearance of low but abnormal levels of albumin in the urine (>30 mg / day or 20 μg / min) (microalbuminuria), followed by albuminuria (>300 mg / 24 hours or 200 μg / min) that develops over a period of 10 to 15 years. In patients with type 1 diabetes, diabetic hypertension typically becomes apparent early, by the time the patient develops microalbuminuria. Once overt nephropathy occurs, glomerular filtration rate (GFR) declines over time, which can take several years, leading to end-stage renal disease (ESRD) in diabetic individuals.

[0136] In one embodiment, the present invention provides a method for treating diabetic neuropathy. Diabetic neuropathy is a family of nerve disorders caused by diabetes. Diabetic neuropathy causes numbness, sometimes pain, and weakness in the hands, arms, feet, and legs. Diabetic neuropathy is classified as peripheral, autonomic, proximal, and focal. Peripheral neuropathy causes pain or loss of sensation in the toes, feet, legs, hands, and arms. Autonomic neuropathy causes changes in digestion, bowel and bladder function, sexual response, and sweating, and may also affect the nerves that serve the heart and control blood pressure. Proximal neuropathy causes pain in the thighs, lower back, or buttocks, leading to leg weakness. Focal neuropathy results in sudden weakness of a single nerve or group of nerves, causing muscle weakness or pain. Nerves anywhere in the body may be affected.

[0137] In one embodiment, the present invention provides a method for treating diabetic retinopathy. The effects of diabetes on the eyes are called diabetic retinopathy. Patients with diabetes are more likely to develop eye problems such as cataracts and glaucoma. The impact of diabetic retinopathy on vision varies greatly depending on the stage of the disease. Some common symptoms of diabetic retinopathy are blurred vision (which is often related to blood sugar levels), floaters and flashing lights, and sudden vision loss.

[0138] The present invention further provides a combination therapy comprising the formulation of the present invention, or the GLP-1 and / or glucagon receptor agonist or a pharmaceutically acceptable salt thereof described herein, and other drugs for the indications described herein.The patient in the method of the present invention may have other diseases and / or conditions.The GLP-1 and / or glucagon receptor agonist formulation of the present invention, or the GLP-1 and / or glucagon receptor agonist or a pharmaceutically acceptable salt thereof described herein, can be administered in combination with one or more additional drugs (e.g., therapeutic agents) used to treat the same or different diseases or conditions as those in the method of the present invention.The GLP-1 and / or glucagon receptor agonist formulation of the present invention, or the GLP-1 and / or glucagon receptor agonist or a pharmaceutically acceptable salt thereof described herein, can be administered simultaneously with, after, or before the administration of the additional drug(s) in the method of the present invention.

[0139] Obese and / or diabetic patients may have other diseases and conditions. In one embodiment, the patient may have chronic kidney disease and may receive renal medications. In one embodiment, the patient may receive diuretics. In one embodiment, the patient may have high blood pressure and may receive high blood pressure medications. In one embodiment, the patient may receive diabetic medications that are not contraindicated. In one embodiment, the patient may have a cardiovascular condition with cardiac medications. In one embodiment, the patient may be vitamin D deficient and may receive vitamin D analogs or supplements.

[0140] Another aspect of the present invention is that the GLP-1 and / or glucagon receptor agonist formulations of the present invention, or the GLP-1 and / or glucagon receptor agonists described herein or pharmaceutically acceptable salts thereof, can be administered together with another therapeutic agent(s) in the methods of the present invention.

[0141] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof, is administered in combination with a drug for treating chronic kidney disease. In one embodiment, the drug for treating chronic kidney disease is a selective androgen receptor modulator (SARM). In one embodiment, the selective androgen receptor modulator is RAYALDEE (25(OH)D3) (for treating secondary hyperparathyroidism (SHPT) in patients with stage 3 or 4 chronic kidney disease (CRO) and others).

[0142] In one embodiment, the agent treating chronic kidney disease is erythropoietin or an analog thereof (e.g., darbepoetin (also known as Aranesp®)). In one embodiment, the agent is a steroid. In one embodiment, the agent is a diuretic, including but not limited to, organomercurials, ethacrynic acid, furosemide, bumetanide, piretanide, muzolimine, chlorothiazide and thiazides, phthalimidine, chlorthalidone, quinazolinones, quinethazone, xipamide, xanthines, aminophylline, carbonic anhydrase inhibitors, acetazolamide mannitol, potassium-sparing diuretics, aldosterone antagonists, pteridines, pyrazines, carboxamide-triamterene, and / or amiloride.

[0143] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulation of the present invention, or the GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof, is administered in combination with a drug for treating a cardiovascular condition. In one embodiment, the drug for treating a cardiovascular condition treats congestive heart failure. The drug for treating congestive heart failure can be an angiotensin-converting enzyme (ACE) inhibitor such as benazepril, captopril, lisinopril, moexipril, perindopril, quinapril, ramipril, or enalaprilat. In one embodiment, the agent treating congestive heart failure is a beta-blocker such as acebutol, atenolol, betaxolol hydrochloride, bisoprolol fumarate, cartelol hydrochloride, hydrochloride, esmolol hydrochloride, labetalol hydrochloride, levobunolol, metipranolol, nadolol, oxprenolol hydrochloride, pindolol, propranolol hydrochloride, sotalol hydrochloride, or timolol maleate. In one embodiment, the agent treating congestive heart failure is digoxin.

[0144] In one embodiment, the agent treating a cardiovascular condition is an antiarrhythmic agent. The antiarrhythmic agent may be a sodium channel blocker, a beta-adrenergic blocker, a calcium channel blocker, or an agent that prolongs repolarization. Sodium channel blockers include, but are not limited to, quinidine, procainamide, disopyramide, lidocaine, tocainide, mexiletine, encainide, or flecainide. In one embodiment, beta-adrenergic blockers include, but are not limited to, propranolol, acebutolol, esmolol, or sotalol. Agents that prolong repolarization include, but are not limited to, sotalol or amiodarone. Calcium channel blockers include, but are not limited to, verapamil, diltiazem, nifedipine, or mibefradil. In one embodiment, the antiarrhythmic agent is adenosine or digoxin.

[0145] In one embodiment, the agent for treating a cardiovascular condition may be a vasoactive or inotropic agent, including, but not limited to, digoxin, dopamine, dobutamine, hydralazine, prazosin, carvedilol, nitroprusside, nitroglycerin, captopril, lisinopril, nifedipine, amlodipine, diltiazem, hydrochlorothiazide, furosemide, spironolactone, AT-1 receptor antagonists (e.g., losartan, irbesartan, valsartan), ET receptor antagonists, dual ET / AII antagonists, neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibitors (dual NEP-ACE inhibitors) (e.g., omapatrilat and gemopatrilat), or nitrates.

[0146] In one embodiment, the agent treating a cardiovascular condition is an anticoagulant, such as a coumarin derivative (e.g., warfarin), or unfractionated heparin, or low molecular weight heparin. In one embodiment, the agent treating a cardiovascular condition is a fibrinolytic agent such as streptokinase, urokinase, alteplase, anistreplase, prourokinase, reteplase, tenecteplase, lanoteplase, staphylokinase, vampire bat saliva, or alfimeplase. In one embodiment, the agent treating a cardiovascular condition is a hypercholesterolemic agent such as niacin-lovastatin, colestipol HCl, fluvastatin sodium, atorvastatin calcium, simvastatin, gemfibrozil, lovastatin, pravastatin sodium, cholestyramine, cholestyramine lite, fenofibrate, colesevelam HCl, or ezetimibe.

[0147] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulations of the present invention, or the GLP-1 and / or glucagon receptor agonists described herein or pharmaceutically acceptable salts thereof, are administered in combination with a diuretic, including but not limited to organic mercurials, ethacrynic acid, furosemide, bumetanide, piretanide, muzolimine, chlorothiazide and thiazides, phthalimidine, chlorthalidone, clorexolone, quinazolinone, quinethazone, metolazone irendensulfonamide, mefruside, chlorobenzamide, clopamide salicylamide, xipamide, xanthine, aminophylline, carbonic anhydrase inhibitors, acetazolamide mannitol, potassium-sparing diuretics, aldosterone antagonists, spironolactone and canrenoate, pteridine, pyrazine, carboxamide-triamterene, or amiloride.

[0148] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulation of the present invention, or the GLP-1 and / or glucagon receptor agonist or a pharmaceutically acceptable salt thereof described herein, is administered in combination with an antidiabetic drug. In one embodiment, the antidiabetic drug is a sulfonylurea. Sulfonylureas include, but are not limited to, tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide, glimepiride, or gliclazide. In one embodiment, the antidiabetic drug is a meglitinide, such as prandin or nateglinide. In one embodiment, the antidiabetic drug is a biguanide, such as metformin. In one embodiment, the antidiabetic drug is a thiazolidinedione, such as rosiglitazone, pioglitazone, or troglitazone. In one embodiment, the antidiabetic drug is an alpha-glucosidase inhibitor, such as miglitol or acarbose. In one embodiment, the antidiabetic agent is a PPARα / γ ligand, a dipeptidyl peptidase 4 (DPP-4) inhibitor, an SGLT (sodium-dependent glucose transporter 1) inhibitor, a glucagon modulator, a glucocorticoid receptor (GR) antagonist, a glucokinase activator (GK), a glycogen phosphorylase inhibitor (GP), an incretin (GLP-1 and GIP) or mimetic, a triacylglycerol lipase inhibitor, an insulin-like growth factor 1 (IGF-1) or analog, an insulin receptor (IR) modulator, a PTP-1B phosphatase (or PTPN1) inhibitor, and / or a modulator of glycogen synthase kinase-3 (GSK3beta).

[0149] In one embodiment, the antidiabetic drug is insulin, for example, an ultrarapid-acting insulin, a short-acting insulin, and / or an intermediate-acting insulin. In one embodiment, the insulin is a long-acting insulin. In one embodiment, the antidiabetic drug is an inhibitor of fatty acid binding protein (aP2), glucagon-like peptide-1 (GLP-1), and dipeptidyl peptidase IV (DPP4) inhibitor.

[0150] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulations of the invention, or the GLP-1 and / or glucagon receptor agonists described herein, or pharmaceutically acceptable salts thereof, are administered in combination with one or more agents treating a metabolic disease, disorder, or condition, which in some embodiments refers to metabolic syndrome. In one embodiment, such agents include, but are not limited to, pancreatic lipase inhibitors such as orlistat or cetilistat, insulin sensitizers such as biguanides (metformin) or PPAR agonists, dual-action PPAR agonists (muraglitazar, tesaglitazar, naveglitazar), PPAR-delta agonists, DPP-IV inhibitors (vildagliptin, sitagliptin), alpha-glucosidase inhibitors (acarbose), glucagon-like peptide-1 analogs (exenatide), , liraglutide), amylin analogs (pramlintide), statins (atorvastatin, simvastatin, rosuvastatin, pravastatin, fluvastatin, lovastatin, pitavastatin), cholesterol absorption inhibitors (ezetimibe), ACE inhibitors (ramipril, captopril, lisinopril), AT-II receptor antagonists (valsartan, telmisartan), cannabinoid receptor antagonists (rimonabant), cholesteryl ester transfer protein, or combinations thereof.

[0151] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulations of the present invention, or the GLP-1 and / or glucagon receptor agonists described herein, or pharmaceutically acceptable salts thereof, are administered in combination with an antihypertensive agent. An antihypertensive agent is any agent that lowers blood pressure, including, but not limited to, calcium channel blockers, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin II receptor antagonists (A-II antagonists), diuretics, beta-adrenergic receptor blockers (β-blockers), vasodilators, and alpha-adrenergic receptor blockers (α-blockers). For example, calcium channel blockers can include amlodipine, bepridil, cretiazem, diltiazem, fendiline, gallopamil, mibefradil, prenylamine, semotiadil, terodiline, verapamil, aranidipine, barnidipine, benidipine, cilnidipine, efonidipine, elgodipine, felodipine, and / or isradipine.

[0152] In one embodiment, angiotensin-converting enzyme inhibitors (ACE inhibitors) may include, but are not limited to, alacepril, benazepril, captopril, lisinopril, quinapril, temocapril, and / or trandolapril. Angiotensin-II receptor antagonists (A-II antagonists) may include, but are not limited to, candesartan, eprosartan, irbesartan, losartan, and / or valsartan. Beta-blockers may include, but are not limited to, alprenolol, bufuralol, epanol, indenol, nebivalol, practolol, sulfinalol, tilisolol, timolol, toliprolol, and / or xybenolol. Alpha-blockers may include, but are not limited to, amosulalol, arotinolol, dapiprazole, fenspiride, indoramin, labetolol, prazosin, tamsulosin, tolazoline, and / or yohimbine.

[0153] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof, is administered together with an agent for treating a metabolic disorder. In some embodiments, the agent for treating a metabolic disorder includes, but is not limited to, vitamins, coenzyme Q10, glucosidase alpha, sodium bicarbonate, bisphosphonates, biotin, allopurinol, levodopa, diazepam, phenobarbital, haloperidol, folic acid, antioxidants, cation channel activators, haptoglobin, and / or carnitine.

[0154] In one embodiment, the agent treating a metabolic disorder is a pancreatic lipase inhibitor such as orlistat or cetilistat, a biguanide, an insulin sensitizer such as a PPAR agonist, a PPAR-delta agonist such as muraglitazar, tesaglitazar, or naveglitazar, a DPP-IV inhibitor such as vildagliptin or sitagliptin, an alpha glucosidase inhibitor such as acarbose, a glucagon-like peptide-1 analogue such as exenatide or liraglutide, a prazolam, or a prazolam. amylin analogues such as lintide; statins such as atorvastatin, simvastatin, rosuvastatin, pravastatin, fluvastatin, lovastatin, or pitavastatin; cholesterol absorption inhibitors such as ezetimibe; ACE inhibitors such as ramipril, captopril, or lisinopril; AT-II receptor antagonists such as valsartan or telmisartan; cannabinoid receptor antagonists such as rimonabant; or beta-3 adrenergic agonists.

[0155] In one embodiment, the GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist described herein, or a pharmaceutically acceptable salt thereof, is administered with a vitamin. In some embodiments, the vitamin includes, but is not limited to, vitamin D, vitamin E, vitamin K, vitamin B, vitamin C, or a combination thereof.

[0156] As used herein, in some embodiments, the terms "administered in combination with" and "administered together" are used interchangeably.

[0157] In one embodiment, the present invention provides a method of administering a stable liquid formulation of the present invention. The method includes a first administration period of a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog) at a first once-weekly (QW) dose, followed by a second administration period at a second once-weekly (QW) dose, where the second QW dose is greater than the first QW dose. In one embodiment, the first QW dose is about 1-20 mg QW. In one embodiment, the second QW dose is about 10-50 mg QW. In one embodiment, the first QW dose in the first administration period is about 20 mg QW, followed by a second administration period providing a second QW dose of 40 mg QW. In one embodiment, the first administration period is 2-6 weeks, or 2-5 weeks, or 3-5 weeks. In another embodiment, the first administration period is 4 weeks. In one embodiment, the second administration period is 2 to 6 weeks, or 2 to 5 weeks, or 3 to 5 weeks. In another embodiment, the second administration period is 4 weeks.

[0158] The formulations of the present invention are substantially free of any impurities. In one embodiment, the formulations of the present invention have about 10% or less of total impurities, or about 8.0% or less of total impurities, or about 7.0% or less of total impurities, or about 6.0% or less of total impurities, or about 5.0% or less of total impurities, or about 4.0% or less of total impurities, or about 3% or less of total impurities, or about 2% or less of total impurities, or about 1% or less of total impurities. In one embodiment, the formulations of the present invention have about 5.0% or less of any one impurity, about 4.0% or less of any one impurity, about 3.5% or less of any one impurity, about 3.0% or less of any one impurity, about 2.5% or less of any one impurity, about 2.0% or less of any one impurity, about 1.5% or less of any one impurity, about 1.0% or less of any one impurity, about 0.5% or less of any one impurity, about 0.25% or less of any one impurity, or about 0.1% or less of any one impurity.

[0159] In one embodiment, the formulation of the present invention is OPK-88003 injection, supplied as a 1 mL (extractable volume) sterile solution packaged in a 2 mL clear flint glass vial. The drug product (Formula 3) is a clear, colorless solution containing 70 mg / mL OPK-88003 (label claim) and the inactive ingredients L-methionine, sorbitol, and sodium acetate trihydrate at a pH of approximately 4.5. A complete list of ingredients and quantitative formulation is provided below.

[0160] [Table 1]

[0161] The present invention further encompasses stable liquid formulations comprising a compound of Formula 3, Formula 4, or Formula 5, or a compound comprising any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or active fragments thereof, wherein the formulation has a pH range of about 3.5 to about 8.0 or about 4.0 to about 7.0. In one embodiment, the formulation is substantially free of sodium chloride. In one embodiment, the formulation of the present invention is substantially free of cysteine. The formulation optionally includes additional excipients selected from organic bases, isotonicity agents, and other excipients that do not destabilize or negatively impact the formulation. Broadly, the compositions or claimed inventions may encompass OXM, any OXM analogs described herein or in the literature and patent literature, as well as related or similar GLP-1 / glucagon receptor agonists. For example, such patent publications or patents include US20150290324, US20110034374, US20150182593, US20150024066, US20150072924, US20150148289, US20090298757, US20140349922, US20150299282 , U.S. Patent No. 8,415,926, U.S. Patent No. 8,415,296, U.S. Patent No. 8,367,607, US20110152182, US20140128318, US20170183383, US20140249299, U.S. Patent No. 8,263,084, US20130281374, US20160317 623, U.S. Patent No. 8,729,017, WO / 2016045400, WO2008076933, US20130059780, US20170226175, US20170349642, US20100121032, US20110136737, US2013137631, U.S. Patent No. 9,663,565, US20150111246, U.S. Patent No. 9,301,938, U.S. Patent No. 9,758,562, US20170128589, US20170088598, US20130085104, US20120183617, US20140142023, all of which are incorporated herein by reference.

[0162] The terms OXM, "oxyntomodulin," "oxyntomodulin analog," "oxyntomodulin peptide analog," or "oxyntomodulin derivative" include native oxyntomodulin and any derivative of such peptide prepared or containing substitutions, additions, deletions, or post-translational modifications (methylation, acylation, ubiquitination, intramolecular covalent linkages) in the amino acid sequence (HSQGTGTSDYSKYLDSRRAQDFVQWLMNKRNRNNIA) (SEQ ID NO: 4), all of which retain and / or have improved properties at either or both the GLP-1 receptor and the glucagon receptor. Such compounds may include those having SEQ ID NOs: 10-42 (corresponding to sequences 4-36 of US20170128589) with at least one optional cysteine ​​residue at positions 38 and / or 39. Amino acids and / or atypical amino acids are available from commercial sources, such as Sigma-Aldrich. Furthermore, such compounds may include derivatives or variants of oxyntomodulin (OXM) peptides obtained through modifications to any amino acid of the oxyntomodulin (OXM) peptide, as well as any side chain or terminus of an amino acid with a functional group or polymer, incorporation of natural or non-natural amino acids and / or their derivatives within the peptide, and / or cross-linking with other peptides.

[0163] The present invention further encompasses the use of stable liquid formulations of the invention, e.g., formulations having a compound of Formula 3, for treating diabetes and / or obesity, and / or excess body weight in patients in need of treatment thereof. The present invention includes stable PEGylated oxyntomodulin-like liquid formulations that effectively lower HbA1c in patients with type 2 diabetes, obesity, or any related medical disorder, disease, or condition that is treated or treatable by a GLP-1 and / or glucagon receptor agonist.

[0164] The present invention encompasses the use of the stable PEGylated oxyntomodulin-like liquid formulations of the present invention to effectively lower HbA1c, further reduce body weight, and / or alter fasting plasma glucose (FPG) in patients with type 2 diabetes, obesity, or any related medical disorder, disease, or condition that is being treated or treatable with a GLP-1 and / or glucagon receptor agonist.

[0165] The present invention encompasses a method for treating type 2 diabetes, obesity, or any related medical disorder, disease, or condition that is treated or treatable by a GLP-1 and / or glucagon receptor agonist in a patient in need of such treatment, comprising administering to the patient a stable liquid formulation of the present invention, for example, a formulation having a PEGylated oxyntomodulin analog of Formula 3.

[0166] As used herein, the term "PEGylation" refers to the attachment of one or more poly(ethylene glycol) (PEG) molecules to an OXM analogue described herein. The PEG group may be covalently attached to the OXM molecule directly or via a linking group, while "PEGylated" refers to an OXM analogue having a PEG substituent attached thereto.

[0167] PEGylation of oxyntomodulin (OXM) peptide analogs can occur via reaction with available amino or sulfhydryl groups, primarily on the OXM component with PEG bearing reactive functional groups, including, but not limited to, azide, carbonate, ester, aldehyde, acrylate, carboxyl, carbodiimide, carbonylimidazole, dichlorotriazine, epoxy, isocyanate, isothiocyanate, maleimide, nitrophenyl carbonate, orthopyridyl disulfide, pyridinyloxycarbonyl, succinimidyl carbonate, succinimidyl glutanate, succinimidyl methyl butanoate, succinimidyl succinate, succinic acid, sulfhydryl, tresylate, vinyl sulfone, and the like.

[0168] The term "PEG" or "PEGylated" refers to any polyethylene glycol molecule of any size or modification. PEG may have a linear, branched, or multi-arm structure and may be monofunctional, bifunctional, or multifunctional. PEG may be substituted or unsubstituted, so long as at least one reactive site is available for substitution or reaction. PEG may have an average molecular weight of 200 to 100,000. PEG as used herein is commercially available (NOP Corporation, Kawasaki, Kanagawa Prefecture, Japan) or can be prepared by polymerizing ethylene glycol according to methods known to those skilled in the art.

[0169] For example, PEG has the formula HO-CH2CH2-(CH2CH2O) n The PEG may be a linear polymer having a terminal hydroxyl group of -CHCH-OH, where n is from about 5 to about 4,000. In one embodiment, n is from about 5 to about 2,500, or from about 8 to about 2,500, or from about 30 to about 2,000, or from about 35 to about 2,000, or from about 35 to about 1,500, or from about 35 to about 1,000, or from about 35 to about 750. The terminal hydrogen may be replaced with a capping group such as an alkyl moiety, e.g., a methyl group, or may be a terminal hydroxyl group that is covalently linked to a linker moiety that can react with oxyntomodulin. Thus, PEG can be linked either directly or indirectly to the oxyntomodulin peptide analog. For example, the linker moiety can be an amide, carbamate, or urea linkage to an amine group of the oxyntomodulin peptide analog (including, but not limited to, the epsilon amine of lysine or the N-terminus). Alternatively, the linker moiety can be a maleimide bond to a thiol group (including, but not limited to, the thiol group of cysteine). In one embodiment, the PEG molecule is mPEG-20kDa maleimide, which reacts with at least one cysteine ​​on the oxyntomodulin analog to form a PEGylated OXM peptide analog for use in a liquid formulation of the invention.

[0170] In one embodiment, linear PEG bearing an activated hydroxyl at each end can be activated at one or both ends with a similarly reactive vinyl sulfone (-SO-CH=CH) or its precursor or derivative (e.g., -SO-CH-CH-X, where X is a halogen) to become bifunctional, as described in U.S. Pat. No. 5,900,461, incorporated herein by reference. PEG vinyl sulfone is stable, isolatable, and highly selective for coupling with thiol moieties on molecules to form thiol-selective and hydrolytically stable linkages.

[0171] PEG can be branched or "multi-armed." For example, as described in U.S. Pat. No. 5,932,462, which is incorporated herein by reference, a branched or multi-arm polymer can have the structure:

[0172] [ka]

[0173] Poly a and Poly b are the above-mentioned HO-CH2CH 2- (CH2CH2O) n represents the -CH2CH2- moiety, and a and Poly b At least one of the groups has an essentially non-reactive end group, e.g., CH3O-CH2CH2-(CH2CH2O) n capped with a -CH2CH2- moiety ("mPEG-"), C represents a carbon, and P and Q may be the same or different and each represent a polymer arm, poly( a and Poly brepresents a linking fragment that connects C to C in the linking fragment by a hydrolytically stable bond, R can be H or a substantially non-reactive moiety, such as an alkyl, and the moiety -Z includes a moiety that has a single site reactive toward a nucleophilic moiety or that can be converted into a site reactive toward a nucleophilic moiety, as well as the reaction product of a nucleophilic moiety and a moiety that has a single site reactive toward a nucleophilic moiety. Such branched or multi-arm PEGs can have the following structure:

[0174] [ka]

[0175] As described in U.S. Pat. No. 7,851,491, which is incorporated herein by reference, the multi-arm PEG can have the following structure:

[0176] [ka]

[0177] R is a hydrocarbon group having 1 to 24 carbon atoms, OA 1 and O.A. 2 are each an oxyethylene group, n represents 0 to 1000, m represents 10 to 1000, and X represents a functional group, e.g., a halogen atom selected from Cl, Br, and I, which can chemically react with an oxyntomodulin (OXM) peptide analog.

[0178] In one embodiment, the branched polyethylene glycol is R[-PEG(linear)-OH] as described in U.S. Pat. No. 6,436,386, which is incorporated herein by reference. m where R represents a core moiety such as glycerol or pentaerythritol, and m represents the number of arms.

[0179] In one embodiment, PEG has the following structure: TL-poly a -R(-Polyb -X) q and a and Poly b is -O-(CH2CH2O) n is —CH2CH2—O—, R is a core molecule such as an amino acid or a polyol (e.g., trimethylolpropane, di-trimethylolpropane, glycerol, pentaerythritol, sorbitol, lysine, and di-lysine), L is a linker, T is a bisphosphonate, Q is an integer from 2 to about 300, and X can be a protected or unprotected chemically reactive group.

[0180] In one embodiment, the bifunctional PEG derivative may have the structure Y-PEG-X, as described in U.S. Patent No. 6,448,369, incorporated herein by reference, where the first functional group X and the second functional group Y are reactive moieties that allow the PEG derivative to react with other molecules, for example, that one desires to conjugate to oxyntomodulin or its analog. Examples of functional groups X and Y include, but are not limited to, mesylate, tosylate, tresylate, -O-(CH) n -CO2H (in the formula, N=1-6), -O-(CH2) n -CO2R 3 (wherein n=1-6 and R 3 is an alkyl group), -NHR 4 (In the formula, R 4 is H or an alkyl or amine protecting group (such as t-Boc and Fmoc), -O-(CH2) n -CH(ZR 5 ) 2 (wherein n is a number from 1 to 6, Z is O or S, and R 5 is H or an alkyl group), -O-(CH2) n -CHO, and O2CCH2CH2CO2R 6 (In the formula, R 6 is H, or NHS, which stands for N-succinimidyl).

[0181] In one embodiment, PEG can comprise a polymer derivative comprising a water-soluble and non-peptidic polymer backbone having at least one terminus attached to the following structure: -WT, where W is -OC-(CH) n -O-, -O-(CH2) n -CO2-(CH2) m -, -O-(CH2) n -CO2-(CH2) m -O-, -(CH2) n -OPO3-(CH2) m - and -O-(CH2) n -CO2-(CHR) m -, wherein each R is H or alkyl, provided that at least one R is alkyl, n is from about 1 to about 5, and m is from about 1 to about 5; and T is a reactive group, such as -COOH, -CO2-succinimidyl, -CO2-sulfosuccinimidyl, -CO2-p-nitrophenyl, and pyridyl disulfide. Such PEGs can be found in U.S. Pat. No. 6,515,100, which is incorporated herein by reference. Examples of such PEGs can include, but are not limited to, the following: CH3-O-PEG-O-(CH2) n -CO2-(CH2) m -COOH. HOOC-(CH2) m -O2C-(CH2) n -O-PEG-O-(CH2) n -CO2-(CH2) m -COOH. NHS-O2C-(CH2) m -O2C-(CH2) n -O-PEG-O-(CH2) n -CO2-(CH2) m -CO2-NHS (NHS is succinimidyl).

[0182] In one embodiment, PEG containing an aldehyde functional group (PEG-aldehyde) spontaneously reacts with the amine and thiol functional groups of cysteine ​​residues in oxyntomodulin peptide analogs, as described in U.S. Patent Publication No. 2010 / 0016550A1, incorporated herein by reference. The newly generated functional group between PEG and oxyntomodulin peptide analogs is 1,3-thiazolidine. Such PEGs can provide site-specific PEGylation of oxyntomodulin peptide analogs.

[0183] In one embodiment, the PEGs used in the present invention may have different molecular weights (e.g., 2-40 kDa), may have linear, branched, and / or multi-arm structures, and may contain one or more functional groups. When PEGs containing two functional groups are used, the final product is an OXM dimer, with the PEG as the linker between them. PEGs with multiple functional groups will produce multimers of PEGylated OXM.

[0184] In one embodiment, the terms "PEGylated oxyntomodulin peptide analog," "PEGylated oxyntomodulin compound," "PEGylated oxyntomodulin," and "PEGylated oxyntomodulin analog" are used interchangeably herein. In one embodiment, oxyntomodulin peptide analog refers to a PEGylated oxyntomodulin peptide analog.

[0185] In one embodiment, the PEGylated oxyntomodulin peptide analog is OPK-88003 (Formula 3). In one embodiment, the PEGylated oxyntomodulin peptide analog is Formula 4. In one embodiment, the PEGylated oxyntomodulin peptide analog is Formula 5.

[0186] The oxyntomodulin peptide analogs used in the formulations herein can be made as described in U.S. Pat. No. 8,367,607 or U.S. Pat. No. 8,415,296, the entire contents of which are incorporated herein by reference. Additionally, compounds of the present invention can be prepared as described in Example 1 and FIG. 1. Generally, Fmoc-Rink amide MBHA resin is deprotected (piperine / DMF), and then the first amino acid (Fmoc-Cys(Trt)-OH) is coupled to the resin in a suitable solvent. The deprotection step is repeated, and the selected protected amino acid is used to prepare the selected polypeptide analog. Cleavage from the resin and deprotection leads to the target polypeptide. In a preferred embodiment, an amide is formed at the C-terminus of the amino acid cleaved from the resin.

[0187] Peptide analogs according to SEQ ID NOS: 1-9, as well as Formulas 3, 4, and 5, were produced by solid-phase peptide synthesis on a Protein Technologies Inc. Symphony or Applied Biosystems 433A automated peptide synthesizer. Synthesis was performed on Fmoc-Rink amide polystyrene resin (Rapp Polymere Tubigen, Germany) at a substitution of approximately 0.7 mmol / g. Synthesis was performed using an Fmoc backbone protecting group strategy. The amino acid side chain derivatives used were Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(boc), Ser(OtBu), Thr(OtBu), Trp(Boc), and Tyr(OtBu). Coupling is performed using approximately 10 equivalents of amino acid activated with diisopropylcarbodiimide (DIC) and hydroxybenzotriazole (HObt) (1:1:1 molar ratio) in dimethylformamide (DMF) or N-methylpyrrolidinone (NMP). Coupling is performed at room temperature for 45-90 minutes.

[0188] Simultaneous cleavage from the resin and removal of side chain protecting groups is carried out in a solution containing trifluoroacetic acid (TFA):triisopropylsilane:3,6-dioxa-1,8-octanedithiol:methanol:anisole 90:4:2:2:2 (v / v) at room temperature for 1.5-2 h. The solution is filtered and concentrated to less than 2 mL, and the peptide is precipitated with cold diethyl ether, redissolved in 30-40 mL of 10% acetonitrile, and centrifuged at a flow rate of 12-15 mL / min. 18 Purify on a reversed-phase high performance liquid chromatography column.

[0189] The sample is eluted with a two-step linear AB gradient of 0–25% B over 20 min, followed by 25–75% B over 100 min (A = 0.05% TFA / water and B = 0.05% TFA / acetonitrile). The product typically elutes at 30–35% acetonitrile. Peptide purity and molecular weight are confirmed using an Agilent 1100 Series liquid chromatography mass spectrometer with a single quadrupole MS detector. The peptide analogs are purified to >95% purity. Salts can be formed from the cleaved polypeptide with the preferred carboxylate salt (e.g., trifluoroacetate). The resulting TFA salt is then used in the final PEGylation step(s). A cation scavenger is utilized to prevent alkylation of the amide from the cleaved resin.

[0190] The resulting peptide is then PEGylated. Typically, lyophilized peptide analogs or salts of the appropriate sequence, such as those shown in SEQ ID NOS: 1-9 and Formulas 3, 4, and 5, or as otherwise set forth herein or in U.S. Pat. No. 8,367,607 or U.S. Pat. No. 8,415,296, are weighed out according to the amount of material required. Small-scale synthesis can begin with, for example, 30-50 mg. The desired molecular weight and chemical structure (e.g., mPEG-20kDa maleimide) (CHO(CHCH)) is then added. nA 2.5-fold molar equivalent of -(CH2)3NHCO(CH2)2 maleimide) is weighed and combined with the peptide of SEQ ID NO: 1 or 2. The reactants are dissolved in a 50 / 50 (v / v) water / acetonitrile mixture to a peptide concentration of approximately 20 mg / mL. The peptide analog solution is diluted 2-fold with 100 mM ammonium acetate, 10 mM ethylenediaminetetraacetic acid (EDTA), pH 7. The resulting mixture is monitored by analytical reverse-phase HPLC, and the reaction is complete after approximately 2 hours. The PEGylated compound is then diluted and purified (ion exchange, reverse-phase HPLC) as described above to form a purified form of the peptide having Formula 3 or the target PEGylated derivative. In a preferred embodiment, the purified form is a salt form, such as an acetate salt. The purified compound may be in crystalline or amorphous form. The reaction is essentially scaled up to obtain sufficient quantities for clinical or commercial use.

[0191] Thus, the present invention provides a process for preparing a pharmaceutically acceptable salt of a PEGylated oxyntomodulin peptide analog. In another embodiment, the present invention provides a process for preparing a PEGylated oxyntomodulin peptide analog.

[0192] For example, the present invention provides a process for preparing an acetate salt of a PEGylated oxyntomodulin peptide analog, (a) dissolving an oxyntomodulin (OXM) peptide analog in a buffer to form an OXM peptide analog solution, wherein the buffer has a pH value of about 6.6 to about 7.0; (b) adding an OXM peptide analog solution to a stirred solution of PEG to produce a reaction mixture comprising a crude PEGylated oxyntomodulin peptide analog product, wherein the solution of PEG is formed by dissolving PEG in the same buffer solution as that in which the OXM peptide analog of step (a) is dissolved; (c) adjusting the reaction mixture to a pH of about 3.95 to about 4.05 using acetic acid; (d) purifying the crude PEGylated oxyntomodulin peptide analog product on an HPLC column with a gradient of acetonitrile and dilute aqueous acetic acid, followed by concentration and lyophilization to provide the acetate salt of the PEGylated oxyntomodulin peptide analog.

[0193] In one embodiment, the HPLC column in step (d) is a reverse-phase HPLC column. In one embodiment, the process of the invention further comprises purifying the crude PEGylated oxyntomodulin peptide analog product on a cation exchange HPLC column prior to step (d). In one embodiment, the PEGylated oxyntomodulin peptide analog product prepared by the process of the invention is a compound of Formula 3, 4, or 5. In other embodiments, the PEGylated oxyntomodulin peptide analog product is a compound of Formula 3.

[0194] In one embodiment, the buffer in the process of the present invention is a solution of acetonitrile and ammonium acetate buffer. In one embodiment, the PEG in the process of the present invention has a molecular weight as described herein. In one embodiment, the PEG in the process of the present invention has a molecular weight of about 20 kDa, or about 30 kDa, or about 40 kDa. In one embodiment, the PEG includes a linker. In one embodiment, the linker is a maleimide linker. In certain embodiments, the PEG is a compound of Formula 1.

[0195] In another aspect, the present invention provides a pharmaceutically acceptable salt of a PEGylated oxyntomodulin peptide analog described herein, for example, a PEGylated oxyntomodulin peptide analog comprising any one of SEQ ID NOs: 1-9 or active fragments thereof, or a PEGylated oxyntomodulin peptide analog represented by Formula 3, Formula 4, or Formula 5.

[0196] Pharmaceutically acceptable salts of the PEGylated oxyntomodulin peptide analogs described herein can be formed by reacting an acid with a basic group of the PEGylated oxyntomodulin peptide analog, such as an amino functional group, or by reacting a base with an acidic group of the PEGylated oxyntomodulin peptide analog, such as a carboxylic acid functional group. In one embodiment, a pharmaceutically acceptable salt of a PEGylated oxyntomodulin peptide analog is an acid addition salt of an amino functional group in the PEGylated oxyntomodulin peptide analog, e.g., the amino group of lysine or other amino acids.

[0197] Suitable pharmaceutically acceptable salts of the amino group of the PEGylated oxyntomodulin peptide analogs described herein can be prepared from inorganic or organic acids. Examples of inorganic salts of amines include, but are not limited to, bisulfate, borate, bromide, chloride, hemisulfate, hydrobromide, hydrochlorate, 2-hydroxyethylsulfonate (hydroxyethanesulfonate), iodate, iodide, isothionate, nitrate, persulfate, phosphate, sulfate, sulfamate, sulfanilate, sulfonic acids (alkylsulfonate, arylsulfonate, halogen-substituted alkylsulfonate, halogen-substituted arylsulfonate), sulfonate, and thiocyanate.

[0198] In one embodiment, examples of organic salts of amines may be selected from, but are not limited to, aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic organic acids, examples of which include acetate, arginine, aspartate, ascorbate, adipate, anthranilate, algenate, alkanecarboxylate, substituted alkanecarboxylate, alginate, benzenesulfonate, benzoate, bisulfate, butyrate, bicarbonate, bitartrate, carboxylate, citrate, camphorate, camphorsulfonate, cinnamate, methyl ... Cyclohexylsulfamate, cyclopentanepropionate, calcium edetate, camsylate, carbonate, clavulanate, cinnamate, dicarboxylate, digluconate, dodecylsulfonate, dihydrochloride, decanoate, enanthate, ethanesulfonate, edetate, edisylate, estolate, esylate, fumarate, formate, fluoride, galacturonate, gluconate, glutamate, glycolate, glucorate, glucoheptanoate, glycerophosphate, glucept Acid salts, glycollylarsanilate, glutarate, glutamate, heptanoate, hexanoate, hydroxymaleate, hydroxycarboxylic acid, hexylresorcinate, hydroxybenzoate, hydroxynaphthoate, hydrofluorate, lactate, lactobionic acid, laurate, malate, maleate, methylenebis(beta-oxynaphthoate), malonate, mandelate, mesylate, methanesulfonate, methyl bromide, methyl nitrate, methyl sulfonate Fonate, monopotassium maleate, mucate, monocarboxylate, nitrate, naphthalenesulfonate, 2-naphthalenesulfonate, nicotinate, napsylate, N-methylglucamine, oxalate, octanoate, oleate, pamoate, phenylacetate, picrate, phenylbenzoate, pivalate, propionate, phthalate, pectinate, phenylpropionate, palmitate, pantothenate, polygalacturonate, pyruvate, quinate, salicylate, succinate, stearate, sulfanilate,These include basic acetate, tartrate, theophylline acetate, p-toluenesulfonate (tosylate), trifluoroacetate, terephthalate, tannate, theoclate, trihaloacetate, triethiodide, tricarboxylate, undecanoate, and valerate.

[0199] In one embodiment, inorganic acids include, but are not limited to, hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids include, but are not limited to, para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, and related inorganic and organic acids. Accordingly, such pharmaceutically acceptable salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-diol ... -dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and other salts.

[0200] In one embodiment, examples of inorganic salts of carboxylic acids or phenols may be selected from alkali metals including ammonium, lithium, sodium, potassium, and cesium, alkaline earth metals including calcium, magnesium, and aluminum, zinc, barium, choline, quaternary ammonium.

[0201] In another embodiment, examples of organic salts of carboxylic acids or phenols may be selected from arginine, organic amines including aliphatic organic amines, alicyclic organic amines, aromatic organic amines, benzathine, t-butylamine, benethamine (N-benzylphenethylamine), dicyclohexylamine, dimethylamine, diethanolamine, ethanolamine, ethylenediamine, hydrabamine, imidazole, lysine, methylamine, meglamine, N-methyl-D-glucamine, N,N'-dibenzylethylenediamine, nicotinamide, ornithine, pyridine, picoline, piperazine, procaine, tris(hydroxymethyl)methylamine, triethylamine, triethanolamine, trimethylamine, tromethamine, and urea.

[0202] As described herein, bases commonly used to form pharmaceutically acceptable salts of GLP-1 and / or glucagon receptor agonists (e.g., PEGylated oxyntomodulin peptide analogs) include, but are not limited to, hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals, such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; organic amines, such as ammonia, unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl; N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(Cl—C6)-alkylamines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids, such as arginine and lysine.

[0203] In one embodiment, the salts may be formed by reacting the free base or free acid form of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water, from which the ions of the existing salt are removed by conventional means, for example, in vacuo, or by lyophilization, or by exchanging the ions of the existing salt with another ion or a suitable ion exchange resin. In one embodiment, the pharmaceutically acceptable salts are prepared by the processes described herein.

[0204] The present invention provides pharmaceutically acceptable salts of GLP-1 and / or glucagon receptor agonists (e.g., PEGylated oxyntomodulin peptide analogs), wherein the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or active fragments thereof, or is represented by Formula 3, Formula 4, or Formula 5. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In one embodiment, the pharmaceutically acceptable salt of the present invention is a hydrochloride, hydrobromide, ascorbate, maleate, or acetate salt. In one embodiment, the salt is an acetate salt. In one embodiment, the salt is a hydrochloride salt.

[0205] In one aspect, the present invention provides a hydrochloride or acetate salt of a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog) in crystalline or amorphous form, wherein the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOs: 1-9 or an active fragment thereof, or is represented by Formula 3, Formula 4, or Formula 5. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In one embodiment, the acetate salt is in crystalline or amorphous form.

[0206] Thus, in another aspect, the present invention provides acetate salts of GLP-1 and / or glucagon receptor agonists (e.g., PEGylated oxyntomodulin peptide analogs) as described herein. For example, the present invention provides acetate salts of compounds of Formula 3. In one embodiment, the present invention provides acetate salts of compounds of Formula 4. In another embodiment, the present invention provides acetate salts of compounds of Formula 5. In one embodiment, the acetate salts of compounds of the present invention are in crystalline or amorphous form. In one embodiment, the acetate salts of compounds of the present invention are in amorphous form.

[0207] Furthermore, the present invention provides a hydrochloride salt of a compound of the present invention. For example, the present invention provides a hydrochloride salt of a compound of formula 3. In one embodiment, the present invention provides a hydrochloride salt of a compound of formula 4. In another embodiment, the present invention provides a hydrochloride salt of a compound of formula 5. In one embodiment, the hydrochloride salt of a compound of the present invention is in crystalline or amorphous form. In one embodiment, the hydrochloride salt of a compound of the present invention is in amorphous form.

[0208] In another aspect, the present invention provides a GLP-1 and / or glucagon receptor agonist formulation comprising an acetate salt of a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog) and a pharmaceutically acceptable excipient, wherein the GLP-1 and / or glucagon receptor agonist comprises any one of SEQ ID NOS: 1-9 or an active fragment thereof, or is represented by Formula 3, Formula 4, or Formula 5. In one embodiment, the GLP-1 and / or glucagon receptor agonist is represented by a compound of Formula 3. In one embodiment, the GLP-1 and / or glucagon receptor agonist is the acetate salt of a compound of Formula 3. In one aspect, the present invention provides a GLP-1 and / or glucagon receptor agonist formulation comprising an acetate salt of a GLP-1 and / or glucagon receptor agonist of the present invention and an aqueous buffer, wherein the formulation has a pH value of about 3.5 to about 8.0 and one or more additional pharmaceutically acceptable excipients.

[0209] As described in U.S. Patent Nos. 8,367,607 and 8,415,296, which are incorporated by reference, GLP-1 and / or glucagon receptor agonists, such as the PEGylated oxyntomodulin analogs described herein, are useful for treating type 2 diabetes, type 1 diabetes, obesity, or any related medical disorder, disease, or condition that is or can be treated by a GLP-1 and / or glucagon receptor agonist. Formulations of the present invention comprising a GLP-1 and / or glucagon receptor agonist (e.g., a PEGylated oxyntomodulin peptide analog) can reduce food intake in patients in need of such treatment and are therefore useful for treating weight gain and / or obesity and / or metabolic disorders. The formulations of the present invention result in improved glucose tolerance and lipid profiles in patients with type 2 diabetes and / or related metabolic diseases or conditions, and / or obesity, or any related medical disorder, disease, or condition that is or can be treated by a GLP-1 and / or glucagon receptor agonist.

[0210] In addition to diabetic and obese patients, patients with impaired glucose tolerance or impaired fasting glucose, patients weighing more than about 25% above normal weight for their height and build, patients with gestational diabetes, and patients with metabolic disorders can also be treated with the improved, stable formulations of the present invention. Other diseases treatable with the claimed formulations include maturity-onset diabetes of the young (MODY), latent autoimmune diabetes, gestational diabetes, metabolic syndrome X, dyslipidemia, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and insulin resistance. The formulations of the present invention may also be useful in treating glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes.

[0211] The preparation of the present invention can be administered parenterally.This includes systemic administration by intramuscular, intravenous, subcutaneous, intradermal or intraperitoneal means.As described herein, the active pharmaceutical moiety in the form of PEGylated analogue is administered to the subject who needs its treatment.Such subject includes mammals such as humans and animals (farm animals, companion animals, etc.).

[0212] In one embodiment, the present invention provides a method for administering a GLP-1 and / or glucagon receptor agonist formulation of the present invention, or a GLP-1 and / or glucagon receptor agonist or a pharmaceutically acceptable salt thereof described herein. In one embodiment, the method comprises administering a stable liquid formulation of the present invention to a patient via a slow dose titration schedule. For example, the method comprises a first once-weekly (QW) dosing period at a first QW dose, followed by a second once-weekly (QW) dosing period providing dose escalation of the compound of the present invention, wherein the second QW dose is greater than the first QW dose. In one embodiment, the second QW dose is at least 0.25-2.0 times (e.g., 0.25, 0.5, 0.75, 1.0, 1.25, 1.50, 1.75, or 2.0 times) greater than the first QW dose. In one embodiment, the second QW dose is at least 0.5 times greater than the first QW dose. In one embodiment, the second QW dose is at least 0.75 times greater than the first QW dose. In one embodiment, the second QW dose is at least 1.0 times greater than the first QW dose. In one embodiment, the second QW dose is at least 1.25 times greater than the first QW dose. In one embodiment, the second QW dose is at least 1.5 times greater than the first QW dose. In one embodiment, the second QW dose is at least 1.75 times greater than the first QW dose.

[0213] In one embodiment, the method further includes at least one more (e.g., a third) dosing period providing a dose escalation of the compound of the present invention to a higher QW dose relative to the second QW dose. For example, the method further includes at least one more dosing period following the second dosing period providing a QW dose that is at least about 0.25-2.0 times (e.g., 0.25, 0.5, 0.75, 1.0, 1.25, 1.50, 1.75, or 2.0) times greater than the second QW dose. In one embodiment, the method further includes at least one more dosing period following the second dosing period providing a QW dose that is at least 0.25 times greater than the second QW dose. In one embodiment, the method further includes at least one more dosing period following the second dosing period providing a QW dose that is at least 0.5 times greater than the second QW dose. In one embodiment, the method further includes at least one more dosing period following the second dosing period providing a QW dose that is at least 0.75 times greater than the second QW dose. In one embodiment, the method further comprises at least one more administration period following the second administration period, providing a QW dose that is at least 1-fold greater than the second QW dose. In one embodiment, the method further comprises at least one more administration period following the second administration period, providing a QW dose that is at least 1.25-fold greater than the second QW dose. In one embodiment, the method further comprises at least one more administration period following the second administration period, providing a QW dose that is at least 1.5-fold greater than the second QW dose.

[0214] The administration of the first QW dose continues for a first administration period of at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks. The administration of the second QW dose continues for a second administration period of at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks. In one embodiment, the second QW dose is greater than the first QW dose. When the method further includes at least one more (e.g., a third) administration period providing a dose escalation of the compound of the present invention to a higher QW dose relative to the second QW dose, the additional (e.g., third) administration period continues for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks.

[0215] When the patient completes the initial titration period with the first low dose once a week, the dose is gradually increased to a maintenance dose.In one embodiment, when the patient completes the initial titration period with the first low dose once a week (QW), the dose is gradually increased to a second (higher than the first) weekly dose, optionally a third (higher than the second) weekly dose, and then to a higher weekly maintenance dose.Those skilled in the art will be able to determine the appropriate dose (in terms of safety and efficacy) for the first or second, or optionally the third weekly dose, and the subsequent weekly maintenance dose.Dose escalation allows the patient to receive therapeutic benefit while gradually being exposed to treatment.This administration schedule improves the patient's tolerance during treatment and reduces the incidence of side effects.

[0216] Generally, dose escalation occurs from a low dose of drug, e.g., about 1 to 30 (e.g., 1, 2, 3, 4, 5, 10, 12, 15, 20, 25, or 30) mg QW, to a higher dose of at least about 15 to 50 (e.g., 15, 20, 25, 30, 35, 40, 45, or 50) mg QW, and further to at least about 40 to 80 (e.g., 40, 45, 50, 55, 60, 65, 70, 75, or 80) mg QW. In one embodiment, the first QW dose is, e.g., about 1 mg QW to about 20 mg QW, followed by a second QW dose of about 10 mg QW to about 50 mg QW, followed by a third QW dose (or maintenance dose) of about 40 mg QW to about 80 mg QW. In one embodiment, a subject receives a dose of 20 mg QW for 4 weeks, followed by a dose of 40 mg QW for 4 weeks, followed by a maintenance dose of 70 mg QW. In one embodiment, a subject receives a dose of 20 mg QW for 4 weeks, followed by a dose of 40 mg QW for 4 weeks, followed by a maintenance dose of 80 mg QW.

[0217] In one embodiment, the first QW dose is about 1 mg QW to about 20 mg QW, e.g., about 3 mg QW to about 20 mg QW, about 5 mg QW to about 20 mg QW, about 8 mg QW to about 20 mg QW, about 10 mg QW to about 20 mg QW, or about 15 mg QW to about 20 mg QW. In one embodiment, the second QW dose is about 10 mg QW to about 50 mg QW, about 15 mg QW to about 50 mg QW, about 20 mg QW to about 50 mg QW, about 25 mg QW to about 50 mg QW, about 30 mg QW to about 50 mg QW, about 35 mg QW to about 50 mg QW, or about 40 mg QW to about 50 mg QW. In one embodiment, the third mg QW dose is about 40 mg QW to about 100 mg QW, about 45 mg QW to about 100 mg QW, about 50 mg QW to about 100 mg QW, about 55 mg QW to about 100 mg QW, about 60 mg QW to about 100 mg QW, about 65 mg QW to about 100 mg QW, about 70 mg QW to about 100 mg QW, about 75 mg QW to about 100 mg QW, about 80 mg QW to about 100 mg QW, about 85 mg QW to about 100 mg QW, or about 90 mg QW to about 100 mg QW. In one embodiment, the third mg QW dose is a mg QW maintenance dose.

[0218] In one embodiment, exemplary dose escalation for a GLP-1 and / or glucagon receptor agonist, or a formulation comprising same, includes, but is not limited to, administering about 10 mg QW followed by about 20 mg QW, or about 10 mg QW followed by about 30 mg QW, or about 10 mg QW followed by about 40 mg QW, or about 10 mg QW followed by about 50 mg QW, or about 10 mg QW followed by about 60 mg QW, or about 10 mg QW followed by about 70 mg QW, or about 10 mg QW followed by about 80 mg QW, about 15 mg QW followed by about 20 mg QW, or about 15 mg QW followed by about 30 mg QW, or about 15 mg QW followed by about 40 mg QW, or about 15 mg QW followed by about 50 mg QW, or about 15 mg QW followed by about 60 mg QW, or about 15 mg QW followed by about 70 mg QW, or about 15 mg QW followed by about 80 mg QW, or about 20 mg QW followed by about 30 mg QW, or about 20 mg QW followed by about 40 mg QW, or about 20 mg QW followed by about 50 mg QW, or about 20 mg QW followed by about 60 mg QW, or about 20 mg QW followed by about 70 mg QW, or about 20 mg QW followed by about 80 mg QW, or about 40 mg QW followed by about 50 mg QW, or about 40 mg QW followed by about 60 mg QW, or about 40 mg QW followed by about 70 mg QW, or about 40 mg QW followed by about 80 mg QW, or about 50 mg QW followed by about 60 mg QW, or about 50 mg QW followed by about 70 mg QW, or about 50 mg QW followed by about 80 mg QW, or about 60 mg QW followed by about 70 mg QW, or about 60 mg QW followed by about 80 mg QW.

[0219] Alternatively, the GLP-1 and / or glucagon receptor agonist formulations of the present invention, or the GLP-1 and / or glucagon receptor agonist or pharmaceutically acceptable salt thereof described herein, may be administered twice a week, every other day, or daily, instead of or in lieu of the once-weekly (QW) dose administration described above. In one embodiment, the GLP-1 and / or glucagon receptor agonist formulations of the present invention, or the GLP-1 and / or glucagon receptor agonist or pharmaceutically acceptable salt thereof described herein, may be administered once a week, twice a week, or at longer dosing intervals.

[0220] In another aspect, the present invention provides oxyntomodulin peptide analogs, and formulations comprising oxyntomodulin peptide analogs, and their use in the treatment of diabetes and / or obesity. U.S. Patent Nos. 8,367,607 and 8,415,296, incorporated herein by reference, claim and disclose several PEGylated derivatives of oxyntomodulin analogs utilized herein and disclose compositions thereof.

[0221] One effective dosing regimen for delivering these molecules to patients is a once-weekly (QD) dosing schedule. The preferred delivery route is via subcutaneous administration using a liquid formulation. In addition to being effective, the specific molecules described herein in liquid formulations must be sterile and chemically and physically stable for both quality / safety / delivery purposes and shelf life. Parameters used to assess chemical and physical stability include, but are not limited to, appearance (color, clarity, etc.), pH, purity, impurities, assay, monomer purity, % aggregates, particulate matter, subvisible particles, peptide mapping, potency (in vitro activity), osmolality, and viscosity. Various stress conditions were applied to attempted liquid formulations of OXM to determine the formulation that provided optimal physical and chemical stability.

[0222] The inventors have discovered that while typical isotonic solutions containing sodium chloride are generally suitable for delivery of drugs in liquid or parenteral formulations, the presence of sodium chloride in solutions containing PEGylated oxyntomodulin analogs unexpectedly causes instability problems. Furthermore, the addition of the PEGylated oxyntomodulin compounds or analogs described herein surprisingly raises the pH above the preferred range. Thus, the inventors have discovered liquid formulations according to the present invention that meet the necessary physical and chemical stability criteria. By discovering the stable liquid formulations herein, the inventors avoid the need to use solid dispersions of the drug (i.e., lyophilized or otherwise) that must be reconstituted with water or saline prior to administration to patients. Furthermore, because the formulations are stable as refrigerated liquids and do not require freezing to maintain stability, they can be stored in prefilled syringes or cartridges rather than vials, making them easier for patients to use at home. Thus, a patient or healthcare professional can "administer" the dosage form directly from the pre-filled syringe, cartridge, or filled vial without the additional step of reconstituting the powder formulation, and / or such an individual could not use a pre-filled syringe (or pre-filled vial) that does not have such a stable liquid formulation.

[0223] The present invention therefore addresses the need to effectively deliver a formulation (e.g., a once-weekly formulation) at a suitable dosage to a patient in need of such treatment, and is directed to effectively reducing each of the criteria for determining type 2 diabetes or obesity or any related medical disorder, disease, or condition that is treated or treatable by a GLP-1 and / or glucagon receptor agonist, including reducing FPG to less than 126 mg / dL and / or reducing 2-hour PG to less than 200 mg / dL, and reducing A1C to less than 6.5% (48 mmol / mol) and reducing PG to less than 200 mg / dL. [Example]

[0224] Example 1: Preparation of acetate salts of compounds of the present invention The compounds of the invention are prepared by solid phase peptide synthesis (SPPS) and chromatographic purification.

[0225] Solid Phase Peptide Synthesis (SPPS) Solid-phase peptide synthesis (SPPS) of compounds is performed according to well-developed standard procedures and protocols that are routinely used worldwide. SPPS is the continuous synthesis of a peptide chain immobilized on a solid support by repeating a cycle that involves the following steps: removal of the N-terminal Fmoc protecting group of the peptide resin (or resin for first deprotection), washing, coupling of the next activated amino acid, capping by acetylation, and washing. This cycle is repeated until the desired peptide sequence is synthesized as described below.

[0226] For the synthesis of the compounds of the present invention, the α-amino group of each amino acid was protected with the base-sensitive 9-fluorenylmethyloxycarbonyl (Fmoc) group, while the side chain functional groups were protected with acid-labile groups. All amino acid derivatives used were commercially available (Figure 1).

[0227] For example, for compound Formula 3, the trifluoroacetate key intermediate was assembled on Fmoc-Rink amide resin. The Fmoc group on the Rink amide resin was removed with piperidine in DMF. After washing with DMF and IPA, a small sample of the resin was removed and subjected to a ninhydrin / Kaiser or chloranil in-process control test, which is a color test that measures residual resin-bound amine groups. The test should be positive to confirm the presence of free amine groups.

[0228] Coupling of Fmoc-protected amino acids was carried out in DMF with variable amino acid equivalents using DIC / HOBt or HBTu / DIPEA / HOBt for activation.

[0229] If at the coupling step the ninhydrin test is found to be negative or slightly positive, indicating a small amount of unreacted amine, the peptide-resin is acetylated using acetic anhydride and DIPEA in DMF. If the test indicates incomplete coupling (positive response), elongation or recoupling of the Fmoc-amino acid is carried out.

[0230] In the Fmoc cleavage step, the N-terminal Fmoc group was cleaved with piperidine in DMF. The solution was filtered, and the peptide resin was washed with DMF, followed by ethyl ether, and dried under vacuum to constant weight.

[0231] Cleavage of peptide from resin and deprotection: The crude peptide was cleaved from the resin by treating the protected peptide resin with TFA in the presence of cation scavengers (TIPS, DTE, and HO). This resulted in the simultaneous cleavage of the peptide from the resin and the removal of the side chain protecting groups from the peptide. The solvent was removed by a concentration step, and the residue was dried after precipitation in ethyl ether to give the crude deprotected peptide.

[0232] Purification of key intermediates The crude peptide solution was purified by preparative HPLC using reversed-phase silica washed with methanol and pre-equilibrated with dilute aqueous TFA. The adsorbed peptide was eluted from the column by applying a gradient of acetonitrile in dilute aqueous TFA (ambient temperature).

[0233] The resulting combined pure fractions were reapplied to the same column, after intermediate washing, pre-equilibrated with dilute aqueous TFA. The adsorbed peptides were eluted from the column by applying a gradient of acetonitrile in 0.1 M sodium perchlorate (pH adjusted to 3.1) and acetonitrile.

[0234] The resulting combined pure fractions were purified one final time using the same column and pre-wash / equilibration sequence as described above. The peptide was eluted using a gradient consisting of aqueous TFA and acetonitrile. Fractions were pooled and isolated by lyophilization.

[0235] Lyophilization of key intermediates: The pool of lyophilized products from the purification step was reconstituted in dilute aqueous TFA containing 5% acetonitrile and lyophilized to the final trifluoroacetate key intermediate of formula 3.

[0236] PEGylation: The trifluoroacetate salt key intermediate of Formula 3 was dissolved in a solution of acetonitrile and ammonium acetate buffer, the pH of which had been adjusted to approximately 6.6-7.0 using ammonium hydroxide or acetic acid, as appropriate. The solution was added to a flask containing a stirred solution of PEG (approximately 20 kDa) maleimide linker dissolved in the same buffer used to dissolve the peptide. Reaction progress was monitored by HPLC. Upon completion, the reaction mixture was then diluted with purified water, the pH adjusted to 3.95-4.05 using acetic acid, and proceeded to the purification step.

[0237] Purification and conversion of the drug substance of formula 3 to acetate salt Ion exchange purification of crude Formula 3 drug substance: The crude drug substance was diluted with purified water and applied to a cation exchange HPLC column. The drug substance was eluted using a gradient of acetonitrile and dilute aqueous NHOAc.

[0238] Desalting and acetate conversion of Formula 3 drug substance: The combined fractions from the ion exchange purification above were diluted with purified water and applied to a preparative reverse-phase HPLC column. The drug substance was eluted using a gradient of acetonitrile and dilute aqueous acetic acid. Fractions were pooled for concentration and lyophilization.

[0239] Lyophilization of acetate salt of drug substance formula 3: The lyophilized drug substance was reconstituted in a dilute aqueous solution of acetonitrile and acetic acid, filtered, and finally lyophilized.

[0240] Example 2: Characterization of the compound of formula 3 Analysis procedure Mass analysis by MS The mass spectrum of Formula 3 was obtained using an ESI ion trap mass spectrometer.

[0241] Intact mass by LC / MS with post-column neutralization The intact mass of formula 3 was determined by LC-MS analysis using post-column neutralization with diethylmethylamine (DEMA). When PEGylated molecules are eluted from RP-HPLC separations with a water / acetonitrile / TFA system, the flow is mixed with a DEMA solution before entering a Water SYNAPT G2-S Q-TOF mass spectrometer.

[0242] Pyrolysis peptide mapping using LC / MS Formula 3 was diluted to 1 mg / mL in 20 mM Tris-HCl buffer (pH 8) and then directly treated with thermolysin solution for 1 hour at 37° C. The digestion solution was directly analyzed using a Waters Acquity UPLC system coupled to a Waters SYNAPT G2-S mass spectrometer.

[0243] Mass spectrometry by MS / MS The amino acid sequence of Formula 3 was investigated by performing MS / MS analysis using both CID and ECD. The MS / MS spectrum was compared with the expected fragment ions for the drug substance. The expected multiply charged b and y fragment ions were calculated using a computer program developed by Croker et al. (Journal of Biomolecular Techniques, 2000, 11(3), 135-141). The fragment ions were assigned to the spectrum.

[0244] [ka]

[0245] There are three different types of bonds that can fragment along the amino acid backbone: NH-CH, CH-CO, and CO-NH. Breaking each bond produces two species, one neutral and the other charged, and only the charged species is monitored by the mass spectrometer. The charge can remain in either of the two fragments, depending on the chemistry and the relative proton affinities of the two species. Thus, there are six possible fragment ions for each amino acid residue, labeled as shown in the diagram above, with the a, b, and c′ ions carrying the charge on the N-terminal fragment and the x, y′, and z ions carrying the charge on the C-terminal fragment. For CID techniques, the most common cleavage site is at the CO-NH bond, producing the b and / or y′ ions, which are the most intense ions and therefore the most frequently used in peptide sequence analysis. The mass difference between two adjacent b or y′ ions indicates a specific amino acid residue. Larger b and y′ fragments are often found as multiply charged ions. For ECD techniques, the most common cleavage site is at the NH-CH bond, which produces c and / or z ions, but the principle for obtaining the amino acid sequence is similar.

[0246] Amino acid analysis by AAA This procedure is used to hydrolyze peptides (USP <1052> The amino acid composition is determined by ion-exchange separation of the resulting free amino acids and quantification by post-column derivatization with ninhydrin. The ion-exchange system is calibrated prior to analysis using a mixture of authenticated amino acids. Results are expressed as relative amino acid ratios expected to match the amino acid composition of the molecule, and as peptide content (quantity), calculated by taking the ratio of the average amino acid recovery to the total amount of sample. The peptide content is then used to calculate the quantity.

[0247] Chiral amino acid analysis by GC-MS Hydrolysis and derivatization The peptides were hydrolyzed in 6N DCl in DO (to compensate for any racemization that may have occurred during hydrolysis). After removing excess reagent with a stream of nitrogen, the dried samples were esterified with DCl in methanol. After cooling, the vials were opened and excess reagent was removed under a stream of nitrogen with slight heating. The residues were dissolved in trifluoroacetic anhydride or pentafluoropropionic anhydride, and the vials were capped and heated. After cooling to ambient temperature, excess reagent was removed with a stream of nitrogen. The residues were dissolved in toluene and subjected to analysis by GC-MS.

[0248] Gas chromatography N(O,S)-fluoroacetyl amino acid esters were separated on a deactivated glass capillary coated with Chirasil-Val. The carrier gas was hydrogen. Peaks were identified by retention time and mass spectrum.

[0249] Determination of acetate and trifluoroacetate by RP-HPLC This procedure is a gradient reverse-phase HPLC method with UV detection at 210 nm designed to determine acetate and trifluoroacetate content as a weight percent.

[0250] GLP-1 activity The GLP-1 activity of Formula 3 is assessed using a cell-based reporter gene assay. This assay utilizes HEK293 cells stably transfected with the GLP-1 receptor and a cAMP response element (CRE)-responsive luciferase gene. In this assay, Formula 3 binds to the GLP-1 receptor on the cell surface, activating cAMP. As a result of this activation, CREB (cAMP response element binding protein) becomes phosphorylated and binds to CRE in the nucleus, which induces luciferase transcription. Light emission from the luciferase reaction is measured and quantified, thereby providing a dose-dependent activation curve of GLP-1.

[0251] Glucagon activity The glucagon activity of Formula 3 is assessed using a cell-based reporter gene assay. This assay utilizes a HEK293 cell line (separate from HEK293, which has a GLP-1 receptor) engineered to stably express the glucagon receptor and a cAMP response element (CRE)-responsive luciferase gene. In this assay, Formula 3 binds to and activates the glucagon receptor, which activates cAMP. As a result of this activation, CREB (cAMP response element binding protein) becomes phosphorylated and binds to CRE in the nucleus, which induces luciferase transcription. Light emission from the luciferase reaction is measured and quantified, thereby generating a dose-dependent activation curve of glucagon.

[0252] Elucidation of structure and other properties Evidence of Chemical Structure Structural elucidation studies were performed on both the peptide intermediate and Formula 3. Tables 1A and 1B list the studies that were performed. All analyses confirm the chemical structures of the peptide intermediate and Formula 3.

[0253] The amino acid sequence and chirality of the peptide intermediates were confirmed by ES-MS, CID- and ECD-MS / MS, amino acid analysis (AAA), and chiral amino acid analysis. The mass ions obtained by ESI-MS for the peptide intermediates were consistent with those predicted. Data from tandem MS / MS were consistent with the expected amino acid sequence. AAA produced amino acid compositions consistent with the theoretical composition, with the exception of Trp and Cys, which were almost completely destroyed during acidic hydrolysis. The chirality of the amino acids in the peptide intermediates was pure (>99%) by chiral amino acid analysis.

[0254] The amino acid sequence of Formula 3 was confirmed by amino acid analysis (AAA), N-terminal sequencing, LC / MS peptide mapping, and intact LC / MS. The amino acid composition from AAA was consistent with the theoretical composition, except for Trp and Cys, which were almost completely destroyed during acidic hydrolysis. Thirty cycles of N-terminal sequencing confirmed the expected sequence of Formula 3. Peptides identified by LC / MS peptide mapping accounted for 35 of the total 39 amino acid residues. The dePEGylated C-terminal Ile-Ala-Cys-Cys peptide of Formula 3 was not detected by LC / MS peptide mapping, indicating that no free peptide intermediates were present in Formula 3. No significant peptide isomers were detected from peptide mapping, indicating that the PEGylation reaction and storage did not alter the amino acid chirality of the peptide. Furthermore, LC / MS peptide mapping detected very low levels of degradation, including Trp29 oxidation and Asp deamidation. Some of the degradation likely occurred during sample preparation.

[0255] Results from MALDI-TOF MS and LC / MS with post-column neutralization of formula 3 with diethylmethylamine were consistent with the peptide backbone and mPEG structure of formula 3 and the mPEG size.

[0256] [Table 2]

[0257] [Table 3]

[0258] Molecular mass determined by MS Peptide Intermediates Electrospray ionization mass spectrometry (ESI MS) was used to determine the mass of the peptide. The deconvoluted mass obtained from each of the multiple charged ions confirmed the correct monoisotopic mass of the peptide intermediate.

[0259] [Table 4]

[0260] Drug substance of formula 3 Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) was used to obtain the molecular mass of the drug substance of Formula 3. The sample from Formula 3 prepared in Example 1 was analyzed.

[0261] A positive ion MALDI-TOF mass spectrum was obtained from the sample, with a broad singly charged quasi-molecular ion cluster observed centered around m / z 48300.

[0262] The accurate molecular mass of the reference standard of Formula 3 was obtained using LC / ESI-MS with post-column neutralization with diethylmethylamine (DEMA). The deconvoluted mass spectrum of Formula 3 is shown in Figure 2. Two series of masses were identified, each containing over 100 masses with a 44-Da mass difference (=1 oxyethylene) between adjacent two masses. The masses of the major series are consistent with the expected structure of Formula 3. For example, one of the major masses at 47,102 Da corresponds to peptide intermediate 1-39 linked to two mPEG chains with 956 oxyethylene units (expected mass: 47,101 Da). The data confirmed the structure of the drug substance of Formula 3. In addition to the major series, the mass of a minor series (most likely corresponding to Formula 3 with one open maleimide ring; see the inset in Figure 2) was 18 Da higher than the mass of the major series.

[0263] N-terminal sequencing Reference standard in Equation 3 The N-terminal amino acid sequence of the reference standard of Formula 3 was determined by 30 cycles of N-terminal amino acid sequencing (Edman degradation).

[0264] The observed sequence was consistent with residues 1–30 of the expected N-terminal sequence of His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-Aib-Gly of formula 3.

[0265] Amino acid sequence by MS / MS Peptide Intermediates The amino acid sequences of the peptide intermediates were investigated by MS / MS analysis using collision-induced dissociation (CID) and electron capture dissociation (ECD). These techniques were used to intentionally fragment intact sample molecules and obtain structural information from the product ion spectra produced by the process. CID-MS / MS analysis and the +5 charge state ([M+5H]) of the peptide intermediates were analyzed. 5+ The results of ECD-MS / MS analysis of m / z 902.0) are summarized in Figures 3 and 4. MS / MS fragmentation and sequence analysis confirmed the primary sequence of the molecule.

[0266] Amino acid sequence by LC / MS peptide mapping drug substance The drug substance sequence of Formula 3 of amino acids was confirmed by LC / MS peptide mapping.

[0267] To recover as many peptide sequences (i.e., digested peptide fragments) as possible, the reference standard of Formula 3 was subjected to LC / MS analysis followed by pyrolytic digestion. The UV chromatogram of the LC / MS analysis of the pyrolytic digestion of Formula 3 is shown in Figure 5, and the results are shown in Table 1D.

[0268] As shown in Table 1D, 35 of the 39 amino acid residues in the Formula 3 peptide backbone were identified by pyrolysis peptide mapping using LC / MS. The PEGylated peptide fragment Formula 3 36–39, eluting at 50.62 min, was not identified by its exact mass because this analysis did not include post-column neutralization. However, the C-terminal PEGylated peptide IACC can be inferred from the mass spectrometry data. The broad peak at approximately 50 min was expected to contain this PEGylated peptide. The combined mass spectrum from this peak showed a series of mass species separated by 44 Da, indicating PEG-containing species. In addition to the expected pyrolysis peptides, several less modified peptides were also identified. These were oxidized Formula 3 peptides 22–25 (Trp 25 oxidation) and deamidated Formula 3 peptides 26–35. While 22-25 of Formula 3 contain only one potential oxidation site, Trp25, 26-35 of Formula 3 contain four Asn residues, and no specific deamidation site has been identified. Some of the degradation may be generated during pyrolytic digestion. No significant peptide isomers were identified, indicating that Formula 3 did not contain significant amino acid racemization, i.e., pure amino acid chirality.

[0269] [Table 5]

[0270] Individual amino acid identities and ratios by AAA Peptide intermediates and drug substances of formula 3 Amino acid analysis was performed on the peptide intermediates, as well as the drug substance, to ensure the identity and correct ratio of the constituent amino acids, demonstrating consistency of the results (see Table 1E). The method involves hydrolyzing the peptide with strong acid, separating the amino acids on an ion-exchange column, and finally detecting the eluate after ninhydrin derivatization.

[0271] Results from amino acid analysis confirm the identity and theoretical relative abundance of the amino acids in the peptide intermediate and drug substance of Formula 3. With the exception of Trp and Cys, which characteristically yield low recoveries for amino acid analysis, amino acid recoveries show good correlation with theoretical relative occurrence.

[0272] [Table 6]

[0273] Identity and chirality of individual amino acids by GC-MS Peptide Intermediates and Drug Substances The peptide portion of the molecule consists of natural amino acids, with the exception of the two 2-aminoisobutyric acid moieties present in the molecule. This amino acid and the two glycines present in the molecule represent the only achiral amino acids. Chiral amino acid analysis of the peptide intermediate and the drug substance of Formula 3 was performed to determine the chiral identity of each constituent amino acid residue. The peptide was hydrolyzed in a deuterated solvent (DCl / DO), appropriately derivatized, and analyzed using gas chromatography-mass spectrometry (GC-MS) to determine each amino acid enantiomer. The results, summarized in Table 1F, confirmed the expected chirality for each amino acid in the drug substance.

[0274] [Table 7]

[0275] Counterion identity and quantity by RP-HPLC drug substance To complete the structural elucidation of the drug substance of formula 3, the acetate content was measured by RP-HPLC to confirm the identity and amount of the counter ion.

[0276] The presence of acetate (measured indirectly as acetic acid) was confirmed. The acetate content was determined to be less than 0.6% (wt / wt), which is consistent with the expected 3 equivalents of acetic acid based on an excess of three basic residues in the peptide.

[0277] Physicochemical properties physical form Peptide intermediates and drug substances of formula 3 Visual inspection was performed to ensure the appearance of the peptide intermediate and the drug substance of Formula 3. The results showed that the appearance of both the peptide intermediate and the drug substance was a white to off-white powder. Both the peptide intermediate and Formula 3 were lyophilized materials and did not form crystalline or polymorphic forms.

[0278] biological activity Oxyntomodulin (OXM) is a peptide hormone released along with GLP-1 from L-cells in the small intestine in proportion to nutrient intake. A distinct receptor for OXM has not been identified. OXM can bind to and activate both the GLP-1 receptor (GLP1R) and the glucagon receptor (GcgR) in vitro. The mechanisms of GLP1R agonism in glucose lowering and weight loss have been well documented. Glucagon has been reported to reduce food intake and increase energy expenditure in Wistar rats. In contrast, anti-glucagon antibodies have been shown to increase meal size and duration in fasting and refeeding experiments. A 7-day human study showed that glucagon reduces food intake. Furthermore, glucagon has been shown to increase FGF21 and decrease PCSK9 expression. Therefore, the combination of GLP1R and GcgR agonism may offer the potential for superior weight loss and improved lipid profiles, as long as GLP1R activity is sufficient to improve glycemic control.

[0279] The biological activity (potency) of Formula 3 reference standard prepared in Example 1 and Formula 3 was evaluated against each other using two cell-based assays (GLP-1 and glucagon). The results are within the variability of the bioassay method and show no difference in potency between the reference standards. Therefore, each is assigned the same value of 100%.

[0280] Impurity profiles of clinical and toxicological batches RP-HPLC chromatograms showing the comparative individual impurity profiles of the clinical and toxicological batches are summarized in Table 1G. The impurity profiles of the clinical batches are comparable to those of the toxicological batches (PG11011 and PG1201).

[0281] [Table 8]

[0282] Due to the purification method (gradient chromatography), as described in the process of Example 1, the presence of organic impurities is highly unlikely. Only organic impurities with properties very similar to those of the peptide can be expected to remain unremoved by these chromatographic steps. Because the progress of the reaction was monitored by HPLC and any such impurities were removed during purification, the presence of any mono-PEGylated impurities is expected to be low. The mono-PEGylated impurity is easily detectable by RP-HPLC, eluting at approximately RRt 0.95 / 0.96 (Table 1G), and may also be a degradation product. Dimers of the mono-PEGylated form (i.e., mPEG-peptide-peptide-mPEG) may arise due to intermolecular cross-linking of cysteine ​​residues and have been previously identified as high-molecular-weight polymers (which may be located to the right of the API on the chromatogram). Potential inorganic impurities, primarily inorganic salts used during purification, are expected to be completely removed during the purification process. The acids used (acetic acid and trifluoroacetic acid) may form salts of the peptide and are included in the analysis of the drug substance. Metal catalytic reagents are not used in the manufacturing process. While stainless steel chromatography columns are used, only trace amounts of metal ions may remain. Mono-PEGylation can also be effected.

[0283] Therefore, the limit for total impurities / degradants in a drug product is 10% and the single largest impurity is 5%. Thus, the formulation of the present invention remains within the limit when samples are stored at 5±3°C for 2 years.

[0284] Example 3: Preparation of formulations of the present invention The formulations of the present invention were initially prepared in aqueous solutions containing approximately 50-85 mg / mL of the active ingredient, Formula 3. The preferred concentration of the active ingredient, PEGylated oxyntomodulin, of Formula 3 is approximately 70 mg / mL. Various compositions containing the active ingredient and a buffer selected from acetate, citrate, and histidine were prepared at various pH levels ranging from 4.0 to 7.0. Solutions of PEGylated oxyntomodulin analog (Formula 3) were stored for 6 days under three storage conditions: 5±3°C, 25±2°C, and 40±2°C, and then analyzed by DLS and RP-HPLC for particle size analysis and purity / impurities, respectively. After adding the PEGylated oxyntomodulin active ingredient at 70 mg / mL to all prepared formulation buffers (acetate, citrate, and histidine), it was discovered that the pH of the buffer increased above the target upon addition of PEGylated oxyntomodulin. Therefore, two additional formulations were prepared in which the pH was adjusted to the target value using a weak acid. These samples were stored at 5±3° C. and also tested for purity / impurities by RP-HPLC. The compositions of the present invention did not show a significant change in pH value for any of the samples between the pH initially measured and the pH measured after 6 days of storage at 5±3° C.

[0285] The particle distribution in the solution was also examined by DLS and showed a multimodal size distribution with polydispersity and low concentrations of particles. Accordingly, the present invention includes PEGylated oxyntomodulin formulations with such characteristics.

[0286] The formulations of the present invention were also tested for related impurities by RP-HPLC and for soluble aggregates by SEC-HPLC under various conditions. The most stable conditions, with respect to minimal increase in impurities, were those using a pH range of about 4.5 to about 5.9 and / or solutions lacking sodium chloride at the tested concentration of 100 mM. An acetate buffer at pH 4.5 was selected as providing the most stability for liquid formulations containing PEGylated oxyntomodulin peptides and analogs thereof.

[0287] Example 4: Preformulation Development Various formulations of the OPK-88003 liquid drug product are being evaluated to develop a stable liquid formulation suitable for use in a pre-filled syringe format for Phase 3 and for eventual commercial administration in conjunction with an auto-injector. Formulation development was conducted in three phases: Phase 1: Preformulation development to narrow down the selection of (1) pH, (2) buffers, (3) surfactants, and (4) excipients; Phase 2: DOE study to assess the stability of the selected combinations using statistical design; Phase 3: Long-term stability of selected lead formulations.

[0288] Phase 1 Preformulation Activity Summary Preformulation development included evaluation of stability as a function of pH, buffer type, and the addition of various excipients, as well as the effect of surfactants. Stability analysis studies included RP-HPLC and SEC-HPLC, DLS, HIAC, and MFI of particles, as well as pH, osmolality, and viscosity.

[0289] Initial Screening: Stability studies of OPK-88003 (Formula 3) evaluated the relative pH and buffer type (acetate, citrate, and histidine). Samples were tested for peptide impurities by RP-HPLC and for the presence of soluble aggregates as measured by DLS. Based on the initial screening data, The optimum pH range is identified as pH 4.5 to 5.0. Acetate or citrate were identified as suitable buffers in this range.

[0290] Excipient Screening: Various excipients were evaluated or experimentally evaluated in acetate buffer (pH 4.5 and 5.0) or citrate buffer (pH 5.5): methionine, arginine, cysteine, glycine, sodium chloride, mannitol, sorbitol, and sucrose. Formulations containing methionine and arginine were noted for improvement and selected for further evaluation in DOE studies. Sorbitol and sucrose are inert, and either is suitable as a tonicity adjuster. Sorbitol was selected for DOE studies due to its potential for less impact on viscosity.

[0291] Surfactant Screening: The effect of various surfactants on particle and aggregate formulations was evaluated in surfactant screening studies. The effect of the following surfactants: polysorbate 20, polysorbate 80, and poloxamer 188 was evaluated in samples subjected to agitation and successive freeze / thaw cycles. Samples were analyzed by SEC-HPLC, DLS, MFI, and HIAC for soluble and insoluble aggregates. Because no chemical degradation or aggregation was observed in samples containing any of the surfactants versus control samples, it was decided to exclude any surfactant from the formulation for further testing.

[0292] Results of the Phase 2 OPK-88003 Formulation Development DOE Study and Rationale for Lead Formulation Selection A design of experiments (DOE) study was designed and executed. Thirty-nine formulations were subjected to accelerated stability (4 weeks at 40°C) and analyzed by RP-HPLC, SEC-HPLC. The results of the DOE study and statistical modeling, as well as the rationale for the selection of the lead formulation for further stability testing in pre-filled syringes, are outlined below.

[0293] Buffer Selection: During preformulation studies, acetate buffer solutions were identified as being similar to citrate buffer solutions when tested in the low pH range. Acetate buffer was selected for the DOE studies and lead formulation because acetate is a better buffer in the pH 4.5-5.0 range. Additionally, a decrease in viscosity was observed in samples prepared in acetate buffer; however, this needs to be confirmed.

[0294] pH Selection: pH 4.5-4.9 was identified as the optimal pH range during preformulation studies for OPK-88003 stability, and this range was selected for testing in the DOE study. Modeling of the results of the DOE study demonstrated that there were statistically significant stability improvements in total aggregates and / or impurities by SEC-HPLC or RP-HPLC. Because statistical improvements were consistently observed in samples at lower pH, it is recommended to select the lowest pH tested for the preferred formulation.

[0295] Methionine: During modeling of the DOE study results, the level of methionine was identified as having a statistically significant effect on the levels of impurities by SEC (total aggregates) and RP-HPLC (total back-peak impurities) after accelerated stability. Specifically, methionine appears to function as a protectant against dePEGylation based on the DOE stability results. Statistical modeling of the DOE results indicates that a potency of approximately 30 mM is the optimal methionine concentration.

[0296] Arginine: Arginine appears to improve stability in certain situations (i.e., in the presence of low levels of methionine), but the difference was not considered statistically significant. There may be some improvement in samples containing arginine under certain conditions, but the optimal pH range may not be optimal here. Furthermore, as a tonicity adjuster, sorbitol is less expensive on a commercial scale than arginine as a pharmaceutical excipient.

[0297] Sorbitol: Sorbitol was chosen as the tonicity adjuster because it is more inert toward amino acids such as arginine and is inexpensive. The maximum strength of sorbitol on the FDA's inactive ingredients database for subcutaneous injection is 4.7%, and 4.0% is required to achieve the target osmolality in the preferred formulation, which is within this range.

[0298] Osmolality: To maintain isotonicity, an osmolality of approximately 300 mOsm / kg is recommended. Osmolality can be adjusted with sorbitol, but the potency of sorbitol is within the range of approved drug precedents, so a target of 300 mOsm / kg is recommended.

[0299] [Table 9]

[0300] Extrapolated Stability: By extrapolating from preliminary accelerated stability data from DOE studies, the liquid formulation should be stable for 2 years (<5% degradation of API) when stored at 2-8°C.

[0301] Formulations: Preferred formulations are listed in Table 2B.

[0302] Formulation 2: Identical formulation to Formulation 1 but at pH 4.7.

[0303] Formulation 3: Arginine (100 mM) replaces sorbitol (230 mM) in Formulation 1. The resulting osmolality is slightly lower (290 mOsm / kg). This formulation demonstrates whether the presence of arginine in the formulation improves long-term stability in any way.

[0304] [Table 10]

[0305] Example 5: Physical and chemical stability of PEGylated OXM peptide analogues A panel of analytical techniques was used to evaluate the physical and chemical stability of the OPK-88003 peptide (Formula 3), including appearance, pH, SEC-HPLC, RP-HPLC, DLS, HIAC, MFI, osmolality, and viscosity. Based on the results obtained, the following formulation conditions are optimal for OPK-88003 (Formula 3) at 70 mg / mL: 10 mM acetate buffer containing 30-35 mM methionine (pH 4.5). In the presence of sorbitol, no effect on OPK-88003 stability was observed, and therefore its use as an isotonic agent is recommended.

[0306] Preformulation development studies are conducted to establish a formulation composition that provides physical and chemical stability, is non-irritating, does not adsorb to glass, and has a viscosity suitable for use in pre-filled syringes or auto-injectors for the OPK-88003 PEGylated oxyntomodulin analog having Formula 3. The concentration of the OPK-88003 peptide was 70 mg / mL in all formulations tested.

[0307] [Table 11]

[0308] Materials and Equipment material ●OPK-88003 PEGylated oxyntomodulin analogue. 0.45 μm Millex-HV PVDF filter, Millipore, P / N SLGV013SL 3mL Norm-Ject silicone oil-free syringe, Fisher P / N 14-817-27 10mL Norm-Ject Silicone Oil-Free Syringe, Fisher P / N 14-817-30 Acetic acid, glacial, Fisher, P / N A491-212 Citric Acid, Anhydrous, JTBaker, P / N 0127-05 ●Filter, 0.2 μm PES membrane, Thermo Scientific, P / N 597-4520 L-histidine, Sigma, P / N H6034-100G Hydrochloric acid, 6N solution, JTBaker, P / N 5619-02 L-Arginine Hydrochloride, JTBaker, P / N 2067-06 L-Cysteine ​​Hydrochloride, Thermo Scientific, P / N 44889 L-Methionine, Sigma, P / N M9625-100G Malvern Zetasizer Nano ZS Poloxamer 188, Spectrum Chemical, P / N P1169 ●PS-20, Sigma, P / N P7949-100ML ●PS-80, JTBaker, P / N 4500-04 Sodium acetate, anhydrous, Sigma, P / N S1429-1KG Sodium chloride, Fisher, P / N S271-500 Sodium chloride, JTBaker, P / N 3627-05 Sodium hydroxide, 6N solution, JTBaker, P / N 5672-02 Sorbitol, Sigma, P / N 85529-1KG Sucrose, JTBaker, P / N 4005-05 ● Trisodium citrate, dihydrate, Sigma, P / N S1804-500G device Agilent 1100 or 1200 HPLC system ●Brookfield DV-III Ultra Programmable Rheometer ●ProteinEasy Micro-Flow Imaging Microscope, DPA 5200 (including Bot1) ●Environmental Specialist ES 2000 Reach-In Chambers Liquid particle counter, HIAC model 9703 Malvern Zetasizer Nano ZS ●Mettler Toledo SevenMulti pH Meter Vapro Vapor Pressure Osmometer, Model 5600

[0309] method pH The pH was tested according to GTM-0015 "Determination of pH" using a calibrated SevenMulti pH Meter.

[0310] exterior Appearance testing was performed according to GTM-0033, "Appearance by Visual Assessment."

[0311] Dynamic Light Scattering Dynamic light scattering was performed using at least 80 μL of undiluted sample in an optical-quality plastic cuvette. Five consecutive measurements were taken for each sample at 25 °C, with at least 3 minutes of equilibration at the beginning of each scan. A protein analysis algorithm (Malvern Zetasizer software) was used as a model for data processing. The viscosity of water at 25 °C (0.8872 mPa·s) was used to calculate the Z-average hydrodynamic diameter from cumulative analysis and the Stokes-Einstein equation. The polydispersity index was obtained from the same cumulative analysis. The modality of the fit was assessed based on a plot of size distribution versus intensity and can be described as monomodal (i.e., one peak) or multimodal (i.e., two or more peaks).

[0312] HIAC The particle concentration is determined by the USP <788> Particulate matter in injection solutions was measured by a liquid particle counting system, HIAC Model 9703 / 9703+, according to the test parameters outlined in [1]. A 0.2 mL injection was performed using a 1 mL syringe equipped with a small sample size probe. Particle counts (2-25 μm size) were reported as cumulative counts / mL per mL of protein solution.

[0313] Reversed-phase chromatography Reverse-phase chromatography was used to determine the purity and related substances of OPK-88003 according to method PRD-P80101-LNCM. This procedure is a gradient reverse-phase HPLC method with UV detection at 214 nm designed to determine the identity, quantity, and related substances of OPK-88003 in drug products. It uses a Zorbax 300Å SB-C18 column. The mobile phase consists of 0.1% v / v trifluoroacetic acid in water (mobile phase A) and 0.1% v / v trifluoroacetic acid in acetonitrile (mobile phase B). The method determines identity by retention time agreement of the main peak or coelution with a reference standard, quantity by comparative determination of the OPK-88003 main peak area to the reference standard, and purity and impurities / related substances as total area percent. The procedure exhibits stability as judged by its ability to resolve known impurities from OPK-88003 and demonstrated by analysis of stressed samples.

[0314] Size exclusion chromatography Monomer purity and related substances were determined by SEC (method PRD-P80102-LNCM).

[0315] This procedure is a size-exclusion HPLC method with UV detection at 214 nm designed to determine the monomeric purity and related substances of OPK-88003 in drug product. It uses a Sepax Zenix SEC-300 column. The mobile phase consists of 60 mM potassium phosphate, 250 mM sodium chloride, 20% ACN at pH 7.0. The method determines monomeric purity and impurities / related substances as total area percent. The procedure exhibits stability as judged by its ability to resolve known impurities from OPK-88003 and demonstrated by analysis of stressed samples.

[0316] MFI Microflow imaging experiments were performed using an MFI 5200 with an automated pipette system to characterize the size, concentration, and morphology of particles present in OPK-88003 samples. Samples were prepared in a laminar flow hood using barrier pipette tips that were rinsed three times to minimize contamination. Analysis was performed in a single measurement using either 700 or 1000 μL of undiluted sample, depending on the amount of sample available. The procedures and respective liquids used for MFI testing are listed below in Table 3A.

[0317] [Table 12]

[0318] The analytical operating parameters used in the method (step number 14) are as shown in Table 3B.

[0319] [Table 13]

[0320] Cumulative counts per mL of particles sized ≥2 μm, ≥5 μm, ≥10 μm, and ≥25 μm were processed from the raw data collected for particles sized between 1 and 100 μm. Additionally, particles sized ≥5 μm with an aspect ratio (AR) of ≥0.85 were processed using morphological classification parameters within the MFI software (MVAS). These morphological classification data represent highly circular particles, which are considered non-proteinaceous, typically either gas bubbles or silicone oil droplets. Proteinaceous particles are typically non-circular and non-crystalline, and therefore have a low aspect ratio according to the MFI software. The circular and non-circular data sets of these morphological classifications are used to determine the circular fraction. The calculated circular fraction is determined by the following equation:

[0321]

number

[0322] The calculated circular fraction therefore provides the ratio of non-proteinaceous to proteinaceous particles found in each test sample. A low circular fraction value indicates that the test material is composed mostly of non-circular, presumably proteinaceous, particles.

[0323] Osmolality The osmolality of OPK-88003 formulation was measured by Vapro Vapor Pressure Osmometer according to the dew point temperature depression method (GTM-0614). Note that due to the higher viscosity of the sample, the osmolality was measured by the dew point temperature depression method instead of the freezing point depression method. The osmometer was calibrated using a series of standard solutions before sample measurement. Samples were analyzed in triplicate using 10 μL of sample for each measurement, and the average value was reported.

[0324] viscosity The viscosity of the OPK-88003 sample was measured using a Brookfield DV-III Ultra Programmable Rheometer according to method GTM-0614. Prior to sample measurement, the performance of the viscometer was verified using a certified viscosity standard. After the standard measurement, 600 μL of the OPK-88003 sample was loaded into the viscometer. Viscosity measurements were performed at multiple torque percentage values ​​to assess Newtonian or non-Newtonian properties.

[0325] A summary of the formulations used for buffer and pH screening, F14 / F15 follow-up studies, excipient screening, and surfactant screening studies is provided in Table 4.

[0326] [Table 14-1]

[0327] [Table 14-2]

[0328] Example 5A: Buffer and pH Screening Studies Thirteen buffer solutions (F1-F13) were evaluated to assess the effect of pH, ranging from approximately 4.0 to 6.5, and buffer type (acetate, citrate, and histidine) on the chemical stability of OPK-88003 (Formula 3) (Table 5). The effect of 100 mM NaCl was also evaluated in formulations F9, F10, and F11. Additionally, formulations F14 and F15 were prepared and tested as part of pre-formulation development studies of pH and buffer type (Table 5).

[0329] [Table 15]

[0330] After preparation of the formulation solutions, the initial pH was determined. It was observed that OPK-88003 API, dissolved at a concentration of 70 mg / mL in the formulation buffer, increased the pH of the solutions. This effect was particularly pronounced in formulations using acetate and citrate buffers, where the pH increased by approximately 0.7–0.9 units compared to the target pH (Table 6). Therefore, two additional formulations were prepared (F12 and F13), and the pH was adjusted to the target value with acetic acid. Based on the observed increasing pH effect, all subsequent formulations were prepared by dissolving OPK-88003 (Formula 3) API in the formulation buffer at a pH 0.5 units lower than the target pH. Additionally, the pH was adjusted to the target pH using an appropriate base or acid if the measured pH was outside the target value.

[0331] Two aliquots of each prepared formulation (F1-F13) were stored for 6 days at the following conditions before testing: 5±3°C, 25±2°C, and 40±2°C. After incubation, pH was measured from one 5±3°C aliquot of each formulation. One aliquot each from the 25±2°C and 40±2°C conditions was analyzed by DLS and RP-HPLC. Additionally, formulations F12 and F13 stored at 5±3°C were tested by RP-HPLC.

[0332] There was no significant change in pH value for any of the samples between the initially measured pH and the sample pH measured after 6 days of storage at 5±3° C. (Table 6).

[0333] [Table 16]

[0334] The particle distribution of OPK-88003 solutions was investigated by DLS. In the DLS study, the formulations were compared in terms of Z-average diameter and PDI. The DLS results showed a multimodal size distribution with polydispersity for all OPK-88003 samples tested (Table 7).

[0335] [Table 17]

[0336] No correlation of PDI between the concentrations tested was observed. The high extinction index of all tested samples indicated low intensity scattered light, suggesting low concentrations of particles in the tested samples.

[0337] The samples were tested by RP-HPLC after incubation for 6 days at 5±3° C. (F12 and F13 samples only), 25±2° C., and 40±2° C., and the results are summarized in Table 8.

[0338] [Table 18]

[0339] A slight but gradual trend in impurity increase was observed as the pH of the samples increased. Impurity accumulation was observed for samples stored at both 25 ± 2°C and 40 ± 2°C. No significant differences in impurity concentrations were observed between different buffer types with similar pH. The presence of NaCl increased impurity levels in all tested formulations compared to those without NaCl, thus indicating a negative effect on OPK-88003 stability. No significant differences in impurity concentrations were observed between formulations F12 and F13 stored at 5 ± 3°C. Based on RP-HPLC results, optimal stability of OPK-88003 was observed in the pH range of approximately 4.5 to 5.9.

[0340] In buffer and pH screening, additional studies were performed on two formulations (F14 and F15). Samples from the initial control (stored at -75±10°C for 2 weeks), 5±3°C control (2 weeks), freeze-thaw (3 cycles), and 40±2°C (2 weeks with one freeze-thaw before testing) were tested using the following methods: pH, RP-HPLC, DLS, HIAC, and MFI.

[0341] The pH was measured in control samples (F14 and F15) stored for 6 days at -75°C and for 17 days at 5±3°C. For both formulations, no significant changes in pH values ​​were observed between the initial and aged samples (Table 6).

[0342] The results of samples tested by RP-HPLC under the conditions and time points mentioned above are summarized in Table 9 below. The most significant change in concentration total impurities was observed for samples stored at 40±2°C compared to the control and freeze-thaw stressed samples at -75°C±10°C and 5±3°C. The accumulation of total impurities in samples stored at 40°C±2°C was slightly higher for formulation F14. This result is in good agreement with the results obtained for formulations F9, F10, and F11 (all containing 100 mM NaCl), in which elevated levels of total impurities were observed.

[0343] The particle distribution in formulations F14 and F15 was investigated by DLS. Similarly, the low intensity of scattered light detected suggested a low concentration of particles in the tested samples, in contrast to the results obtained for formulations F1-F13. DLS results showed a polydisperse, multimodal size distribution for all OPK-88003 samples tested (Table 10).

[0344] The number and size of subvisible particles were measured by HIAC and MFI. No significant differences in particle counts were observed for F14 and F15 formulations stored at -75°C ± 10°C and the control sample at 5 ± 3°C (Table 11).

[0345] A slight increase in particle count was observed for both formulations subjected to freeze-thaw stress. The highest particle count increase was observed for samples stored at 40 ± 2°C, with increases across all particle sizes observed for formulation F15. In contrast to HIAC, no clear trend in particle increase was observed by MFI for both formulations F14 and FF15 (Table 12). Based on the RP-HPLC results, buffers in the pH range of 4.5 to 5.9, but particularly around pH 4.5, exhibited the most positive effect on OPK-88003 stability. The presence of NaCl in the tested formulations increased the accumulation of total impurities and was therefore not further tested in preformulation development studies.

[0346] [Table 19]

[0347] [Table 20]

[0348] [Table 21]

[0349] [Table 22]

[0350] The above results demonstrate that the preferred pH range is 4.5-5.9, with the most preferred being about 4.5, and that the presence of sodium chloride has a negative effect on the accumulation of total impurities.

[0351] Example 5B: Excipient Screening Study Based on the results of the buffer and pH screening studies, 15 formulations (F16–F30) were evaluated to assess the effect of four different excipients (arginine, methionine, sorbitol, or sucrose) in two buffers (acetate and citrate) on the stability of OPK-88003 (Table 13).

[0352] [Table 23]

[0353] All four excipients were tested in acetate buffer at pH 4.5 and 5.0, and citrate buffer at pH 5.5. Three formulations without excipients (F16, F21, and F26) were tested as controls. All formulations prepared according to protocol TD06315 were divided into three aliquots and incubated at the following temperatures: 5±3°C, 40±2°C, and 50°C. After two weeks of incubation, pH was measured from the 5±3°C aliquot of each formulation. Samples stored at 5±3°C, 40±2°C, and 50°C were tested by RP-HPLC and DLS. Viscosity was performed only on the 5±3°C samples containing sorbitol or sucrose (F19, F20, F24, F25, F29, and F30).

[0354] There was no significant change in pH observed between the initial sample and the sample stored for 2 weeks at 5 ± 3°C. The only exception was formulation F16, which changed from pH 4.57 to pH 4.22 (approximately 0.4 units) (Table 6). A similar formulation (F1) measured at pH 4.90 and stored for 6 days at 5 ± 3°C, and all other excipient screening formulations, did not show a similar change; therefore, the observed differences are due to method variability rather than an actual change in pH.

[0355] The stability of OPK-88003 was determined by RP-HPLC chromatography. Samples were tested under the conditions and time points described above, and the results are summarized in Table 14. The lowest accumulation of OPK-88003 impurities was observed in acetate buffers containing methionine or arginine subjected to 40±2°C at pH 4.5 and 5.0 at 50°C compared to the corresponding control formulations stored at the same temperatures. A slight beneficial effect of the presence of methionine on OPK-88003 stability was also observed in all formulations stored at 5±3°C. The presence of sorbitol or sucrose did not have a clear protective effect on OPK-88003 stability in any of the tested formulations subjected to 40±2°C and 50°C. Minor differences in the accumulation of OPK-88003 degradants were observed with respect to pH or buffer type. In general, the concentrations of total impurities in acetate buffers at pH 4.5 and 5.0 subjected to 40±2° C. were lower compared to citrate buffer at pH 5.5. However, this trend was not observed for samples stored at 50° C.

[0356] The particle distribution was investigated by DLS. Similarly, in contrast to the results obtained in the buffer and pH screening studies, the low intensity of scattered light detected suggests a low concentration of particles in the tested samples. DLS results showed a polydisperse, multimodal size distribution for all OPK-88003 samples tested (Table 15).

[0357] Viscosity measurements were collected at variable shear rates and a constant temperature of 22°C and then averaged (Table 16). Comparable viscosities (range 20.1-22.6 cP) were measured for acetate buffers containing sorbitol or sucrose (F19, F20, F24, and F25) at pH 4.5 and 5.0. Significantly higher viscosities of 30.9 and 29.1 cP, respectively, were observed for citrate buffers containing sorbitol or sucrose (F29 and F30) at pH 5.5. This indicates that the viscosity in the tested formulations is dependent on pH or, more likely, the type of buffer. Tables 14-16 show the results of a study of various excipients on the formulations, and based on this data, methionine was selected as the preferred excipient, along with sorbitol and acetate buffer, with a pH of approximately 4.5 being the preferred buffer.

[0358] [Table 24]

[0359] [Table 25-1]

[0360] [Table 25-2]

[0361] [Table 26]

[0362] Example 5C: Surfactant Screening Studies The protective effect of nonionic surfactants on the stability of OPK-88003 was evaluated for PS-20, PS-80, and poloxamer 188 in temperature stress, freeze-thaw, and agitation tests. Two buffers, pH 5.0 acetate and pH 5.5 citrate containing 0.1% of each surfactant, were tested. Two corresponding buffers (F21 and F26) without surfactant were used as controls. Additionally, two formulations (F24 and F37) were prepared to evaluate the effect of 0.1% PS-80 on the stability of OPK-88003 in pH 5.0 acetate buffer containing 250 mM sorbitol. A total of 10 formulations (F21–F37) were screened in the surfactant screening study (Table 17).

[0363] [Table 27]

[0364] Samples were prepared by mixing various excipients (F21, F24, F26, and F31-F37). For the temperature stress test, samples were stored at 50°C for one week. Matching controls were stored at 5±3°C for the same period. For the agitation test, samples were agitated (50 rpm, room temperature) for approximately 50 hours. Matching controls without agitation were stored nearby at room temperature. For the freeze-thaw test, samples were subjected to three rapid freeze-thaw cycles. Vials were frozen at 75±10°C for at least overnight. Samples were thawed by placing the vials on the lab bench at room temperature for at least two hours. The vials were refrozen, and the freezing procedure was repeated a total of three times. Stress and control samples were analyzed by visual inspection (appearance), SEC-HPLC, and DLS methods. RP-HPLC analysis was performed only on samples stored at 50°C.

[0365] In the studies carried out, all stress samples remained unchanged in terms of color, transparency, and particle content compared to the control samples.

[0366] By SEC-HPLC, no significant changes in % total impurities were detected for freeze-thawed and agitated samples compared to the corresponding controls in both acetate and citrate buffers. Similarly, no beneficial effect of any of the tested surfactants was observed on OPK-88003 stability compared to the corresponding controls without surfactant. The area percent of total impurities was nearly identical for all samples tested (Tables 18 and 19).

[0367] DLS studies detected low intensity scattered light for all tested samples, suggesting low particle concentrations. DLS results showed polydisperse, multimodal size distributions for all tested OPK-88003 samples (Table 20).

[0368] The RP-HPLC results obtained for samples stored at 50°C are summarized in Table 21. No beneficial effect of any of the tested surfactants was observed compared to the corresponding surfactant-free controls (i.e., F21, F24, F26) for both acetate and citrate buffers. The determined % total impurities were comparable for all samples tested (7.2-8.2%).

[0369] In conclusion, in the temperature stress, freeze-thaw, and agitation tests performed, no beneficial effect of any of the tested surfactants on OPK-88003 stability was observed.

[0370] [Table 28-1]

[0371] [Table 28-2]

[0372] [Table 29]

[0373] [Table 30-1]

[0374] [Table 30-2]

[0375] [Table 31]

[0376] Example 5D: Design of Experimental Studies and Additional Formulation Tests Based on the results from the buffer and pH, excipient, and surfactant screening studies, design components were selected for evaluation in a DOE study. The design components included pH, methionine, arginine, and sorbitol and were evaluated in acetate buffer at an OPK-88003 concentration of 70 mg / mL. A total of 36 formulations (F DOE 1st floor to 1st floor DOE 36) were evaluated (Table 22). In addition, three formulations (F DOE 37, F DOE 38, and F DOE 39) were prepared to evaluate the effects of cysteine, acetate buffer at 20 mM concentration, and methionine and arginine at concentrations different from those selected for the DOE study. The results obtained for these formulations were not included in the statistical analysis. Designed center-point formulations (FD OE 20:70 mg / mL OPK-88003, 25 mM methionine, 65 mM arginine, 95 mM sorbitol, pH 4.7) was prepared and tested in triplicate.

[0377] [Table 32]

[0378] DOE samples were subjected to unstressed (5±3°C) and stressed (40±2°C / 75%±5RH) conditions for 4 weeks prior to analysis. Statistical optimization was performed, which resulted in the selection of candidate formulations for the OPK-88003 product. In addition to the DOE formulations, three additional formulations (F DOE 37, F DOE 38, and F DOE 39) were prepared and tested simultaneously with the DOE samples. The results obtained for these formulations were not included in the statistical analysis.

[0379] All initial sample aliquots were tested by pH, RP-HPLC, and SE-HPLC. Aliquots of each formulation were incubated at 5±3°C and 40±2°C for 4 weeks and then tested by pH, RP-HPLC, and SE-HPLC methods. Osmolality was measured by the initial F DOE Only 20 (center point) samples were measured (Table 26). Additionally, for formulation F39, viscosity was measured at the initial time point (and HIAC), and MFI analysis was performed at the initial time point and after storing the samples for 4 weeks at 25±2°C.

[0380] For the DOE formulation samples (including F37 and F38 samples), no significant changes in pH values ​​were observed after 4 weeks of storage at 5±3°C and 40±2°C when compared to the initial samples (Table 23). For the F39 samples, no significant changes in pH values ​​were observed after 4 weeks of storage at 5±3°C, 25±2°C, and 40±2°C when compared to the initial samples (Table 23). A maximum difference of 0.15 units was observed between the initial F38 samples when stored at 5±3°C for 4 weeks.

[0381] F DOE 37, F DOE 38, and F DOE The effect of temperature stress on OPK-88003 stability in 39 samples was evaluated by SEC analysis. The results at the initial and 4-week time points are summarized in Tables 24A and 24B. The results at the initial time point and at 5±3°C and 25±2°C (F DOE 39) analyzed after 4 weeks of storage. DOE 37, F DOE38, and F DOE No significant changes in the % total impurities content were observed among the 39 samples. F stored at 40±2°C for 4 weeks DOE 37 and F DOE A slight increase in % Total Impurities (1.2-1.3%) was observed for 39 samples compared to the initial time point. Sample F38 (formulated with 25 mM cysteine) stored at 40 ± 2°C was significantly affected with an increase in % Total Impurities of 47.7% compared to the initial time point (Figure 6).

[0382] F DOE 37, FDOE 38, and F DOE The RP-HPLC results obtained for 39 samples correspond to the SEC data. The results at the initial and 4-week time points are summarized in Tables 25A and 25B. F analyzed at the initial time point and after 4 weeks of storage at 5±3°C. DOE 37 and F DOE No significant changes in the % total impurities were observed among the 39 samples. DOE 39) and F analyzed after 4 weeks of storage at 40 ± 2°C. DOE 37 and F DOE Significant differences in % Total Impurities were observed among the 39 samples. A substantial increase in % Total Impurities (8.5%) was observed for the F38 sample stored at 5±3°C for 4 weeks. Sample F38 stored at 40±2°C for 4 weeks was highly degraded with a 51.7% higher % Total Impurities compared to the initial time point. An overlaid chromatogram of the F38 sample (formulated with 25 mM cysteine) is shown in Figure 7.

[0383] Center point sample (F DOE 20), the mean osmolality value of the three measurements was 290 mOsm / kg (Table 26).

[0384] F DOE The 39 HIAC results showed an increase in all particle sizes for samples stored at 25±2° C. when compared to the initial time point samples (Table 11).

[0385] F DOEThe 39 MFI results showed a slight increase in all particle sizes for samples stored at 25±2° C. when compared to the initial time point samples (Table 12).

[0386] F DOE The viscosity measured for 39 was 18.8 cP, comparable to the values ​​obtained for other formulations using acetate buffer (Table 16).

[0387] [Table 33-1]

[0388] [Table 33-2]

[0389] [Table 33-3]

[0390] [Table 34]

[0391] [Table 35-1]

[0392] [Table 35-2]

[0393] [Table 36-1]

[0394] [Table 36-2]

[0395] [Table 37-1]

[0396] [Table 37-2]

[0397] [Table 38]

[0398] [Table 39-1]

[0399] [Table 39-2]

[0400] The DOE results obtained for 4-week samples by SEC (Table 24B) and RP-HPLC (Table 25B) were statistically analyzed to design an optimized formulation for OPK-88003. The experimental design was generated using Design-Expert® version 9.0.1 from Stat-Ease, Inc. The significance of the responses was assessed using the ANOVA method at a 95% confidence interval (a p-value of less than 0.05 indicated a statistically significant correlation). Responses that met this significance threshold were analyzed by the software in a reduced quadratic model (design model) (Table 28), as shown in Table 27.

[0401] [Table 40]

[0402] The model F-score of 4.72 suggests that the model is significant. There is only a 0.09% chance that this large F-score is caused by noise.

[0403] DOE calculations were performed using four factors: pH, methionine concentration, arginine concentration, and sorbitol concentration, and two responses: % accumulation of back main peak impurities (RP-HPLC) and % accumulation of total aggregate impurities (SE-HPLC). Calculations were performed using pH values ​​obtained for samples stored at 40 ± 2°C for 4 weeks.

[0404] The DOE model generated for the accumulation of back-main peak impurities (%) detected by RP-HPLC analysis (the difference between the back-main peak impurities % obtained for samples stored for 4 weeks at 5 ± 3 °C and 40 ± 2 °C) showed a gradual increase in impurities as a function of pH (Figures 8 and 9). The lowest accumulation of back-main peak impurities (%) was determined at pH 4.5. The accumulation of back-main peak impurities (%) also depended on the methionine concentration, with the lowest determined at approximately 30-35 mM. The presence of arginine and sorbitol did not significantly affect the back-main impurities (%) in the tested formulations. Similar results were obtained for the accumulation of total aggregate impurities (%) detected by SEC analysis (the difference between the total aggregate impurities % obtained for samples stored for 4 weeks at 5 ± 3 °C and 40 ± 2 °C). The DOE model showed a gradual increase in impurities as a function of pH, with the lowest accumulation of % total aggregate impurities at pH 4.5 and a methionine concentration of approximately 30-35 mM (Figures 10 and 11). The presence of arginine and sorbitol had no significant effect on % total aggregate impurities in the formulations tested.

[0405] Results from the DOE showed the highest stability of OPK-88003 in formulations containing approximately 30-35 mM methionine at pH 4.5.

[0406] An optimal liquid formulation was achieved in which OPK-88003 was soluble and physical and chemical degradation was minimized. Several buffer types, pH conditions, excipients, and the presence / absence of surfactants were evaluated as part of these formulation screening and accelerated stability studies. Various biophysical and analytical tools were then used to detect and identify chemical and physical changes in the API under stress conditions. The following formulation conditions are recommended for the most preferred formulation of OPK-88003 at a concentration of 70 mg / mL: 10 mM acetate buffer with 30-35 mM methionine, pH 4.5. Since no effect on OPK-88003 stability was observed in the presence of sorbitol, its use as an isotonicity agent is recommended. Other isotonicity agents may also be used if they do not negatively impact the formulation's stability.

[0407] Example 6: Weekly Subcutaneous Dose Study OPK-88003 was studied in 402 subjects with T2DM in Phase 1 and 2 studies at a subcutaneous (SC) dose once weekly (QW). Single and multiple ascending doses up to 60 mg QW were tested in a two-part, double-blind, randomized, placebo-controlled Phase 1 clinical pharmacology study. Doses ranging from 10 mg to 50 mg per week were tested in a 24-week, double-blind (first 12 weeks), randomized, placebo-, and active comparator (2 mg exenatide ER)-controlled Phase 2 study. Supratherapeutic multiple SC doses of 40 mg once daily for 7 days, and titration doses of 15 to 60 mg over 6 days were also tested in a double-blind, placebo-controlled clinical pharmacology study.

[0408] Clinical data from a Phase 1 study showed that QW SC administration of OPK-88003 produced improvements in HbA1c and fasting glucose and resulted in progressive weight loss. Changes from baseline in weight after 5 weeks of treatment in obese non-diabetic patients with T2DM ranged from -1.55 kg to -2.23 kg compared with -0.81 kg in the placebo group at the 25 mg to 60 mg dose levels. Glucose tolerance and fasting plasma glucose (FPG) were significantly improved in subjects with T2DM.

[0409] In a phase 2 study, administration of OPK-88003 at 10, 15, 30, and 50 mg QW doses for 6 months in subjects with T2DM resulted in dose-dependent effects on blood glucose and weight loss. Statistically significant reductions in mean HbA1c (up to 1.43%) were observed for all doses at 12 and 24 weeks. OPK-88003 significantly reduced HbA1c levels compared to placebo and was non-inferior to 2 mg of exenatide ER at 30 mg and 50 mg doses (p=0.994 and 0.628, respectively). The significant reduction in FGF with OPK-88003 resulted in similar 7-point self-monitored blood glucose (SMBG) profile values ​​compared to treatment with exenatide ER and lower values ​​compared to placebo. The pharmaceutical composition used in this study was Formula 3 (herein referred to as OPK-88003) as a lyophilized formulation in a glass vial, which, in addition to the active ingredient, contained sodium citrate dihydrate (buffer), citric acid (buffer), sucrose (stabilizer), and mannitol (bulking agent), and finally sterile water for pre-injection. The vial contained 25 mg of Formula 3 at a pH of approximately 5.5. The vial provides a solid lyophilized formulation that is reconstituted for injection with 1.1 mL of sterile water for injection.

[0410] OPK-88003 also produced a dose-dependent weight loss of up to 3.3 kg by week 24. Compared to placebo and exenatide ER, OPK-88003 50 mg produced a statistically significant percent change in body weight from baseline at weeks 12 (p<0.001 and p=0.011, respectively) and 24 (p=0.007 and p=0.05, respectively). By week 24, at least twice as many subjects on OPK-88003-50 mg (35.5%) lost ≥5% of their body weight compared to placebo (11.8%) (p=0.004) and exenatide ER (18.3%) (p=0.025).

[0411] Example 7: Dose Escalation Study A randomized, double-blind, dose-escalation, placebo-controlled trial was conducted in approximately 110 subjects with type 2 diabetes mellitus (T2DM) controlled by diet and exercise alone or a stable dose of metformin. The study aimed to treat T2DM and obesity. The dose-escalation regimen was designed to improve glucose control and increase weight loss. The study had four phases: a screening / baseline phase (up to 2 weeks before the first dose), an 8-week dose-escalation phase, and a 22-week target dose phase, with a 30-week treatment period, and a 4-week follow-up period. Subjects were randomly assigned to receive OPK-88003 (Formula 3 in a buffered solution) or placebo QW (once weekly). The study evaluated the effects of a dose-escalation regimen of OPK-88003 on HbA1c, weight loss, and safety over 30 weeks in adult T2DM patients with inadequate glucose control with metformin and / or diet and exercise.

[0412] Dose-escalation phase 2 study OPK-88003 or placebo was administered as a weekly SC injection. OPK-88003 or placebo was administered initially at 20 mg QW for 4 weeks, followed by 40 mg QW for 4 weeks. After completing an 8-week dose escalation, subjects received a target dose of 70 mg QW for 22 weeks. The control group received matching placebo SC injections QW for 30 weeks.

[0413] A total of 113 subjects were randomized to OPK-88003 and placebo in a 1.75:1 ratio.

[0414] The primary efficacy outcome of change in HbA1c from baseline to 30 weeks was performed on the mITT population. The primary efficacy outcome was analyzed using a linear construct of the analysis of covariance (ANCOVA) model, with treatment group and BMI strata as factors and baseline HbA1c value as the covariate. An additional supportive analysis of the primary efficacy outcome was a mixed model repeated measures (MMRM). The factors in the model were BMI strata, treatment group, baseline value, visit, and treatment group by visit interaction. Additional covariates could be added. The mean percent weight change from baseline at 30 weeks was analyzed using a dose-response model similar to the primary analysis. A logistic regression analysis was performed for the percentage of subjects with a 5% or greater weight loss, with treatment, HbA1c, and BMI strata as fixed effects and baseline weight as the covariate. Comparisons between treatment groups for the number and percentage of subjects achieving an HbA1c of 6.5% or less were made for response after 30 weeks of treatment by LOCF based on a logistic regression model including factors for BMI strata and treatment group with baseline HbA1c as covariates.

[0415] The list of definitions of abbreviations and terms used herein includes the following:

[0416] [Table 41]

[0417] The primary objective of the study was to evaluate the effect of dose escalation of QW SC OPK-88003 versus placebo injections on absolute change in HbA1c from baseline to 30 weeks in subjects with type 2 diabetes mellitus (T2DM) inadequately controlled with diet and exercise alone or treated with a stable dose of metformin. Patients included in the study had T2DM for at least 6 months according to the American Diabetes Association2 disease diagnostic criteria. Diabetes Classification and Diagnosis: Standards of Medical Care in Diabetes-2018 guidelines (Diabetes Care 2018:41(Suppl 1):S13-S27).

[0418] Secondary endpoints include the mean percent weight change from baseline after the entire treatment period (30 weeks), the percent of subjects with a weight loss of 5% or more after 30 weeks of treatment, the change in FPG from baseline to after 30 weeks of treatment, and the number and percentage of subjects achieving an HbA1c of 6.5% or less (note that 7.6% HbA1c is 60 mmol / mol and 7.2% HbA1c is 55 mmol / mol).

[0419] Other objectives include changes from baseline to 30 weeks of treatment in HDL-C, total cholesterol, triglycerides, and LDL-C, fasting FGF-21, adiponectin, beta-hydroxybutyrate, glucagon, and insulin levels, incidence and rate of GI and CV events, immunogenicity of OPK-88003 to assess the PK of OPK-88003, and PD endpoints including, but not limited to, efficacy (glucose, HbA1c, weight), tolerability (e.g., nausea and vomiting), safety (e.g., QTc, HR), and biomarkers (e.g., insulin, glucagon).

[0420] Patients included in the study were men or women aged 18–80 years, with HbA1c levels of ≥7.0% and ≤10.5% at screening, and BMIs of ≥27 and ≥45 kg / m at screening. 2The appropriate doses described herein in stable formulations administered directly to T2DM patients in need of treatment for T2DM lower HbA1c by less than the patient's pre-regimen (baseline) state.

[0421] The drug product / formulation is a sterile solution for injection (1 mL extractable volume in a 2 mL glass vial). The vial contains 70 mg / mL of OPK-88003 and inactive ingredients selected from L-methionine (antioxidant), sorbitol (isotonicity agent), and sodium acetate trihydrate (buffer) at a pH of approximately 4.5. The pH is adjusted with acid or base. In one embodiment, the formulation includes: OPK-88003 70mg / mL ●L-methionine 4.48mg / mL Sorbitol 38.26mg / mL Sodium acetate trihydrate 1.36 mg / mL ●Water appropriate amount ~1mL

[0422] The placebo product composition is identical to the OPK-88003 drug product without the OPK-88003 active ingredient.

[0423] Previous research findings Similar effects on HbA1c and greater weight loss were observed with OPK-88003 compared with exenatide ER in a previous phase 2 study. These findings are consistent with the hypothesis that the additional glucagon activity associated with OPK-88003 provides equivalent glycemic control and superior weight loss compared with GLP-1 modulation alone. Because it has been demonstrated that additional weight loss can be achieved by increasing the dose of GLP-1 agonists (Davies et al., JAMA, 2015, 314:687-699), increasing the dose of OPK-88003 may demonstrate additional weight loss benefits. A previous phase 2 study investigated the dose-response relationship of OPK-88003 versus placebo and the positive control exenatide ER and established that QW injections over 24 weeks were able to reduce HbA1c by up to 1.43% and body weight by up to 3.3 kg. The severity of the AE profile observed to date with OPK-88003, including at the 50 mg QW dose tested in the Phase 2 study, does not outweigh the expected favorable benefits on glucose and body weight. Therefore, the balance of benefits and risks of OPK-88003 supports further clinical trials of OPK-88003 at doses higher than 50 mg QW.

[0424] Previous human studies also demonstrated a dose-dependent increase in GI side effects with higher OPK-88003 doses; however, these effects were essentially transient. In efforts to optimize OPK-88003 for glucose control and weight loss, dose titration, from gradual dose escalation to the target dose level, may be key to reducing GI side effects. Other GLP-1 agonists, such as liraglutide QD (once daily) and semaglutide QW, have shown that when slow dose titration is implemented, higher doses can be achieved and the rate of patients reporting GI problems can be significantly reduced. In the multiple-ascending-dose portion of the DPO-101 Phase 1 trial for diabetes, the use of a 60 mg QW titration regimen improved the GI tolerability of OPK-88003. Therefore, it is reasonable to predict that a slower dose-escalation regimen would exhibit improved tolerability of nausea and vomiting at doses higher than the 50 mg dose administered in the previous Phase 2 trial. Therefore, the study will employ a slow dose titration schedule over 8 weeks to help mitigate gastrointestinal AEs.

[0425] Dose selection This study includes a target dose of OPK-88003 of 70 mg administered weekly. Based on data from subjects with T2DM in a previous Phase 2 study, the 70 mg QW dose is predicted to produce the greatest reductions in fasting glucose and HbA1c and greater weight loss than the 50 mg QW dose from the dose-range-finding Phase 2 study. The 70 mg QW dose level will be administered using a fixed dose-escalation schedule over 8 weeks. Plasma levels of OPK-88003 reach a steady state over 5 weeks, with a maximum cumulative Cmax and area under the curve (AUC) ratio of 2.6-fold. Therefore, slow dose titration, with dose escalation every 4 weeks, is reasonable. For the first 4 weeks of dose escalation, subjects received 20 mg, followed by 40 mg QW for 4 weeks.

[0426] Safety and tolerability from a previous Phase 2 study support the evaluation of the planned dose levels over an 8-week dose escalation period.

[0427] A population PK model with first-order absorption and elimination, along with interindividual variability for all PK parameters, was used to simulate the proposed 70 mg QW dose. This dose is 33% higher (Cmax and AUC) when compared to the 50 mg dose, which had acceptable safety and tolerability over 24 weeks. Simulated PK exposures derived from a previous Phase 2 study model, combined with a Phase 1 clinical pharmacology study with supratherapeutic exposures and nonclinical safety margins, support multiple doses of 70 mg administered QW in the target population.

[0428] Due to the long terminal half-life of OPK-88003, supratherapeutic exposure in clinical pharmacology studies lasted approximately 3 weeks after the final dose. The once-daily dosing regimen resulted in a high rate of moderately severe nausea and vomiting requiring concomitant medication; however, most events resolved within 1 week of the final dose. The intensity and / or duration of these events are expected to be reduced by slow dose titration in the current study. In previous OPK-88003 studies, there were no reported safety concerns based on clinical laboratory assessments and ECGs, and there were no serious or serious adverse events (SAEs) at supratherapeutic exposure.

[0429] Safety data from human studies confirm dose-related, monitorable AEs related to GI tolerability. Most events were single episodes that were mild in severity, transient, and self-limited. These events most commonly occurred after the first dose, and then the incidence decreased to near-control levels by the fourth dose, consistent with a GLP-1R agonist effect.

[0430] Furthermore, studies on molecules with similar mechanisms have shown improved tolerability with slower dose titration and longer duration of administration, with no new unexpected toxicities noted with longer duration of administration.

[0431] The toxicology program for OPK-88003 provides continuous plasma exposure in rats and monkeys on a BIW dosing schedule, supporting the proposed dosing regimen. Based on exposure (AUC) derived from population PK modeling in humans and NOAELs for organ toxicity determined in rats (3 mg / kg) and monkeys (1.5 mg / kg) after chronic dosing, the safety margin for the 70 mg dose corresponds to a 0.7-fold and 10-fold exposure factor, respectively. Considering clinical efficacy and tolerability, the 70 mg QW dose is predicted to be tolerable with slow dose escalation and provide benefits in glycemic control and weight loss. Data from this study, along with data from a previous Phase 2 study, will enable a robust benefit-risk assessment in T2DM, form the basis for evaluating the effects of dose escalation on OPK-88003's efficacy, safety, and tolerability, and support the selection of dose(s) and dose regimen to be included in Phase 3 studies.

[0432] Study design Subjects followed a fixed-dose escalation regimen with dose escalation over 8 weeks. The target dose of 70 mg QW was achieved after 4 weeks of 20 mg followed by 4 weeks of 40 mg QW. Individual subjects were on study for a maximum of 36 weeks. Blinding was maintained throughout the study.

[0433] This study is designed to establish the effect of a dose-escalation regimen on the safety, tolerability, and efficacy of 70 mg OPK-88003 compared with placebo in subjects with T2DM. The study duration, target population, and efficacy endpoints are typical of Phase 2 trials and consistent with FDA guidance. A 4-week follow-up period after the treatment period ensures adequate time to assess the reversibility of any clinical or laboratory abnormalities. Subjects who develop AD during follow-up will undergo additional follow-up visits approximately every 4 months after their last dose. This clinical study enrolled subjects with inadequate glycemic control based on HbA1c values ​​ranging from 7.0 to 10.5%, inclusive, at the beginning and end of the study. Similar screening HbA1c ranges have been used in many studies of T2DM treatment. Subjects treated with diet and exercise alone or in combination with stable metformin monotherapy (≥1000 mg / day) were enrolled. Subjects taking a second oral antihyperglycemic agent (OAM) may also be eligible if the second OAM was discontinued more than 3 months prior to Visit 1. At least 3 months of stable metformin treatment is required to minimize baseline glucose drift prior to study enrollment.

[0434] The study duration for each individual subject was up to 36 weeks and consisted of four phases: screening (up to 2 weeks before the first dose), dose escalation (8 weeks), target dose / maintenance (22 weeks), and follow-up (4 weeks).

[0435] result Data were analyzed for the modified intention to treat patient population (mITT, 108 of 113 patients) using mixed model repeated measures (MMRM). The mITT population includes all patients who received at least one dose of the drug and one after the baseline assessment.

[0436] A top-line analysis of the study's results showed that OPK88003 met its primary objective with a statistically significant reduction in hemoglobin A1c (HbA1c) after 30 weeks of treatment versus placebo, as well as a statistically significant weight loss versus placebo, a key secondary endpoint.

[0437] OPK88003 demonstrated strong, clinically meaningful reductions in HbA1c at 30 weeks (-1.30% vs. placebo, mean absolute reduction of -0.09%, p<0.0001). Additionally, 50% of OPK88003-treated patients achieved an HbA1c ≤ 6.5% compared to 13.8% of placebo-treated controls (p=0.0008).

[0438] Tables 29-33 show the analysis of the change in HbA1c from baseline to Weeks 4, 8, 12, 22, and 30 / LOCFh in the MMRM mITT population. Table 34 shows the analysis of the change in HbA1c from baseline to Week 30 / LOCFall in the ANCOVA mITT population.

[0439] Patients treated with OPK88003 achieved significant weight loss at 30 weeks (-4.4 kg vs. -1.8 kg compared with placebo, p=0.01). Approximately 38% of treated patients achieved a weight loss of 5% or more compared with 13% of placebo-treated patients (p=0.008).

[0440] Table 35 shows an analysis of weight change from baseline to week 30 in the MMRM mITT population. Table 36 shows an analysis of subjects achieving >=5% weight loss in the mITT population.

[0441] OPK88003-treated patients demonstrated a significant reduction in blood triglycerides from baseline. The triglyceride reduction in the OPK88003-treated group was -31.2 mg / dL (p=0.005) compared with -11.6 mg / dL (p=0.44) for placebo. OPK88003 treatment demonstrated a safety and tolerability profile expected for the GLP-1 receptor agonist class. The most frequent adverse events were nausea, vomiting, and diarrhea. These were mostly mild, occurred primarily during the titration period, and resolved over time. No serious adverse events were observed.

[0442] Table 37 shows the analysis of the change in triglycerides from baseline to week 30 in the MMRM mITT population.

[0443] [Table 42-1]

[0444] [Table 42-2]

[0445] [Table 43-1]

[0446] [Table 43-2]

[0447] [Table 44-1]

[0448] [Table 44-2]

[0449] [Table 45-1]

[0450] Table 45-2

[0451] Table 46-1

[0452] Table 46-2

[0453] Table 47-1

[0454] Table 47-2

[0455] Table 48-1

[0456] Table 48-2

[0457] Table 49-1

[0458] Table 49-2

[0459] Table 50-1

[0460] [Table 50-2]

[0461] Example 8: Solubility, Viscosity, Syringability, Temperature Excursion, and Stability Initial formulation studies were performed on OPK-88003 peptide formulated at 60, 70, and 80 mg / mL in 10 mM citrate buffer (pH 5.5). The following methods were used to test OPK-88003 samples: MFI, HIAC, DLS, viscosity, and syringeability. Additionally, solubility studies were performed on OPK-88003 samples formulated at 40, 60, and 80 mg / mL in 10 mM citrate buffer (pH 5.5).

[0462] Solubility Studies: Lyophilized OPK-88003 dry powder was completely soluble in acetate buffer at room temperature at all concentrations tested (40, 60, and 80 mg / mL). Dissolution times were approximately 1-2 minutes for the 40 mg / mL, 2-3 minutes for the 60 mg / mL, and approximately 3-4 minutes for the 80 mg / mL samples. All solutions were clear and colorless. The solubility of OPK-88003 solutions was evaluated after storing samples at room temperature, 5°C, and -5°C for 18 hours. No phase separation or peptide precipitation was observed under all conditions tested.

[0463] [Table 51]

[0464] [Table 52]

[0465] Viscosity: As expected, the viscosity in the formulations tested increased with increasing OPK-88003 concentration. In all formulations, the viscosity remained unchanged with increasing shear rate, indicating Newtonian behavior.

[0466] [Table 53]

[0467] Needle Penetration: The breaking relaxation force and sliding equilibrium stress were measured for three OPK-88003 solutions using six different needle types. No significant differences in breaking relaxation force were observed between the OPK-88003 solutions and needle types tested. Clear trends in sliding equilibrium stress were observed based on needle type and OPK-88003 concentration.

[0468] [Table 54]

[0469] [Table 55]

[0470] [Table 56]

[0471] Stability Studies: Additional stability studies were performed on 70 mg / mL acetate buffer with methionine pH 4.5 in 1 mL long BD Neopak™ prefillable syringes.

[0472] [Table 57]

[0473] Example 9: Evaluation of local tolerance of the formulation of the present invention This study was designed to evaluate the local tolerance following a single subcutaneous dose of OPK-88003 at two different concentrations and in two different vehicle formulations, as well as their respective vehicle controls, when administered to New Zealand White rabbits.

[0474] Rabbits were assigned to four treatment groups (2 females per group), and each animal received two single subcutaneous injections: one containing a vehicle control and one containing an OPK-88003 test article formulation. Rabbits were subcutaneously injected with 1 mL of either OPK-88003 acetate buffer-based vehicle control 1 and 25 or 75 mg / mL OPK-88003 formulated in an acetate buffer-based vehicle, or citrate buffer-based vehicle control 2 and 25 or 75 mg / mL OPK-88003 formulated in a citrate buffer-based vehicle, as outlined below. Vehicle (control) 1 = 10 mM acetate buffer, 30 mM methionine, 230 mM sorbitol, pH 4.5 Vehicle (control) 2 = 30 mM citrate, 20 mg / mL mannitol, 5 mg / mL sucrose, pH 5.5 Injection site 1 was in a different location than injection site 2 Each volume was 1 mL.

[0475] result All animals survived until scheduled euthanasia at the end of the study. Other than the local injection site reactions described below, no clinical findings were observed related to the vehicle control or test article formulations. Individual Draize scoring results are presented in Table 36. A summary of the mean Draize edema scores is presented in Table 37.

[0476] Erythema: During the study, no animals exhibited erythema at the injection site after administration of either the two vehicle control or test article formulations at 25 or 75 mg / mL.

[0477] Edema Associated with Vehicle Control: Very slight edema (score of 1) was observed at the injection site of acetate buffer-based vehicle (Vehicle Control 1) in one animal (#002) by 30 minutes post-dose and at the injection site of citrate buffer-based vehicle (Vehicle Control 2) in two animals (#006, #007) (score of 1) by 77 minutes post-dose. The mean edema scores for acetate buffer-based vehicle injection sites were 0.25 and 0 at 15 minutes post-dose and the last scoring time point (beginning and end of study, respectively). The mean edema scores for citrate buffer-based vehicle injection sites were 0.5 and 0.5 at the beginning and end of the study, respectively.

[0478] Edema Associated with 25 and 70 mg / mL OPK-88003: As early as 15 minutes after administration, all animals had very slight (score 1) to slight (score 2) edema at the injection site of administration of the test article formulation of OPK-88003, which either remained the same or decreased in severity by the end of the study. Animals in Groups 1 and 3 (administered test article at 25 mg / animal formulated in acetate and citrate buffer-based vehicles, respectively) similarly exhibited mean edema scores of 2 and 1.5 at the beginning and end of the study, respectively.

[0479] Group 2 animals (administered test substance at 70 mg / animal formulated in an acetate buffer-based vehicle) exhibited mean edema scores of 2 and 1 at the beginning and end of the study, respectively. Group 4 animals (administered test substance at 70 mg / animal formulated in a citrate buffer-based vehicle) exhibited mean edema scores of 1 and 0.5 at the beginning and end of the study, respectively. The slightly greater degree of edema associated with the OPK-88003 formulation compared with the vehicle control may be due, in part, to the higher viscosity of these solutions (approximately 22 cP), which occurs in the presence of OPK-88003 but not in the vehicle control alone. Given the absence of any visible inflammation at the injection site at necropsy, edema due to the high viscosity of the OPK-88003 formulation is likely.

[0480] In summary, the degree of localized edema at the injection site was slightly greater with OPK-88003 administration compared with vehicle control, regardless of the vehicle used in formulation. The injection site edema associated with 25 mg / mL OPK-88003 was similar regardless of whether it was formulated in an acetate or citrate buffer-based vehicle. The injection site edema associated with 70 mg / mL OPK-88003 was slightly greater with acetate compared with citrate buffer-based vehicles. However, the degree of injection site edema and any differences observed between treatment groups were minimal and therefore not considered adverse or significant.

[0481] [Table 58-1]

[0482] [Table 58-2]

[0483] [Table 59]

[0484] Gross Pathology: No vehicle control or OPK-88003 test article formulation-related gross findings were observed at the injection sites. Bruising outside the area of ​​the marked injection site was observed in the following animals during gross necropsy: #001 (Group 1): Left side presented with bruising outside the area of ​​the marked injection site. ●#006 (Group 3): The left side presented with bruising outside the area of ​​the marked injection site. #007 (Group 4): The right side presented with bruising outside the area of ​​the marked injection site. #008 (Group 4): Left and right sides presented with bruising outside the area of ​​the marked injection site.

[0485] However, this bruising was determined to be a result of the subcutaneous injection treatment and not related to the vehicle control or test substance.

[0486] In conclusion, a single subcutaneous injection of OPK-880033 formulated in either an acetate or citrate buffer-based vehicle at dose levels of 25 or 70 mg / animal was well tolerated in female New Zealand White rabbits and was associated with only very slight localized edema at the injection site. Edema associated with the OPK-88003 formulation, likely due to its higher viscosity, was slightly greater than that seen with the vehicle control alone. However, edema was not dose-dependent, was minimal, and was not considered adverse. Therefore, we conclude that there were no significant differences in local injection site edema between the two vehicle controls or between the dose levels of the OPK-88003 formulations.

[0487] The inventors discovered that while typical isotonic solutions containing sodium chloride are generally suitable for drug delivery in liquid or parenteral formulations, the presence of sodium chloride in solutions containing PEGylated oxyntomodulin analogs unexpectedly caused instability issues. Furthermore, the addition of the PEGylated oxyntomodulin compounds or analogs described herein surprisingly caused the pH to rise above the preferred range. Thus, the inventors discovered liquid formulations according to the present invention that meet the necessary physical and chemical stability criteria. By discovering the stable liquid formulations herein, the inventors avoided the need to use solid dispersions of the drug (i.e., lyophilized or otherwise) that must be reconstituted with water or saline prior to administration to patients. Furthermore, because the formulations are stable as refrigerated liquids and do not require freezing to maintain stability, they can be stored in prefilled syringes or cartridges rather than vials, making them easier for patients to use at home. Thus, a patient or healthcare professional can "administer" the dosage form directly from the pre-filled syringe, cartridge, or filled vial without the additional step of reconstituting the powder formulation, and / or such an individual could not use a pre-filled syringe (or pre-filled vial) that does not have such a stable liquid formulation.

[0488] The above examples are non-limiting in nature, and such formulations can include any PEGylated oxyntomodulin analog and any suitable buffer and / or isotonicity agent, and / or stabilizer, provided that such liquid formulations made therefrom are substantially free of impurities, suitable for sterile manufacture, stable, and meet the required delivery and physical properties, including flow, viscosity, and compatibility with delivery methods including pre-filled syringes, auto-injectors, and the like for single and multiple use.

[0489] All of the features described in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above-described aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. While preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, and the like, can be made without departing from the spirit of the invention, and therefore are deemed to be within the scope of the invention as defined in the following claims.

Claims

1. 1. A PEGylated GLP-1 and / or glucagon receptor agonist liquid formulation comprising: a pharmaceutically effective amount of a PEGylated GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof; methionine present in an amount of 30 mM to 35 mM; an aqueous acetate buffer present in an amount of 10 mM; the formulation has a pH value of 4.5, A formulation wherein the PEGylated GLP-1 and / or glucagon receptor agonist is present at a concentration of 70 mg / mL and comprises a compound of formula 3: (Formula 3)

2. A PEGylated GLP-1 and / or glucagon receptor agonist liquid formulation, comprising: a pharmaceutically effective amount of a PEGylated GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof; methionine present in an amount between 0 mM and 35 mM; an aqueous acetate buffer present in an amount of 10 mM; the formulation has a pH value of 4.5, The GLP-1 and / or glucagon receptor agonist is present at a concentration of 70 mg / mL and has the amino acid sequence: His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Gly-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa ３８ -Xaa ３９ (SEQ ID NO:1), comprising Xaa ３８ is Cys-PEG, Xaa ３９ is Cys-PEG, The C-terminal amino acid is amidated.

3. 3. The formulation of claim 1 or 2, wherein the formulation contains less than or equal to 10.0% total impurities as measured by RP-HPLC or size exclusion chromatography.

4. 4. The formulation of any one of claims 1 to 3, wherein the formulation comprises cysteine ​​in an amount less than 25 mM.

5. 5. The formulation of any one of claims 1 to 4, wherein the formulation contains sodium chloride in an amount less than 100 mM.

6. The formulation of claim 1, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, ascorbate, maleate, or acetate.

7. (a) the GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, is present in a suitable vial; (b) the GLP-1 and / or glucagon receptor agonist, or a pharmaceutically acceptable salt thereof, is present in a single-use vial; (c) the GLP-1 and / or glucagon receptor agonist is present at a concentration of 70 mg / mL; (d) the formulation has an osmolality of 500 mOsm / kg or less. (e) the formulation has a viscosity of 10 cP to 60 cP; (f) the preparation has a syringeability of a breaking relaxation force of less than 10 N and a sliding equilibrium stress of less than 30 N; (g) the formulation is stable for at least five freeze / thaw cycles, and / or (h) the formulation is stable for at least 48 hours at 30°C / 65% RH and for 12 months at 5°C; The formulation according to any one of claims 1 to 6.

8. The formulation of claim 1, wherein the PEGylated GLP-1 and / or glucagon receptor agonist is in the form of an acetate salt.

9. The formulation of claim 8, wherein the acetate buffer is sodium acetate trihydrate.

10. A formulation according to any one of claims 1 to 9, wherein the formulation causes no or little injection site reaction.

11. The formulation according to any one of claims 1 to 10, further comprising an isotonic agent.

12. 12. The formulation of claim 11, wherein the isotonicity agent comprises sucrose, mannitol, or sorbitol.

13. 13. The formulation of claim 12, wherein the isotonicity agent is sorbitol and is present in an amount of 230 mM to 250 mM.

14. (a) the formulation further comprises 0.001% (w / v) to 1% (w / v) of one or more preservatives, based on the total volume of the formulation; (b) the formulation further comprises 0.001% (w / v) to 1% (w / v) of one or more preservatives based on the total volume of the formulation, and the preservative is m-cresol, phenol, and / or benzyl alcohol; and / or (c) the formulation further comprises one or more additional pharmaceutically acceptable excipients; The formulation according to any one of claims 1 to 13.

15. further comprising one or more additional pharmaceutically acceptable excipients; The formulation of any one of claims 1 to 14, wherein the additional excipient comprises methionine or arginine.

16. 16. The formulation of claim 15, wherein the additional excipient is methionine, and the methionine is L-methionine.

17. A formulation according to any one of claims 1 to 16 for use in treating patients with type 2 diabetes.

18. 17. The formulation according to any one of claims 1 to 16, for use in treating a patient in need of lowering HbA1c or a patient in need of lowering body weight.

19. 19. The formulation of claim 18, wherein the patient's HbA1c level is reduced by 1.30% compared to baseline or the patient's weight is reduced by 4.4 kg compared to baseline.

20. A formulation according to any one of claims 1 to 16 for use in treating a patient in need of lowering blood triglycerides.

21. 21. The formulation of claim 20, wherein the patient's blood triglycerides are reduced by 31.2 mg / dL compared to baseline.

22. 18. The formulation of any one of claims 1 to 17 for use in treating a patient having a disease or condition selected from the group consisting of obesity, type 1 diabetes, type 2 diabetes, impaired glucose metabolism, impaired glucose tolerance, impaired fasting glucose, hyperinsulinemia, insulin resistance, hypertension, fatty liver disease, non-alcoholic steatohepatitis (NASH), diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy.

23. (a) the formulation is administered twice weekly, or once weekly, or at longer intervals; (b) the formulation is administered once a week; (c) the formulation is substantially free of sodium chloride; (d) the formulation is substantially free of cysteine; (e) the GLP-1 and / or glucagon receptor agonist is administered for 30 weeks; (f) the pH of the liquid dose is 4.5; (g) the dosage administered to the patient is in the range of 20 to 150 mg / week. (h) the dosage administered to the patient is in the range of 0.5 to 1.5 mg / kg / week; (i) the formulation is administered from a pre-filled syringe, cartridge, pen, auto-injector, or vial; and / or (j) the formulation is administered from a pre-filled syringe, cartridge, pen, auto-injector, or vial, and the pre-filled syringe, cartridge, pen, auto-injector, or vial contains a buffer solution having 70 mg / mL of the GLP-1 and / or glucagon receptor agonist; The formulation according to any one of claims 17 to 22.

24. a first administration period of a GLP-1 and / or glucagon receptor agonist at a first once-weekly (QW) dose, followed by a second administration period at a second once-weekly (QW) dose, prior to administration of a pharmaceutically effective amount of the formulation; the second once-weekly (QW) dose is greater than the first once-weekly (QW) dose; The formulation according to any one of claims 17 to 23.

25. (a) the first once weekly (QW) dose is 1 to 20 mg QW; (b) the second once weekly (QW) dose is 10-50 mg QW; (c) the first administration period is from 2 weeks to 6 weeks, or from 2 weeks to 5 weeks, or from 3 weeks to 5 weeks; (d) the first administration period is 4 weeks; (e) the second administration period is from 2 weeks to 6 weeks, or from 2 weeks to 5 weeks, or from 3 weeks to 5 weeks; and / or (f) the second administration period is 4 weeks.

25. The formulation of claim 24.

26. further comprising a third dosing period with a third once weekly (QW) dose; 26. The formulation of claim 24 or 25.

27. (a) the third once-weekly (QW) dose is 50-80 mg QW; (b) the third once-weekly (QW) dose is 70 mg QW; (c) the third administration period is from 2 weeks to 6 weeks, or from 2 weeks to 5 weeks, or from 3 weeks to 5 weeks; and / or (d) the third administration period is 4 weeks.

27. The formulation of claim 26.

28. A pre-filled syringe containing the formulation according to any one of claims 1 to 16.

29. A multi-dose container containing a formulation according to any one of claims 1 to 16.

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