Formulations of Glucagon-like Peptide 2 (GLP-2) Analogs

Abstract

Describes a liquid formulation of a GLP-2 analogue that renders the GLP-2 analogue suitable for long-term storage as a liquid and / or renders the GLP-2 analogue particularly suitable for delivery by a drug delivery device. Also described is a solid composition comprising an acetate of a glucagon-like peptide 2 (GLP-2) analogue that can be used to prepare the liquid formulation. The development of these liquid formulations is based on the following findings: the acetate present in the formulation derived from the GLP-2 analogue has an effect on the viscosity of the formulation; during long-term storage of the GLP-2 analogue at 2 to 8 °C, the concentration-dependent negative correlation of covalent oligomer formation depends on the increase in the GLP-2 analogue concentration; and the GLP-2 analogue used in the formulation is incompatible with the phosphate buffer commonly used in the prior art for reconstituted powdered or lyophilized GLP-2 compositions.

Description

Technical Field

[0001] The present invention relates to preparations of glucagon-like peptide-2 (GLP-2) analogs and their medical uses, for example, in the treatment and / or prevention of gastric and intestinal disorders and for ameliorating the side effects of chemotherapy and radiation therapy. Furthermore, a solid composition comprising an acetate salt of a glucagon-like peptide-2 (GLP-2) analog that can be used to prepare a liquid formulation is also described. Background Art

[0002] Human GLP-2 is a 33-amino acid peptide with the following sequence:

[0003] Hy-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH. It is derived from specific post-translational processing of proglucagon in enteroendocrine L cells of the intestine and in specialized regions of the brainstem. GLP-2 binds to a single G protein-coupled receptor belonging to the class II glucagon secretin family.

[0004] GLP-2 has been reported to induce significant growth of the small intestinal mucosal epithelium by stimulating stem cell proliferation in the crypts and by inhibiting apoptosis in the villi (Drucker et al., 1996, Proc. Natl. Acad. Sci. USA 93:7911-7916). GLP-2 also has growth effects on the colon. In addition, GLP-2 inhibits gastric emptying and gastric acid secretion (Wojdemann et al., 1999, J. Clin. Endocrinol. Metab. 84: 2513-2517), enhances intestinal barrier function (Benjamin et al., 2000, Gut 47: 112-119), stimulates intestinal hexose transport by upregulating glucose transporters (Cheeseman, 1997, Am. J. Physiol. R 1965-71), and increases intestinal blood flow (Guan et al., 2003, Gastroenterology, 125: 136-147).

[0005] It is recognized in the art that glucagon-like peptide-2 receptor analogs have therapeutic potential for treating intestinal diseases. However, native hGLP-2 (a 33 amino acid gastrointestinal peptide) is not useful in clinical settings due to its very short half-life in humans (about 7 minutes [1-33] for full-length GLP-2 and 27 minutes [3-33] for truncated GLP-2). To a large extent, the short half-life is due to degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, attempts have been made in the art to develop GLP-2 receptor agonists with better pharmacokinetic properties, in particular to improve the half-life of the GLP-2 molecule. For example, GLP-2 analogs with substitutions have been proposed, such as, for example, GLP-2 analogs containing a Gly substitution at position 2 ([hGly2]GLP-2, teduglutide), which increase the half-life from 7 minutes (native GLP-2) to about 2 hours. Acylation of peptide drugs with fatty acid chains has also been shown to be beneficial for extending systemic circulation and improving enzyme stability without compromising biological efficacy. However, although these attempts have improved the pharmacokinetics of GLP-2 analogs, and they are sometimes described in the art as "long-acting," it must be remembered that this is compared to native hGLP-2, which has a half-life of about hours rather than minutes. In turn, this means that the GLP-2 analogs still need to be administered to patients once or more daily.

[0006] US 5,789,379 discloses GLP-2 analogs for administration by injection. The analogs are provided as powdered peptides and are mixed with phosphate-buffered saline (PBS) at a pH of 7.3 to 7.4 at a GLP-2 concentration of 130 mg / ml prior to injection. In some cases, the GLP-2 / PBS composition is mixed with gelatin to provide a depot formed by a solution of 130 mg / l GLP-2 in PBS / 15% gelatin. US 5,789,379 does not disclose a stable aqueous liquid formulation of the GLP-2 analog, and the GLP-2 analog is typically reconstituted from a powder prior to injection.

[0007] In WO 97 / 39031 and US 6,184,201, GLP-2 analogs [Gly 2 ] GLP-2. Here, the alanine at position 2 has been replaced by glycine to render the peptide resistant to DPP IV cleavage. As in US Pat. No. 5,789,379, the GLP-2 analog is provided as a powdered peptide and is mixed with saline, PBS, or 5% dextrose prior to injection, optionally with the addition of acetic acid as a solubility enhancer.

[0008] WO 02 / 066511 describes GLP-2 analogues with extended half-lives in vivo and their use as pharmaceuticals for the treatment of gastrointestinal disorders, such as inflammatory bowel disease. The GLP-2 analogues are stored in lyophilized form and reconstituted for administration in a medium, such as saline or PBS.

[0009] WO 01 / 41779 describes h[Gly 2 ] Use of GLP-2 as a pretreatment for inhibiting chemotherapy-induced apoptosis and promoting cell survival. h[Gly 2 ] GLP-2 is delivered by subcutaneous or intravenous injection or infusion following reconstitution of the analog in PBS.

[0010] WO 2001 / 049314 relates to formulations of GLP-2 peptides and analogs thereof that exhibit excellent stability after storage and / or exposure to elevated temperatures. The GLP-2 composition comprises a GLP-2 peptide or analog thereof, a phosphate buffer, L-histidine and mannitol.

[0011] WO 2006 / 117565 describes GLP-2 analogs that are compatible with [hGly 2 ] comprises one of a plurality of substitutions compared to GLP-2, and which has increased in vivo biological activity and / or increased chemical stability, for example as assessed in an in vitro stability assay. In particular, GLP-2 analogues are described which have substitutions at one or more of positions 8, 16, 24 and / or 28 of the wild-type GLP-2 sequence, optionally in combination with further substitutions at one or more of positions 3, 5, 7, 10 and 11 and at position 2 and / or deletion of one or more of amino acids 31 to 33. These substitutions may also be combined with the addition of N-terminal or C-terminal stabilizing peptide sequences. Daily or twice-daily administration of these GLP-2 analogues is also described. Among the molecules disclosed in WO 2006 / 117565, glepaglutide (ZP1848) has been designed to be stable in a liquid formulation and is typically administered by daily dosing using an injection pen.

[0012] Improving the formulation of GLP-2 analogs, particularly providing stable liquid formulations that can be stored for extended periods without excessive physical or chemical degradation of the active monomeric form of the peptide, remains a challenge in the field. In liquid formulations of peptide drugs, chemical pathways that can arise include the formation of covalently linked dimers and oligomers of the peptide, which reduces the amount of the active monomeric form of the peptide by forming these covalently linked, high-molecular-weight oligomeric products. The law of mass action means that, generally, the higher the concentration of the peptide drug in a formulation, the higher the likelihood of forming covalently linked oligomeric products.

[0013] It is also a goal in the art of GLP-2 analog formulations to provide formulations in which the viscosity of the formulation is controlled within a range that makes it suitable for use in a delivery device such as a prefilled syringe, an infusion pump, a wearable syringe, or an autoinjector. SUMMARY OF THE INVENTION

[0015] Broadly speaking, the present invention is based on studies reported in the Examples which led to some unexpected findings relating to liquid formulations of GLP-2 analogues which render them suitable for long term storage as liquids and / or which render them particularly suitable for delivery via drug delivery devices.

[0016] In a first study, the inventors discovered that acetate derived from a GLP-2 analogue present in the formulation has an effect on the viscosity of the formulation. This opens up the possibility of controlling the viscosity of the formulation by varying and / or controlling the acetate concentration. Liquid formulations with a low viscosity range can be used clinically because they offer advantages in the development and manufacture of drug delivery devices by potentially reducing breakage, dosing failure, inaccurate dosing, and other malfunctions during drug product manufacturing and / or patient use. In addition, low viscosity can allow for faster injections and / or the use of needles with narrower bores (i.e., higher gauges), which in turn can reduce injection discomfort. This opens up the possibility of providing GLP-2 analogue formulations in the form of drug delivery devices (e.g., prefilled syringes, adjustable dose auto-injectors, disposable auto-injectors, wearable syringes, or infusion pumps), thereby providing patients with ready-to-use formulations in simpler, safer, and more patient-friendly devices. Controlling the formulation to a higher viscosity may be applicable to other drug delivery devices.

[0017] In a second study, the inventors found that the formation of covalently bound oligomers during long-term storage of ZP1848 (glepaglutide) at 2 to 8°C was concentration-dependent. However, contrary to the general observation that the law of mass action suggests that covalent oligomer formation increases with increasing peptide drug concentration, the inventors found that the concentration dependence of oligomer formation was inversely correlated with increasing GLP-2 analog concentration. Without wishing to be bound by any particular theory, the inventors believe that the decrease in covalently linked oligomer formation with increasing GLP-2 analog concentration is a result of the lysine tail of the GLP-2 analog promoting the formation of self-associating structural assemblies of the native peptide, which hinders the formation of covalently bound oligomers in the formulation. This means that weakly self-associating species can dissociate after administration to a patient to release biologically active monomers, rather than resulting in loss of active material, as occurs when covalently bound oligomers are formed.

[0018] In a third study, the inventors discovered that the GLP-2 analogs used in the formulations of the present invention were incompatible with the phosphate buffers commonly used in the prior art for reconstituted powdered or lyophilized GLP-2 compositions. This study found that only some buffers were compatible with formulating these GLP-2 analogs, making them suitable for long-term storage in liquid form.

[0019] Thus, in a first aspect, the present invention provides a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the following formula:

[0020] R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0021] in:

[0022] R 1 It is hydrogen, C 1-4 an alkyl group (e.g., methyl), an acetyl group, a formyl group, a benzoyl group, or a trifluoroacetyl group;

[0023] X5 is Ser or Thr;

[0024] X11 is Ala or Ser;

[0025] R 2 is NH2 or OH; and

[0026] Z 1 and Z 2 independently absent or a peptide sequence having 1 to 6 Lys amino acid units;

[0027] wherein the preparation comprises:

[0028] (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL;

[0029] (b) a buffer selected from the group consisting of a histidine buffer, a methanesulfonate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-Tris buffer, and a MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM;

[0030] (c) a non-ionic tonicity adjusting agent selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0031] (d) Arginine in an amount to provide a formulation with a pH of about 6.6 to about 7.4.

[0032] In some embodiments, the formulation comprises 5% or less of the GLP-2 analogue in the form of covalently linked oligomer products. Alternatively or additionally, the total acetate concentration generated by the GLP-2 analogue in the formulation is less than or equal to 11% acetate / mg of GLP-2 analogue. Alternatively or additionally, the formation of covalently linked oligomers of the GLP-2 analogue is inversely dependent on the concentration of the GLP-2 analogue in the formulation.

[0033] The components of the formulation and their amounts provide a formulation that has at least 90% of the GLP-2 analogue content and less than 10% of chemical degradation products when stored at 2 to 8°C for at least 18 months.

[0034] In another aspect, the present invention provides an article of manufacture or kit comprising a container holding the stable pharmaceutical formulation of the present invention.

[0035] In another aspect, the present invention provides a delivery device comprising a liquid formulation comprising a GLP-2 analogue of the present invention.

[0036] In another aspect, the present invention provides a formulation of a glucagon-like peptide 2 (GLP-2) analogue of the invention for use in therapy.

[0037] In another aspect, the present invention provides formulations of the glucagon-like peptide 2 (GLP-2) analogs of the present invention for use in a method of treating and / or preventing gastric and intestinal related disorders in a human patient.

[0038] In another aspect, the present invention provides a method for producing a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analog is represented by the formula:

[0039] R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0040] in:

[0041] R 1 It is hydrogen, C 1-4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl

[0042] X5 is Ser or Thr

[0043] X11 is Ala or Ser

[0044] R 2 It is NH2 or OH;

[0045] Z 1 and Z 2 independently absent or a peptide sequence having 1 to 6 Lys amino acid units;

[0046] wherein the method comprises formulating (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL with: (b) a buffer selected from the group consisting of histidine buffer, methanesulfonate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer, and MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity adjuster selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, said non-ionic tonicity adjuster being present at a concentration of about 90 mM to about 360 mM; and (d) arginine in an amount to provide a formulation having a pH of about 6.6 to about 7.4;

[0047] wherein the formulation comprises 5% or less of the GLP-2 analogue as covalently bonded oligomeric products.

[0048] In another aspect, the present invention provides the use of a formulation comprising a glucagon-like peptide 2 (GLP-2) analogue, or a pharmaceutically acceptable salt or derivative thereof, for providing a liquid pharmaceutical formulation that is stable for 24 months when stored at 2 to 8°C, wherein the GLP-2 analogue is represented by the formula:

[0049] R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0050] in:

[0051] R 1 It is hydrogen, C 1-4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl

[0052] X5 is Ser or Thr

[0053] X11 is Ala or Ser

[0054] R 2 It is NH2 or OH;

[0055] Z 1 and Z 2 independently absent or a peptide sequence having 1 to 6 Lys amino acid units;

[0056] wherein the preparation comprises:

[0057] (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL;

[0058] (b) a buffer selected from the group consisting of a histidine buffer, a methanesulfonate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-Tris buffer, and a MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM;

[0059] (c) a non-ionic tonicity adjusting agent selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0060] (d) Arginine in an amount to provide a formulation with a pH of about 6.6 to about 7.4.

[0061] In another aspect, the present invention provides a method for regulating the viscosity of a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analog is represented by the formula:

[0062] R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0063] in:

[0064] R 1 It is hydrogen, C 1-4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl

[0065] X5 is Ser or Thr

[0066] X11 is A1a or Ser

[0067] R 2 It is NH2 or OH;

[0068] Z 1 and Z 2 independently absent or a peptide sequence having 1 to 6 Lys amino acid units;

[0069] wherein the method comprises formulating (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL with: (b) a buffer selected from a histidine buffer, a methanesulfonate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-Tris buffer, or a MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity adjuster selected from mannitol, sucrose, glycerol, sorbitol, and trehalose, said non-ionic tonicity adjuster being present at a concentration of about 90 mM to about 360 mM; and (d) arginine in an amount to provide a formulation having a pH of about 6.6 to about 7.4;

[0070] wherein the total acetate concentration resulting from the GLP2 analog in the formulation is less than or equal to 11% acetate / mg GLP-2 analog, and wherein the viscosity of the formulation measured at 25°C is greater than 0.8 and less than or equal to 2.0 mPa / second.

[0071] In another aspect, the present invention provides a method for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analog in a stable liquid pharmaceutical formulation comprising a GLP-2 analog represented by the formula:

[0072] R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Ihr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0073] in:

[0074] R 1 It is hydrogen, C 1-4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl

[0075] X5 is Ser or Thr

[0076] X11 is Ala or Ser

[0077] R 2 It is NH2 or OH;

[0078] Z 1 and Z 2 independently absent or a peptide sequence having 1 to 6 Lys amino acid units;

[0079] wherein the method comprises formulating (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL with: (b) a buffer selected from a histidine buffer, a methanesulfonate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-Tris buffer, or a MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity adjuster selected from mannitol, sucrose, glycerol, sorbitol, and trehalose, said non-ionic tonicity adjuster being present at a concentration of about 90 mM to about 360 mM; and (d) arginine in an amount to provide a formulation having a pH of about 6.6 to about 7.4;

[0080] wherein the formulation comprises 5% or less of the GLP-2 analogue as covalently linked oligomer products. In some cases, in this aspect of the invention, the formation of covalently linked oligomers of the GLP-2 analogue is inversely dependent on the concentration of the GLP-2 analogue in the formulation.

[0081] In another aspect, the present invention provides use of a formulation for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue, or a pharmaceutically acceptable salt or derivative thereof, in a liquid pharmaceutical formulation that is stable for 24 months when stored at 2 to 8°C, wherein the GLP-2 analogue is represented by the formula:

[0082] R 1 -Z 1-His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0083] in:

[0084] R 1 It is hydrogen, C 1-4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl

[0085] X5 is Ser or Thr

[0086] X11 is Ala or Ser

[0087] R 2 It is NH2 or OH;

[0088] Z 1 and Z 2 independently absent or a peptide sequence having 1 to 6 Lys amino acid units;

[0089] wherein the preparation comprises:

[0090] (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL;

[0091] (b) a buffer selected from the group consisting of a histidine buffer, a methanesulfonate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-Tris buffer, and a MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM;

[0092] (c) a non-ionic tonicity adjusting agent selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0093] (d) arginine in an amount to provide a formulation with a pH of about 6.6 to about 7.4;

[0094] wherein the formulation comprises 5% or less of the GLP-2 analogue as covalently linked oligomer products. In some cases, in this aspect of the invention, the formation of covalently linked oligomers of the GLP-2 analogue is inversely dependent on the concentration of the GLP-2 analogue in the formulation.

[0095] In another aspect, the present invention provides use of a formulation for regulating the viscosity of a liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue, or a pharmaceutically acceptable salt or derivative thereof, wherein the liquid pharmaceutical formulation is stable for 24 months when stored at 2 to 8°C, wherein the GLP-2 analogue is represented by the following formula:

[0096] R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0097] in:

[0098] R 1 It is hydrogen, C 1-4 Alkyl (e.g., methyl), acetyl, formyl, benzoyl, or trifluoroacetyl

[0099] X5 is Ser or Thr

[0100] X11 is Ala or Ser

[0101] R 2 It is NH2 or OH;

[0102] Z 1 and Z 2 independently absent or a peptide sequence having 1 to 6 Lys amino acid units;

[0103] wherein the preparation comprises:

[0104] (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL;

[0105] (b) a buffer selected from the group consisting of a histidine buffer, a methanesulfonate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-Tris buffer, and a MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM;

[0106] (c) a non-ionic tonicity adjusting agent selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0107] (d) arginine in an amount to provide a formulation with a pH of about 6.6 to about 7.4;

[0108] wherein the total acetate concentration generated by the GLP2 analog in the formulation is less than or equal to 11% acetate / mg GLP-2 analog, and the viscosity of the formulation measured at 25° C. is 0.8 to 2.0 mPa / s.

[0109] In another aspect, the present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula:

[0110] (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH), wherein x is 1.0 to 8.0.

[0111] In another aspect, the present invention provides a stable aqueous pharmaceutical formulation comprising:

[0112] (a) a solid composition of the present invention having a concentration of about 2 mg / mL to about 30 mg / mL;

[0113] (b) a buffer selected from the group consisting of a histidine buffer, a methanesulfonate buffer, an acetate buffer, a glycine buffer, a lysine buffer, a TRIS buffer, a Bis-Tris buffer, and a MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM;

[0114] (c) a non-ionic tonicity adjusting agent selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0115] (d) arginine in an amount to provide a formulation with a pH of about 6.6 to about 7.4;

[0116] wherein the formulation comprises 5% or less of the GLP-2 analogue as a covalently bonded oligomeric product, and wherein the formulation has a viscosity of 0.8 to 2.0 mPa / second measured at 25°C.

[0117] In all aspects of the invention described herein, the buffer may be selected from the group consisting of a histidine buffer, a methanesulfonate buffer and an acetate buffer.

[0118] In all aspects of the invention described herein, the non-ionic tonicity adjusting agent may be selected from mannitol, sucrose, glycerol and sorbitol.

[0119] In some embodiments, the formulation comprises 5% or less of the GLP-2 analogue in the form of covalently linked oligomer products. Alternatively or additionally, the total acetate concentration generated by the GLP-2 analogue in the formulation is less than or equal to 11% acetate / mg of GLP-2 analogue. Alternatively or additionally, the formation of covalently linked oligomers of the GLP-2 analogue is inversely dependent on the concentration of the GLP-2 analogue in the formulation.

[0120] In another aspect, the present invention relates to a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the following formula:

[0121] R 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0122] in:

[0123] R 1 It is hydrogen, C 1-4 an alkyl group (e.g., methyl), an acetyl group, a formyl group, a benzoyl group, or a trifluoroacetyl group;

[0124] X5 is Ser or Thr;

[0125] X11 is Ala or Ser;

[0126] R 2 is NH2 or OH; and

[0127] Z 2 It is a peptide sequence with 6 Lys amino acid units;

[0128] The formulation comprises the components as set out in any one of the aspects of the invention as set out herein.

[0129] In this aspect of the invention, formulations comprising a glucagon-like peptide 2 (GLP-2) analog or a salt thereof can be used to treat and / or prevent stomach and intestinal related disorders, such as ulcers, digestive disorders, malabsorption syndrome, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue (e.g., caused by gluten-induced enteropathy or celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, Crohn's disease, ulcerative colitis, small intestinal damage, or short bowel syndrome (SBS). Alternatively or in addition, glucagon-like peptide 2 (GLP-2) analogs can be used to treat and / or prevent gastric and intestinal disorders, such as radiation enteritis, infectious or post-infectious enteritis, or small intestinal damage caused by toxic agents or other chemotherapeutic agents. In this case, treatment with GLP-2 analogs can optionally be combined with one or more anti-cancer therapies and thus can include administering one or more chemotherapeutic agents to the patient or treating the patient with radiation therapy.

[0130] In some embodiments of the present invention, in the above formula, X5 is Thr and / or X11 is Ala. Some examples of these glucagon-like peptide 2 (GLP-2) analogs include:

[0131] ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1)

[0132] ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO: 2);

[0133] ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3);

[0134] ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4);

[0135] ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 5); or

[0136] ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6).

[0137] In some embodiments of the present invention, in the above formula, X5 is Ser and / or X11 is Ser. Some examples of these glucagon-like peptide 2 (GLP-2) analogs include:

[0138] ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7);

[0139] ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 8); or

[0140] ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9).

[0141] Some embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying drawings. However, numerous other aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure.

[0142] As used herein, "and / or" is considered to be specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" is considered to be specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually listed herein.

[0143] Unless the context indicates otherwise, the descriptions and limitations of the features listed above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 A typical chromatogram showing the separation of oligomers from the ZP1848 peptide is shown.

[0146] Figure 2Shown are how viscosity (squares) and hydrodynamic radius (z-average) (circles) change as a function of acetate concentration after formulation manufacture. The data indicate that above 11% acetate, viscosity and hydrodynamic radius (z-average) begin to increase.

[0147] Figure 3 Shown are stability evaluations at 20 mg / mL using different buffers at 40°C for 0 to 3 weeks (normalized to 100% at the start).

[0148] Figure 4 Stability at 2 mg / ml (normalized to 100% at the start) is shown for 0 to 3 weeks at 40°C using different buffers.

[0149] Figure 5 Shown are stability evaluations at 20 mg / mL using different tonicity agents for 0 to 3 weeks at 40°C (normalized to 100% at the start).

[0150] Figure 6 Shown are stability evaluations at 2 mg / mL using different tonicity agents for 0 to 3 weeks at 40°C (normalized to 100% at the start).

[0151] Figure 7 Shown are the purity of Formulations 1 to 5 using different concentrations of ZP1848 acetate, different salt forms, different tonicity agents, and different buffers.

[0152] Figure 8 The stability of the peptides in combination with different preservatives is shown for 13 weeks at 25°C.

[0153] Figure 9 Shown are the HPLC purities of the investigated preparations at 25°C (accelerated conditions). Detailed Description of the Invention

[0155] definition

[0156] Unless otherwise stated, the following definitions are provided for specific terms used in the above written description.

[0157] Throughout the specification and claims, conventional one-letter and three-letter codes for naturally occurring amino acids are used.All amino acid residues in the peptides of the invention are preferably in the L-configuration, however, D-configuration amino acids may also be present.

[0158] Preferred compounds of the present invention have at least one GLP-2 biological activity, in particular in causing intestinal growth. This can be assessed in an in vivo assay, for example as described in the Examples of, for example, WO 2006 / 117565, in which the mass of the intestine or part thereof is determined after the test animal has been treated or exposed to a GLP-2 analogue.

[0159] In some aspects of the invention, the liquid formulation comprising the GLP-2 analog has a total acetate concentration in the formulation of less than or equal to 11% acetate / mg of the GLP-2 analog, and more preferably less than or equal to 10% acetate / mg of the GLP-2 analog, more preferably less than or equal to 9% acetate / mg of the GLP-2 analog, more preferably less than or equal to 8% acetate / mg of the GLP-2 analog, more preferably less than or equal to 7% acetate / mg of the GLP-2 analog, more preferably less than or equal to 6% acetate / mg of the GLP-2 analog, more preferably less than or equal to 5% acetate / mg of the GLP-2 analog, more preferably less than or equal to 4% acetate / mg of the GLP-2 analog, more preferably less than or equal to 3% acetate / mg of the GLP-2 analog, and more preferably less than or equal to 2% acetate / mg of the GLP-2 analog. The acetate concentration in the lyophilized drug substance can be controlled by adjusting the concentration of acetic acid in the mobile phase used during the final chromatography step. This results in a drug substance having an acetate content of less than 11%. Thus, for example, for a formulation having 20 mg / mL of a GLP-2 analog, the total acetate concentration would be less than or equal to 37 mM. For reference, 10% total acetate concentration equals 34 mM, 9% equals 30 mM, 8% equals 27 mM, 7% equals 24 mM, and 6% equals 20 mM. The total acetate concentration can be determined using methods known in the art (e.g., HPLC).

[0160] In the following examples, it is shown that the viscosity of the liquid formulations of the present invention depends on the total acetate concentration. Preferably, the viscosity of the formulation measured at 25°C is between 0.8 and 2.0 mPa / second. Conveniently, the viscosity can be measured using a microVISC TM In parallel, the hydrodynamic radius can be measured using a dynamic light scattering DLS plate reader (Wyatt DynaPro II). Samples of drug substance (DS) of a GLP-2 analogue containing 6% acetate were prepared, as well as simulated DS with 7.8 to 15% acetate, followed by addition of acetate. Data from formulations manufactured with acetate concentrations varying from 6.7 to 15% are shown below. Figure 2 The effect of controlling the total acetate concentration is that the injectability of the formulations of the invention can be adjusted, for example by reducing the total acetate concentration to provide a less viscous formulation that can be injected more easily.

[0161] The liquid formulation according to the present invention is preferably an isotonic liquid formulation. "Isotonic" means that the formulation of the present invention has the same or similar osmotic pressure as body fluids. Preferably, the osmotic concentration of the formulation of the present invention is about 300 ± 60 mOsm as measured by an osmometer.

[0162] Additionally or alternatively, the present invention demonstrates that the formation of covalently linked oligomers of the GLP-2 analog is inversely dependent on the concentration of the GLP-2 analog in the formulation. As shown in the Examples, the amount of covalently linked oligomers can be determined using size exclusion chromatography and determining the area under the peaks for monomeric GLP-2 analogs and oligomers, respectively. This can be performed using a Dionex Ultimate 3000 HPLC system with a linear gradient, at a flow rate of 0.5 mL / min for analysis. The mobile phase consists of 45% acetonitrile and 55% 0.1% TFA in Milli-Q water. Detection is performed at a wavelength of 215 nm. This means that the formulations of the present invention typically contain the GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL, more preferably at a concentration of about 15 mg / mL to about 25 mg / mL, and most preferably at a concentration of about 20 mg / mL. In other embodiments, the present invention typically contains the GLP-2 analog at a concentration of about 2 mg / mL, 5 mg / mL, 10 mg / mL, or 20 mg / mL. In some aspects of the invention, the concentration of the GLP-2 analogue is preferably selected such that the formulation contains 10% or less, more preferably 5% or less, more preferably 4% or less, more preferably 3% or less, and most preferably 2% or less of the GLP-2 analogue in the form of covalently bound oligomeric products, preferably after 18 months of storage. By way of example, the amount of covalently bound oligomeric products may be in the range of 2% to 5%, more preferably in the range of 2% to 4%, and most preferably in the range of 2% to 3%.

[0163] In some cases, the formulations of the present invention may be used in a once- or twice-daily dosing regimen. In some cases, the formulations of the present invention may be used in a once- or twice-weekly dosing regimen. Alternatively or additionally, the dosing regimen of the GLP-2 analogs of the present invention may include several doses or courses of doses separated in time by 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In a preferred embodiment, the doses are separated in time by 3, 3.5, 4, 5, 6, 7, or 8 days. In a preferred embodiment, the doses are separated in time by 3, 3.5, 4, or 7 days. As will be understood in the art, the timing between doses may vary to some extent such that each and every dose is not separated by exactly the same time. This will generally be guided by the physician's discretion. Thus, the doses may be separated in time by a clinically acceptable time range, for example, from about 2 days to about 10 days, or from about 3 or 4 days to about 7 or 8 days.

[0164] The formulations of the present invention are stable liquid pharmaceutical formulations of GLP-2 analogs. A "stable" formulation is one in which the peptide therein substantially maintains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the formulation substantially maintains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the expected shelf life of the formulation. The formulations of the present invention are provided as stable liquid formulations, such as stable aqueous liquid formulations. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). In the present invention, a "stable" formulation includes a formulation in which at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% of the GLP-2 analogue in the formulation is active after the formulation has been stored at 2 to 8°C for at least 18 months.

[0165] Stability can be measured at a selected temperature for a selected time, for example, using an elevated temperature to reduce the time spanned by the test formulation. Typically, storage at a temperature of 2 to 8°C means storage under normal refrigeration conditions. In certain embodiments, the formulation is stable under such conditions for at least 12 months, more preferably at least 18 months, more preferably at least 24 months. Stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including evaluating aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity and / or by visual inspection); assessing charge heterogeneity by using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; evaluating the biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, differences in glycosylation, etc.

[0166] A peptide "retains its physical stability" in a pharmaceutical formulation and is considered to retain its biological activity if it shows no (or minimal) signs of aggregation, precipitation, and / or denaturation, e.g., when inspected visually for color and / or clarity, or as measured by UV light scattering, dynamic light scattering, circular dichroism, or by size exclusion chromatography.

[0167] A peptide "retains its chemical stability" in a pharmaceutical formulation if the chemical stability at a given time is such that the peptide is considered to still retain its biological activity, as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the peptide. Chemical alterations can involve isomerization, oxidation, size changes (e.g., tailoring), which can be evaluated using, for example, HPLC or size exclusion chromatography, SDS-PAGE, and / or mass spectrometry. Other types of chemical alterations include charge changes n (e.g., due to deamidation), which can be evaluated, for example, by HPLC or ion exchange chromatography or icIEF.

[0168] GLP-2 analogs

[0169] Compared to native GLP-2 and as defined above, the GLP-2 analogs present in the formulations of the present invention have one or more amino acid substitutions, deletions, inversions, or additions. This definition also includes the synonymous terms GLP-2 mimetic and / or GLP-2 agonist. Furthermore, the analogs of the present invention may additionally have chemical modifications to one or more of their amino acid side groups, α-carbon atoms, terminal amino groups, or terminal carboxylic acid groups. Chemical modifications include, but are not limited to, the addition of chemical moieties, the creation of new bonds, and the removal of chemical moieties. Modifications at amino acid side groups include, but are not limited to, acylation of the ε-amino group of lysine, N-alkylation of arginine, histidine, or lysine, alkylation of the carboxylic acid groups of glutamic acid or aspartic acid, and deamidation of glutamine or asparagine. Modifications of the terminal amino group include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxyl group include, but are not limited to, amide, lower alkylamide, dialkylamide, and lower alkyl ester modifications. Preferred lower alkyl groups herein are C1-C4 alkyl. Additionally, one or more side or terminal groups may be protected by protecting groups known to the ordinarily skilled peptide chemist.The α-carbon of the amino acid may be mono- or dimethylated.

[0170] In some aspects, the liquid formulations of the present invention utilize a glucagon-like peptide 2 (GLP-2) analog represented by the following formula, or a pharmaceutically acceptable salt or derivative thereof:

[0171] R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-lle-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-lle-Ala-Trp-Leu-lle-Ala-Thr-Lys-lle-Thr-Asp-Z 2 -R 2

[0172] in:

[0173] R 1 It is hydrogen, C 1-4 an alkyl group (e.g., methyl), an acetyl group, a formyl group, a benzoyl group, or a trifluoroacetyl group;

[0174] X5 is Ser or Thr;

[0175] X11 is Ala or Ser;

[0176] R 2 is NH2 or OH; and

[0177] Z 1 and Z 2Independently absent, or a peptide sequence having 1 to 6 Lys amino acid units.

[0178] In some embodiments of the present invention, in the above formula, X5 is Thr and / or X11 is Ala. Some examples of these glucagon-like peptide 2 (GLP-2) analogs include:

[0179] ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1)

[0180] ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO: 2);

[0181] ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3);

[0182] ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4);

[0183] ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 5); or

[0184] ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6).

[0185] In one embodiment of the present invention, the glucagon-like peptide 2 (GLP-2) analog is ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1).

[0186] In some embodiments of the present invention, in the above formula, X5 is Ser and / or X11 is Ser. Some examples of these glucagon-like peptide 2 (GLP-2) analogs include:

[0187] ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7);

[0188] ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 8); or

[0189] ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9).

[0190] In one embodiment of the present invention, the glucagon-like peptide 2 (GLP-2) analog is ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7).

[0191] It will be appreciated that the peptides (drug substances) of the present invention may also be provided in the form of salts or other derivatives. Salts include pharmaceutically acceptable salts, such as acid addition salts and basic salts. Some examples of acid addition salts include hydrochlorides, citrates, chloride salts, and acetate salts. Preferably, the salt is an acetate salt. Generally, it is preferred that the salt is not a chloride salt. Some examples of basic salts include salts wherein the cation is selected from the group consisting of: alkali metals (e.g., sodium and potassium); alkaline earth metals (e.g., calcium); and ammonium ions. + N(R 3 )3(R 4 ), where R 3 and R 4 independently represents an optionally substituted C 1-6 Alkyl, optionally substituted C 2-6 Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 and more recent editions, and in Encyclopaedia of Pharmaceutical Technology.

[0192] In some preferred embodiments, the acetate salt of the GLP-2 analog of the present invention is selected from ZP1848-acetate, ZP2949-acetate, ZP2711-acetate, ZP2469-acetate, ZP1857-acetate, ZP2530-acetate, ZP1846-acetate, ZP1855-acetate, and ZP2242-acetate. In the context of the present invention, the term "ZP1848-acetate" refers to the ZP1848 molecule in the form of an acetate salt. The acetate salt of the GLP-2 analog can be represented by the formula (GLP-2 analog), x(CH3COOH), where x is 1.0 to 8.0, i.e., where x is 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0. In any composition of the acetate salt of the GLP-2 analog, molecules with different numbers of acetate molecules may be present, such that x is not necessarily an integer. In some cases, x is from 4.0 to 8.0, x is from 6.0 to 8.0, or x is from 4.0 to 6.5. In some cases, x is from 4.0 to 6.0, x is from 2.0 to 7.0, x is from 3.0 to 6.0, x is from 4.0 to 6.0, or x is from 4.0 to 8.0.

[0193] In a preferred embodiment, the GLP-2 analog is ZP1848-acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (SEQ ID NO: 1) or (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH), wherein x is 1.0 to 8.0.

[0194] Therefore, in another aspect, the present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog. The solid composition can be formulated with an excipient for preparing a liquid formulation of the present invention. In one embodiment, the present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula:

[0195] (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH), wherein x is 1.0 to 8.0.

[0196] The upper limit of 8.0 acetate molecules per GLP-2 analogue equates to an acetate content of less than 11% acetate and can be formulated to have a viscosity of 0.8 to 2.0 mPa / second measured at 25°C.

[0197] The range of the number of acetate molecules associated with each molecule of the GLP-2 analog defines the molecular weight range of the formulation components. For example, for the acetate salt of ZP1848, the range of the number of acetate molecules associated with each molecule of the GLP-2 analog defines the molecular weight range of ZP1848-acetate. For example, 1 acetate equivalent per molecule of ZP1848 provides a molecular weight = 4316 + 60 = 4376 Da. Thus, for increasing acetate equivalents of ZP1848, the molecular weights are as follows: 1 acetate equivalent = 4376 Da; 2 acetate equivalents = 4436 Da; 3 acetate equivalents = 4496 Da; 4 acetate equivalents = 4556 Da; 5 acetate equivalents = 4616 Da; 6 acetate equivalents = 4676 Da; 7 acetate equivalents = 4736 Da; and 8 acetate equivalents = 4796 Da. This in turn defines the following molecular weight ranges: 1 to 8 acetate equivalents = 4376 Da to 4796 Da; 4 to 8 acetate equivalents = 4556 Da to 4796 Da and 6 to 8 acetate equivalents = 4676 Da to 4796 Da.

[0198] Other derivatives of the GLP-2 analogs of the present invention include those with metal ions (such as Mn 2+ and Zn 2+ ) coordination complex, ester (such as an in vivo hydrolyzable ester), free acid or base, hydrate, prodrug or lipid. Using techniques well known in the art, esters can be formed between the hydroxyl or carboxylic acid groups present in the compound and a suitable carboxylic acid or alcohol reaction partner (reaction partner). Derivatives that are prodrugs of the compound can be converted into one of the parent compounds in vivo or in vitro. Generally speaking, at least one biological activity of the compound will be reduced in the prodrug form of the compound and can be activated by conversion of the prodrug to release the compound or its metabolite. Some examples of prodrugs include the use of protecting groups that can be removed in situ to release the active compound or to inhibit the clearance of the drug in the body.

[0199] Z 1 and Z 2 The present and / or absent sequence is independently present or is a peptide sequence having 1 to 6 Lys amino acid units (i.e., 1, 2, 3, 4, 5, or 6 Lys residues). The Lys residues may have a D-configuration or an L-configuration, but have an L-configuration. Particularly preferred sequences Z are sequences having 4, 5, or 6 consecutive lysine residues, and in particular 6 consecutive lysine residues. Exemplary sequences Z are shown in WO 01 / 04156. In certain embodiments, Z 1 does not exist. In this case, Z 2 May be present or absent.

[0200] Preparation of GLP-2 analogues

[0201] The formulation of the GLP-2 analog is a ready-to-use formulation. As used herein, the term "ready-to-use" refers to a formulation that does not require constitution or dilution with a prescribed amount of diluent (e.g., water for injection or other suitable diluent) prior to use via a designated route of administration.

[0202] As described herein, the liquid formulations of the GLP-2 analogs of the present invention comprise a buffer, a nonionic tonicity regulator, and an appropriate amount of arginine to adjust the pH of the final formulation. According to conventional pharmaceutical practice, the formulations of the present invention are sterile and / or do not contain a reducing agent. In some cases, the liquid formulations of the present invention are aqueous liquid formulations. In some cases, the liquid formulations of the present invention are non-aqueous liquid formulations.

[0203] As used herein, the term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. Screening experiments in the Examples indicate that the formulations of the present invention preferably contain a buffer selected from the group consisting of histidine buffer, methanesulfonate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer, and MOPS buffer, as these buffers provide stable formulations in which the GLP-2 analog dissolves without becoming viscous, turbid, or precipitating the peptide drug. In some preferred embodiments, the buffer is a histidine buffer, such as L-histidine. Typically, the buffer will be present at a concentration of about 5 mM to about 50 mM, more preferably at a concentration of about 5 mM to about 25 mM, and most preferably at a concentration of about 15 mM. Based on the experiments in this application, preferably, the buffer is not a phosphate buffer, a citrate buffer, a citrate / Tris buffer, and / or a succinate buffer.

[0204] The term "tonicity regulator" as used herein refers to a pharmaceutically acceptable tonicity regulator for adjusting the tension of a formulation. The formulation of the present invention is preferably isotonic, i.e., it has an osmotic pressure substantially the same as that of human serum. The tonicity regulator used in the formulation is preferably a non-ionic tonicity regulator, and is preferably selected from mannitol, sucrose, glycerol, sorbitol, and trehalose. A preferred non-ionic tonicity regulator is mannitol, such as D-mannitol. The concentration of the tonicity regulator will depend on the concentrations of the other components of the formulation, especially when the formulation is intended to be isotonic. Generally speaking, the non-ionic tonicity regulator will be used at a concentration of about 90 mM to about 360 mM, more preferably at a concentration of about 150 mM to about 250 mM, and most preferably at a concentration of about 230 mM.

[0205] Usually, select component and the amount of liquid preparation of the present invention to provide pH to be approximately 6.6 to approximately 7.4, more preferably pH is approximately 6.8 to approximately 7.2, and most preferably pH is about 7.0 preparation. Can add arginine in right amount (quantum sufficient, qs) to regulate pH, make it in the pH range of expectation. According to the experiment shown in the embodiment, preferably do not use hydrochloric acid or sodium hydroxide to carry out pH regulator.

[0206] In one embodiment, the liquid formulation of the present invention consists of: a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL; a buffer selected from the group consisting of histidine buffer, methanesulfonate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer, and MOPS buffer, wherein the buffer is present at a concentration of about 5 mM to about 50 mM; a non-ionic tonicity adjuster selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, wherein the concentration is about 90 mM to about 360 mM; and an amount of arginine to provide a pH of about 6.6 to about 7.4.

[0207] In one embodiment, the liquid formulation of the present invention consists of: a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL; a buffer selected from a histidine buffer, a methanesulfonate buffer, and an acetate buffer, wherein the buffer is present at a concentration of about 5 mM to about 50 mM; a non-ionic tonicity adjuster selected from mannitol, sucrose, glycerol, and sorbitol, wherein the concentration is about 90 mM to about 360 mM; and arginine in an amount to provide a pH of about 6.6 to about 7.4.

[0208] In another embodiment, the liquid formulation of the present invention comprises a GLP-2 analog at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, a mannitol at a concentration of about 230 mM, and an amount of arginine to provide a pH of about 7.0.

[0209] In another embodiment, the liquid formulation of the invention comprises a GLP-2 analog at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, a mannitol at a concentration of about 230 mM, and a pH of about 7.0.

[0210] In another embodiment, the liquid formulation of the present invention comprises ZP1848-acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (SEQ ID NO: 1) at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, mannitol at a concentration of about 230 mM, and an amount of arginine to provide a pH of about 7.0.

[0211] In another embodiment, the liquid formulation of the present invention comprises ZP1848-acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2acetate (SEQ ID NO: 1) at a concentration of about 20 mg / mL, histidine buffer at a concentration of about 15 mM, mannitol at a concentration of about 230 mM, and a pH of about 7.0.

[0212] In another embodiment, a liquid formulation of the present invention comprises an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH) (wherein x is 1.0 to 8.0) at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, a mannitol concentration of about 230 mM, and a pH of about 7.0.

[0213] In another embodiment, a liquid formulation of the invention comprises an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH) (wherein x is 1.0 to 8.0) at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, a mannitol at a concentration of about 230 mM, and a pH of about 7.0, for a once or twice daily dosing regimen.

[0214] In another embodiment, a liquid formulation of the invention comprises an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH) (wherein x is 1.0 to 8.0) at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, a mannitol at a concentration of about 230 mM, and a pH of about 7.0, for a once or twice weekly dosing regimen.

[0215] In another embodiment, the liquid formulation of the present invention comprises ZP1846H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7) at a concentration of about 20 mg / mL; a histidine buffer at a concentration of about 15 mM, a mannitol at a concentration of about 230 mM, and an amount of arginine to provide a pH of about 7.0.

[0216] In another embodiment, the liquid formulation of the present invention comprises ZP1846H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7) at a concentration of about 20 mg / mL; histidine buffer at a concentration of about 15 mM, mannitol at a concentration of about 230 mM, and a pH of about 7.0.

[0217] In some cases, the liquid formulations of the present invention further comprise a preservative. In some cases, the preservative is selected from the group consisting of benzalkonium chloride, chlorobutanol, methylparaben, and potassium sorbate. Typically, the preservative is present in a concentration of about 0.1% to about 1% of the final formulation volume.

[0218] In another embodiment, the liquid formulation is selected from the group consisting of an aqueous liquid formulation, a liquid formulation in various hydrophilic or hydrophobic solvents, an emulsion and a liquid suspension. In a preferred embodiment, the liquid formulation is an aqueous liquid formulation.

[0219] For example, the liquid formulations of the present invention can be prepared by mixing stock solutions of the GLP-2 analog, buffer, nonionic tonicity adjuster, and optional preservative in water, optionally diluting the resulting solution, and adjusting to the target pH. Conveniently, the solutions of buffer and nonionic tonicity adjuster can be mixed first to provide the desired concentration of each excipient. The solution of the GLP-2 analog can then be added, and the pH adjusted, if necessary, for example, using acetic acid / 0.5 M L-arginine. Water is added to the final volume.

[0220] Preferably, the glucagon-like peptide 2 (GLP-2) analog is administered to the patient parenterally, preferably by injection, most typically by subcutaneous injection, intramuscular injection, intravenous injection or intraperitoneal injection. Preferably, the administration is by subcutaneous injection. The injection can be performed by a physician, nurse or other health care professional, or can be self-administered by the patient. As set out herein, in some aspects, the formulations of the present invention have a viscosity that facilitates loading the formulation into a pre-filled syringe, injection pen or other injection device. This can have the advantage of pre-determining the dose of the formulation administered to the patient, for example without the need for measuring through a multi-use vial. Therefore, in other aspects, the present invention provides an article of manufacture or a kit comprising a container containing a stable (e.g., aqueous stable) pharmaceutical formulation of a GLP-2 analog according to the present invention; or a pre-filled syringe or injection device or injection pen comprising an aqueous liquid formulation comprising a GLP-2 analog according to the present invention.

[0221] Medical conditions

[0222] The GLP-2 analog formulations of the present invention can be used as pharmaceutical agents for the prevention or treatment of individuals suffering from gastrointestinal disorders (including the upper gastrointestinal tract of the esophagus) by administering an effective amount of a GLP-2 analog or salt thereof as described herein. Gastric and intestinal related disorders include: ulcers of any etiology (e.g., peptic ulcers, drug-induced ulcers, ulcers associated with infections or other pathogens), digestive disorders, malabsorption syndrome, short bowel syndrome, cul-de-sac syndrome, inflammatory bowel disease, abdominal spongiosa (e.g., caused by gluten-induced enteropathy or celiac disease), tropical spongiosa, hypogammaglobulinemic spongiosa, enteritis, ulcerative colitis, small intestinal damage, and chemotherapy-induced diarrhea / mucositis (CID).

[0223] As described above, generally speaking, individuals who would benefit from improved small intestinal mass and subsequent maintenance of normal small intestinal mucosal structure and function and / or normal small intestinal mucosal structure and function are candidates for treatment with the GLP-2 analogs of the present invention. Specific conditions that can be treated with GLP-2 analogs include various forms of splenomegaly, including: abdominal splenomegaly, which is caused by a toxic reaction to α-gliadins from wheat and may be a result of gluten-induced enteropathy or celiac disease and is marked by a significant loss of small intestinal villi; tropical splenomegaly, which is caused by infection and is marked by partial flattening of the villi; and hypogammaglobulinemic splenomegaly, which is typically observed in patients with common variable immunodeficiency or hypogammaglobulinemia and is marked by a significant decrease in villus height. The efficacy of GLP-2 analog therapy can be monitored by intestinal biopsy to examine villus morphology, by biochemical assessment of nutrient absorption, by patient weight gain, or by improvement in symptoms associated with these conditions.

[0224] Another specific condition that can be treated with the GLP-2 analogs of the present invention or for which the GLP-2 analogs may be useful therapeutically and / or preventively is short bowel syndrome (SBS), also known as short gut syndrome or simply short gut, which results from surgical resection, congenital defects, or disease-related intestinal absorption loss, where the patient is subsequently unable to maintain a balance of fluids, electrolytes, and nutrients on a regular diet. Although adaptation typically occurs over the two years following resection, SBS patients experience reduced dietary intake and fluid losses.

[0225] Other conditions that can be treated with or for which the GLP-2 analogs of the invention are useful prophylactically include, in addition to the radiation enteritis described above, infectious or post-infectious enteritis and small bowel damage due to cancer chemotherapeutic or toxic agents.

[0226] GLP-2 analogs can also be used to treat nutritional deficiencies (eg, cachexia and anorexia).

[0227] A specific embodiment of the present invention is about using the peptides of the present invention for preventing and / or treating intestinal damage and dysfunction. Such damage and dysfunction are well-known side effects of cancer chemotherapy. Administration of chemotherapy is often associated with undesirable side effects related to the gastrointestinal system, such as mucositis, diarrhea, bacterial translocation, malabsorption, abdominal cramps, gastrointestinal bleeding and vomiting. These side effects are clinical consequences of structural and functional damage to the intestinal epithelium and often require a reduction in the dosage and frequency of chemotherapy.

[0228] Administration of the GLP-2 peptide analogs of the present invention can enhance the trophic effects of intestinal crypts and rapidly provide new cells to replace damaged intestinal epithelium following chemotherapy. The ultimate goal achieved by administering the peptides of the present invention is to reduce the morbidity associated with gastrointestinal damage in patients undergoing chemotherapy while simultaneously optimizing chemotherapeutic regimens for treating cancer. Concomitant prophylactic or therapeutic treatments can be provided according to the present invention to patients undergoing or about to undergo radiation therapy.

[0229] The stem cells of the small intestinal mucosa are particularly susceptible to the cytotoxic effects of chemotherapy due to their rapid proliferation rate (Keefe et al., Gut, 47: 632-7, 2000). The damage induced by chemotherapy to the small intestinal mucosa is clinically commonly referred to as gastrointestinal mucositis and is characterized by absorptive damage and barrier damage to the small intestine. For example, it has been shown that the widely used chemotherapeutic agents 5-FU, irinotecan and methotrexate increase apoptosis in the small intestine of rodents, leading to villous atrophy and crypt dysplasia (Keefe et al., Gut 47: 632-7, 2000; Gibson et al., J Gastroenterol. Hepatol. Sep; 18 (9): 1095-1100, 2003; Tamaki et al., J. Int. Med. Res. 31 (1): 6-16, 2003). In humans, chemotherapeutic agents have been shown to increase apoptosis in intestinal crypts 24 hours after administration and subsequently reduce villus area, crypt length, mitotic counts per crypt, and intestinal epithelial cell height three days after chemotherapy (Keefe et al., Gut, 47:632-7, 2000). Thus, structural changes in the small intestine directly contribute to intestinal dysfunction and, in some cases, diarrhea.

[0230] Gastrointestinal mucositis following cancer chemotherapy is an increasingly serious problem that, although it gradually remits, is essentially incurable once established. Studies with the commonly used cytostatic cancer drugs 5-FU and irinotecan have shown that effective chemotherapy with these drugs primarily affects the structural integrity and function of the small intestine, while the colon is less sensitive and responds primarily with increased mucus formation (Gibson et al., J. Gastroenterol. Hepatol. Sep; 18(9): 1095-1100, 2003; Tamaki et al., J Int. Med. Res. 31(1): 6-16, 2003).

[0231] The formulations of the present invention comprising GLP-2 analogs can be used to prevent and / or treat gastrointestinal damage and side effects of chemotherapeutic agents. This potentially important therapeutic application can be applied to currently used chemotherapeutic agents, such as, but not limited to: 5-FU, altretamine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxycarbamide, Idarubicin, Ifosfamide, Irinotecan, Liposomal Doxorubicindoxorubicin, leucovorin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, streptozocin, tegafur-uracil, temozolomide, thiotepa, tioguanine / thioguanine, topotecan Topotecan, Treosulfan, Vinblastine, Vincristine, Vindesine, Vinorelbine, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Asparaginase, Cyclophosphamide, Cytarabine, Dacarbazine, Actinomycin D, Daunorubicin, Docetaxel, Doxorubicin amphetamine, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, folinic acid, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, streptozotocin, tegafur-uracil, temozolomide, thiotepa, thioguanine, topotecan, thiosulfate, vinblastine, vincristine, vindesine, and vinorelbine.

[0232] Delivery of formulations

[0233] In some aspects, the present invention relates to ready-to-use formulations of GLP-2 analogs intended for parenteral administration and suitable for use in, for example, a vial, prefilled syringe, infusion pump, wearable injector, disposable autoinjector, or adjustable dose autoinjector. Example

[0234] The following examples are provided to illustrate some preferred aspects of the present invention and are not intended to limit the scope of the invention. The GLP-2 analogs administered according to the dosage regimen described herein can be prepared according to the solid phase peptide synthesis method described, for example, in WO 2006 / 117565, the contents of which are expressly incorporated by reference in their entirety.

[0235] Example 1. Synthesis of ZP1848-acetate and similar GLP-2 analogs

[0236] The ZP1848-acetate peptide was synthesized using the Fmoc Solid Phase Peptide Synthesis (SPPS) method under standard coupling conditions. After completion of the synthesis, the peptide sequence was deprotected and cleaved from the solid support, and the crude peptide was purified using preparative reverse phase HPLC. The peptide was converted to the desired acetate form by applying a mobile phase with an appropriate concentration of acetic acid during the final chromatography step and then lyophilized. The resulting drug substance products had an acetate content of less than 11% or less than 8 equivalents of acetate: Batch 1 (6% acetate, 4.6 equivalents of acetate), Batch 2 (7% acetate, 5.4 equivalents of acetate) and Batch 3 (6% acetate, 4.6 equivalents of acetate). This synthesis and purification scheme can be adapted to prepare other GLP-2 analogs used in the formulations of the present invention.

[0237] Example 2. Study of covalently bound oligomers in pharmaceutical formulations of the GLP-2 analog ZP1848-acetate form

[0238] Materials and Methods

[0239] For the detection of covalently linked oligomers, a Dionex Ultimate3000 HPLC system with a linear gradient was used for analysis at a flow rate of 0.5 mL / min. The mobile phase consisted of 0.1% TFA in 45% acetonitrile and 55% Milli-Q water. Detection was performed at a wavelength of 215 nm. The injection volume was 4 μg of peptide. The column used to separate the covalently formed peptides was a TSKgel SuperSW2000 (TSK BioScience) with a particle size of 4 μm and a size of 300*4.6 mm. The overall run time was 25 minutes. For the chemical stability evaluation of the peptide monomers, a C18 column with an acidic mobile phase and an acetonitrile gradient was used.

[0240] Prepare stock solutions of mannitol (700 mM), L-histidine (200 mM), and ZP1848 peptide (acetate; 60 mg / mL) in water (Milli-Q). Mix the mannitol and histidine solutions in appropriate amounts to obtain 230 mM mannitol and 15 mM histidine. Add the peptide stock solution to final concentrations of 0.2, 2, and 20 mg / mL, respectively. Add water to 90% of the final volume. If necessary, adjust the pH to pH 7 using 1 M acetic acid / 0.5 M L-arginine. Add water to the final volume.

[0241] Results and Discussion

[0242] It is known in the art that increasing the concentration of a peptide or protein drug in a liquid formulation increases the concentration of dimers, trimers, and higher-order oligomers due to mass action effects leading to a higher probability of covalent reactions (see van Maarschalkerweerd et al., Intrinsically Disord. Proteins. 2015; 3(1): e1071302). Consequently, the formation of covalent high molecular weight degradation products (cHMWDPs) increases as a function of drug substance concentration and has the effect of reducing the amount of bioactive monomeric peptide available in the formulation. Therefore, this was investigated in the formulation of the GLP-2 analog ZP1848-acetate.

[0243] A typical chromatogram showing the separation of oligomers from ZP1848-acetate monomers is shown in Figure 1 ZP1848-acetate oligomers are well separated from ZP1848-acetate monomers and are all integrated into a single peak. Peak area percentage is used to quantify oligomers, specifically the amount of covalently linked dimers and trimers.

[0244] After 24 months of storage at 2 to 8°C, formulations containing 0.2, 2, and 20 mg / mL of ZP1848 were analyzed. Dimer formation (two covalently linked ZP1848-acetate molecules) was predominant, but trimer formation was also observed to some extent (confirmed by LC-MS). The formulation containing 0.2 mg / mL had 2.6% oligomers, 2 mg / mL had 1.91%, and 20.0 mg / mL had 1.35%. The initial value for the amount of oligomers was less than 0.1%.

[0245] Table 1: Formation of covalently linked oligomers at long-term stability at 2 to 8°C after 24 months

[0246] Drug product concentration, ZP1848-acetate Covalently linked oligomers 0.2 mg / mL 2.60％ 2mg / mL 1.91％ 20 mg / mL 1.35％

[0247] During long-term storage of ZP1848-acetate (glepaglutide) at 2 to 8°C, it was unexpectedly found that the formation of covalently bound oligomers was concentration-dependent, but contrary to general expectations, the concentration-dependence of oligomer formation was inversely correlated with increasing GLP-2 analog concentration. Without wishing to be bound by any particular theory, the present inventors believe that the decrease in covalently bound oligomer formation with increasing drug concentration is a result of the lysine tail of the GLP-2 analog promoting a competitive reaction, which results in the formation of higher-order species in which GLP-2 analog molecules are weakly associated rather than covalently linked. This means that these weakly associated species are able to dissociate to release biologically active monomers, rather than resulting in loss of active material, as occurs when covalently bound oligomers are formed.

[0248] Example 3: Buffer Screening for Formulation of GLP-2 Analog ZP1848 Acetate

[0249] A study was conducted to examine the effect of different buffer salts on the stability of a ZP1848-acetate (4 mg / mL) formulation. The total buffer concentration in the formulation was 20 mM.

[0250] Materials and Methods

[0251] Prepare the buffer solutions listed in Table 2 below. Adjust the pH of the buffer with 1 M HCl / 1 M NaOH. Dissolve the ZP1848 peptide (acetate) in the relevant buffer at 80% of the final sample volume to give 4 mg / mL in the final formulation. If necessary, adjust the pH to the desired formulation pH using 200 mM acetic acid or 100 mM L-arginine. Add buffer solution to the final volume. Fill each formulation into appropriate vials (1 mL / vial) for stability testing.

[0252] Results and Discussion

[0253] Visual appearance indicated that all formulations containing citrate buffer, citrate / Tris buffer, or succinate buffer were viscous and / or turbid (see Table 2). Acetate buffer (20 mM, pH 5), methanesulfonate buffer (20 mM, pH 6), histidine buffer (15 mM, pH 7), and histidine-arginine (15+5 mM, pH 7) produced formulations that were clear and non-viscous by visual inspection.

[0254] Table 2: Screening of formulations with different buffering agents

[0255]

[0256] Example 4: Incompatibility of Phosphate Buffer with the GLP-2 Analog ZP1848-Acetate

[0257] Materials and Methods

[0258] Prepare stock solutions of mannitol (700 mM), phosphate buffer (200 mM), and ZP1848-acetate peptide (60.2 mg / mL) in water (Milli-Q). Mix the stock solutions in appropriate amounts to obtain the formulations shown in Table 3 below. Add water to 90% of the final volume. If necessary, adjust the pH to the desired formulation pH using 1 M acetic acid / 0.5 M L-arginine. Add water to the final volume. After 24 hours at room temperature, visually inspect the sample container for clarity and viscosity.

[0259] Table 3: Formulations tested for the effect of phosphate buffer

[0260]

[0261] Results and Discussion

[0262] Visual inspection indicated that the 20 mg / mL ZP1848-acetate formulations containing 20 to 50 mM phosphate buffer, pH 6.5 to 7.5, were turbid and / or highly viscous after 24 hours at room temperature. It was therefore concluded that the phosphate buffer was incompatible with ZP1848-acetate in these formulations.

[0263] Example 5: Effect of Acetate Content on the Viscosity of GLP-2 Analog ZP1848-Acetate Formulations

[0264] A study was conducted to determine the effect of acetate content on the viscosity of ZP1848-acetate formulations.

[0265] Materials and Methods

[0266] Samples were prepared using the drug substance (DS) of the GLP-2 analogue ZP1848-acetate containing 6% acetate. Acetate was added to explore the effect of increasing acetate content in the range of 7.8% to 15% acetate (see Table 4).

[0267] Prepare stock solutions of mannitol (700 mM), acetic acid (1000 mM), histidine (200 mM), and ZP1848-acetate peptide (60 mg / mL) in Milli-Q water. Mix the stock solutions in appropriate amounts to obtain the formulations shown in Table 4 below. Add water to 90% of the final volume. If necessary, adjust the pH to the desired formulation pH using 250 mM arginine. Add water to the final volume. Fill each formulation into appropriate vials for stability testing.

[0268] Visually inspect the vial for clarity and viscosity using a microVISC TMViscosity was measured using a viscometer. Hydrodynamic radius was measured using a Wyatt DynaPro II dynamic light scattering (DLS) plate reader. The sample size loaded on the plate was 170 μl.

[0269] Table 4: Formulations containing 20 mg / ml ZP1848-acetate at pH 7 with different acetate concentrations

[0270]

[0271] Results and Discussion

[0272] The viscosity and hydrodynamic radius of the formulations with different acetate concentrations are shown in Figure 2 The results show that at higher acetate concentrations, the viscosity of the ZP1848-acetate formulation unexpectedly increases in a nonlinear manner. Therefore, if the total acetate concentration in the formulation is less than or equal to 11% acetate / mg GLP-2 analog, it is advantageous to control the viscosity at a low / constant level because this opens up the possibility of providing the formulation of the GLP-2 analog in the form of a drug delivery device.

[0273] Example 6: Stabilization of Formulations of the GLP-2 Analog ZP1848-Acetate at 2 and 20 mg / mL by Buffer Salts The role of sex

[0274] A study was conducted to examine the effect of different buffer salts on the stability of ZP1848-acetate (2 and 20 mg / mL) formulations. All buffers were used at a concentration of 15 mM.

[0275] Materials and Methods

[0276] Prepare stock solutions of mannitol (700 mM), L-histidine (200 mM), glycine (400 mM), lysine (200 mM), TRIS (200 mM), bis-TRIS (200 mM), MOPS (100 mM), succinate (200 mM), MES (2-(N-morpholino)ethanesulfonic acid) (200 mM), methanesulfonate (200 mM), phosphate (200 mM), and ZP1848 peptide (acetate; approximately 50 mg / ml) in water (Milli-Q). Mix the excipient solutions in appropriate amounts to give the formulations shown in Tables 5 and 6 below. All formulations contained 230 mM mannitol and 15 mM buffer. Add the peptide stock solution. Add water to 90% of the final volume. If necessary, adjust the pH to pH 7 using 1 M acetic acid / 0.5 M L-arginine. Add water to the final volume. The formulations were filled in vials and placed in a stability study at 40°C.

[0277] Results and Discussion

[0278] Based on the observed results, 15 mM concentrations of the buffers histidine, glycine, lysine, TRIS, Bis-TRIS, MOPS, methanesulfonate, and MES can be used in ZP1848-acetate formulations at 2 mg / mL and 20 mg / mL peptide at pH 7.0.

[0279] Table 5: Stability of formulations prepared with different buffers

[0280]

[0281]

[0282] Table 6: Formation of covalently linked oligomers in formulations using different buffers

[0283]

[0284]

[0285] The formation of covalently linked oligomers was evaluated for different buffers (Table 6). At 20 mg / mL, succinic acid formed a gel after 1 week and could not be evaluated at 2 and 3 weeks of the stability study. At 2 mg / mL, the same buffer had a significantly higher covalently linked oligomer formation (2.1%). The overall trend was that after accelerated storage for 3 weeks at 40°C, the 2 mg / mL formulation had a higher covalently linked oligomer formation than the 20 mg / mL formulation.

[0286] In these formulations, phosphate buffer and succinate buffer were incompatible with ZP1848-acetate at 2 and 20 mg / mL.

[0287] The stability of the peptide monomer was evaluated by determining the HPLC purity during a stability study at 40°C for 3 weeks. The results are presented in Figure 3 and Table 7. Due to the gel formation described previously, succinic acid could only be evaluated at the first time point at 20 mg / mL. Results were available for 2 mg / mL over three test weeks. Only small, insignificant differences could be detected between the buffers evaluated. Therefore, the choice of buffer did not appear to affect the stability of the peptide monomer.

[0288] Table 7: Formation of covalently linked oligomers using different buffers at 40°C for 0 to 3 weeks

[0289]

[0290] Example 7: Effect of Tonicity Modifiers on the Stability of Formulations of the GLP-2 Analog ZP1848-Acetate at 2 and 20 mg / mL Qualitative role

[0291] A study was conducted to examine the effects of different tonicity modifiers on the stability of ZP1848-acetate (2 and 20 mg / mL) formulations.

[0292] Materials and Methods

[0293] Stock solutions of L-histidine (200 mM), sucrose (730 mM), glycerol (977 mM), D-sorbitol (801 mM), D-(+) trehalose dehydrate (500 mM), D-mannitol (700 mM) and ZP1848-acetate peptide (acetate; approximately 50 mg / ml) were prepared in water (Milli-Q). The excipient solutions were mixed in appropriate amounts to give the formulations shown in Table 8 below. All formulations contained 15 mM histidine. Peptide stock solutions were added as needed to give the peptide contents shown in Table 8. Water was added to 90% of the final volume. If necessary, the pH was adjusted to pH 7 using 1 M acetic acid / 0.5 M L-arginine. Water was added to the final volume. Each formulation was filled in a vial and placed in a stability study at 40°C. The sample containers were visually inspected for clarity and viscosity, and the hydrodynamic radius was analyzed using DLS (dynamic light scattering) analysis.

[0294] Results and Discussion

[0295] Based on the observations shown in Table 8, mannitol, sucrose, glycerol, sorbitol, and trehalose can be used in these formulations with 2 mg / ml and 20 mg / mL of ZP1848-acetate and pH 7.0.

[0296] Table 8: Formulations prepared using different tonicity modifiers

[0297]

[0298] The formation of covalent oligomers was measured for formulations 1 to 10 at 40°C for up to 3 weeks. The results are shown in Table 9. After 1 week of stability testing, differences in the formation of covalent oligomers were seen for the 10 formulations. In addition, the rate (slope) was quite consistent throughout the test period. The formation of covalently linked oligomers was significantly higher for formulations 3 (20 mg / mL-glycerol), 7 (2 mg / mL-sucrose), 8 (2 mg / mL-glycerol), and 9 (2 mg / mL-sorbitol) than for the other formulations. Mannitol minimized the formation of covalently linked oligomers. The overall trend was that for all tonicity agents studied, the formation of covalently linked oligomers was higher at 2 mg / mL compared to 20 mg / mL.

[0299] Table 9: Formation of covalently linked oligomers for Formulations 1 to 10 from 0 to 3 weeks at 40°C

[0300]

[0301] Peptide monomer stability was evaluated by determining HPLC purity during a 3-week stability study at 40°C. Similar to covalently linked oligomers, chemical stability was poor when glycerol was used and differed from that of other tonicity agents. Results are presented in Figure 5 and Figure 6 middle.

[0302] Example 8: Physical Stabilization of Acids and Bases for pH Adjustment in Formulations of the GLP-2 Analog ZP1848-Acetate Sexual influence

[0303] Materials and Methods

[0304] Prepare stock solutions of mannitol, histidine, and ZP1848-acetate peptide in water. Add the stock solutions of mannitol and histidine to water and mix, and add the peptide solution so that the final peptide content is 10 mg / mL. Add water until 90% of the final volume. Then adjust the pH to pH 7 using 250mM arginine / 1M AcOH or 1M NaOH / 1M HCl (see Table 10). Add water until the final volume. Each formulation is filled in a vial for stability testing and placed in a stability study at 5°C, 25°C, and 40°C. Visually inspect the sample containers for clarity and viscosity.

[0305] Results and Discussion

[0306] The results shown in Table 10 indicate that pH adjustment using 1 M NaOH / 1 M HCl had a negative effect on the physical stability of the ZP1848-acetate formulation.

[0307] Table 10: Effect of acid / base on physical stability

[0308]

[0309] Example 9: ZP1848 peptide acetate and ZP1848 peptide chloride salts for GLP-2 analog ZP1848 peptide formulation use

[0310] A study was conducted to examine the effects of using ZP1848 peptide acetate and ZP1848 peptide hydrochloride salts in selected ZP1848 formulations. The effects of salt type, concentration, buffer, and tonicity modifier were examined after accelerated storage at 40°C. An attempt was made to synthesize the sodium salt of ZP1848 peptide, but it was found to be unavailable.

[0311] Materials and Methods

[0312] Prepare stock solutions of mannitol (700 mM), histidine (200 mM), sorbitol (700 mM), mesylate (200 mM) and ZP1848 peptide solution (chloride salt; approximately 50 mg / mL) in Milli-Q water. The excipient solutions were mixed in appropriate amounts to give the formulations shown in Tables 11 and 12 below. The peptide stock solutions were added to give the desired final peptide content. Water was added to 90% of the final volume. If necessary, the pH was then adjusted to the desired formulation pH using 1 M acetic acid / 0.5 M L-arginine. Water was added to the final volume. Each formulation was filled in vials and placed in a stability study at 40°C. The sample containers were visually inspected for clarity and viscosity, and the hydrodynamic radius was analyzed using DLS.

[0313] Results and Discussion

[0314] The results shown in Tables 11 and 12 indicate that after 3 weeks at 40° C., the Z-average values, viscosities, and visual appearance of Formulations 1, 2, 3, and 4 had unchanged stability, as assessed by visual appearance and DLS. Formulation 5 showed changes in stability over time, as assessed by Z-average values, viscosity, and visual appearance.

[0315] Table 11: Effect of peptide salt type

[0316]

[0317] Table 12: Effect of peptide salt type

[0318]

[0319]

[0320] The chemical stability of Formulations 1 to 5 was followed at 40°C for up to 4 weeks. The purity obtained was normalized to 100% at the time of release. The results are shown in the figure. No significant differences in the chemical stability of the peptide monomer were observed for Formulations 1, 2, 4, and 5. Formulation 3 showed slightly lower, but acceptable, chemical stability after 4 weeks, and this was most likely due to the lower concentration of this formulation.

[0321] The formation of covalent oligomers was measured for Formulations 1 to 5 at 40°C for up to 4 weeks. The results are shown in Table 13. Even after 1 week of stability testing, differences in covalent oligomer formation were still visible among the five formulations. In addition, the rates (slopes) were quite consistent throughout the testing period. Formulation 1 (20 mg / mL, acetate salt of ZP1848, histidine as a tonicity agent) was the most stable formulation, forming approximately 1.1% covalent oligomers after 4 weeks of stability studies at 40°C. Formulation 5 (20 mg / mL, chloride salt of ZP1848, methanesulfonate as a tonicity agent) was the second most stable formulation, having approximately 2.1% covalent oligomer formation after 4 weeks of stability studies at 40°C. Formulation 3 (2 mg / mL, chloride salt of ZP1848, histidine as a tonicity agent) was the third most stable. Formulation 2 (20 mg / mL, chloride salt of ZP1848, histidine as a tonicity agent) was the fourth most stable. The least stable formulation was Formulation 4 (20 mg / mL, chloride salt of ZP1848, sorbitol as a tonicity agent).

[0322] For the acetate salt of ZP1848 after 3 weeks at 40°C, a trend towards slightly reduced stability was previously seen when using sorbitol compared to mannitol (0.9% for mannitol and 1.1% for sorbitol), see Example 7. When comparing the acetate and chloride salts, the difference between the sorbitol-containing formulation and the mannitol-containing formulation was more pronounced, with the 20 mg / mL formulation containing the chloride salt and sorbitol having approximately 3.9% covalent oligomer formation. When comparing the 2 mg / mL and 20 mg / mL chloride salt formulations, after 4 weeks at 40°C, covalent oligomer formation was 2.4% for 2 mg / mL (an increase of 0.53% per week) and 3.3% for 20 mg / mL (an increase of 0.75% per week). This was surprising as this was not the case with the acetate salt. For the acetate salt at 20 mg / mL, the formation of covalent oligomers after 3 weeks at 40°C was 0.9% (increasing by 0.21% per week), while for 2 mg / mL it was 1.2% (increasing by 0.27% per week). This higher formation of covalently linked oligomers at lower acetate concentrations is also consistent with what is seen during long-term stability. However, for the chloride salts, the situation was reversed, with higher formation of covalently linked oligomers as the ZP1848 concentration increased.

[0323] Table 13: Formation of covalently linked oligomers for Formulations 1 to 5 at 40°C for 0 to 4 weeks

[0324]

[0325] Example 10: Formulation of ZP1848 peptide acetate and preservatives for 20 mg / mL of the GLP-2 analog ZP1848 peptide Uses

[0326] A study was conducted to examine the compatibility of ZP1848 peptide acetate with commonly used preservatives. The effects of preservatives and temperature were examined after accelerated storage.

[0327] Materials and Methods

[0328] Prepare stock solutions of mannitol (700 mM), histidine (200 mM), and ZP1848 peptide solution (acetate; approximately 50 mg / mL) in Milli-Q water. The final concentration of the peptide is 20 mg / mL, mannitol is 230 mM, and histidine is 15 mM. The preservative solutions are mixed in appropriate amounts to obtain the formulations shown in Table 14 below. The peptide stock solution is added to obtain the desired final peptide content. Water is added to 90% of the final volume. If necessary, the pH is then adjusted to the desired formulation pH using 1 M acetic acid / 0.5 M L-arginine. Water is added to the final volume. Each formulation is filled in a vial. The sample containers are visually inspected for clarity and viscosity, and analyzed for covalently linked oligomers by SEC and for peptide monomer stability by HPLC.

[0329] Results and Discussion

[0330] The results from this study are listed in Tables 14, 15 and Figure 8 Compared to Formulation 1, in which no preservative was added, the formulation appeared to be unaffected by the addition of the preservative.

[0331] Chemical stability was evaluated by determining the stability (purity) of covalently linked oligomers and peptide monomers. Formulation 4 (potassium sorbate) had higher covalently linked oligomer formation, but within an acceptable range. All other formulations had similar amounts of covalently linked oligomers. Normalized purity after 13 weeks at 25°C showed that ZP1848-acetate again had similar stability to Formulation 4, which had slightly lower, but acceptable, purity.

[0332] Table 14: Effect of preservative screen at 25°C for 26 weeks

[0333]

[0334] Table 15: Formation of covalently linked oligomers with different preservatives at 25°C for 13 weeks

[0335]

[0336] Example 11: ZP1848 peptide acetate and preservatives for 2 and 20 mg / mL of the GLP-2 analog ZP1848 peptide Uses of preparations

[0337] Materials and Methods

[0338] Prepare stock solutions of mannitol (700mM), histidine (200mM), and ZP1848 peptide solution (acetate; approximately 50mg / mL) in Milli-Q water. The final concentrations of the peptide were 20 and 2mg / mL, mannitol was 230mM, and histidine was 15mM. Preservative solutions (m-cresol and phenol) were mixed in appropriate amounts to obtain the formulations shown in Table 14 below. Water was added to 90% of the final volume. If necessary, the pH was then adjusted to the desired formulation pH (7.0) using 1M acetic acid / 0.5M L-arginine. Water was added to the final volume. Each formulation was filled in a vial. The sample containers were visually inspected for clarity and viscosity, and the peptide monomer stability was analyzed by HPLC.

[0339] Results and Discussion

[0340] The results from this study are listed below in Table 16. All formulations were tested for long-term stability at 5°C for 52 weeks. All solutions tested remained clear and non-viscous throughout the timeframe of the study.

[0341] Table 16: Physical appearance of test formulations after 52 weeks stability study at 5°C

[0342]

[0343]

[0344] Evaluation of ZP1848-acetate by HPLC after accelerated stability study at 25°C is shown in Figure 9 For the phenol-containing formulations, slightly lower chemical stability was observed, and the chemical stability of m-cresol was similar to that of the unpreserved formulation. In the long-term stability study at 5°C, no significant differences were seen for the samples after 12 months of stability study, and the normalized ZP1848-acetate purity of all samples was greater than 94% (data not shown). Therefore, all formulations studied were stable in the long-term stability study for at least 52 weeks.

[0345] Although the present invention has been described in conjunction with the above-mentioned embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when present disclosure is given. Therefore, some embodiments of the present invention set forth are considered to be illustrative and not restrictive. Without departing from the spirit and scope of the present invention, various changes for the described embodiments may be made. All documents cited herein are expressly incorporated by reference in their entirety for all purposes. Sequence Listing <110> ZEALAND PHARMA A / S <120> Preparations of glucagon-like peptide 2 (GLP-2) analogs <130> 007551930 <140> PCT <141> 2019-09-27 <140> EP 18197755.4 <141> 2018-09-28 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 1 His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp Lys Lys Lys Lys Lys Lys 35 <210> 2 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 2 His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp Lys Lys Lys 35 <210> 3 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 3 His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp Lys Lys 35 <210> 4 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 4 His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp Lys <210> 5 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 5 His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp <210> 6 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 6 His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp <210> 7 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 7 His Gly Glu Gly Ser Phe Ser Ser Glu Leu Ser Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp Lys Lys Lys Lys Lys Lys 35 <210> 8 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 8 His Gly Glu Gly Ser Phe Ser Ser Glu Leu Ser Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp <210> 9 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <400> 9 His Gly Glu Gly Ser Phe Ser Ser Glu Leu Ser Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp Lys <210> 10 <211> 33 <212> PRT <213> Homo sapiens <400> 10 His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 11 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide sequence <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa = Ser or Thr <220> <221> MISC_FEATURE <222> (11)..(11) <223> Xaa = Ala or Ser <400> 11 His Gly Glu Gly Xaa Phe Ser Ser Glu Leu Xaa Thr Ile Leu Asp Ala 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr 20 25 30 Asp

Claims

1. A method for reducing the viscosity of a stable liquid pharmaceutical formulation comprising a pharmaceutically acceptable acetate of a glucagon-like peptide 2 (GLP-2) analogue, wherein the GLP-2 analogue is: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1) wherein the method comprises formulating (a) a pharmaceutically acceptable acetate of the GLP-2 analogue at a concentration of 2 mg / mL to 30 mg / mL together with: (b) a buffer selected from histidine buffer, methanesulfonate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer or MOPS buffer, the buffer being present at a concentration of 5 mM to 50 mM; (c) a non-ionic tonicity regulator selected from mannitol, sucrose, sorbitol and trehalose, the non-ionic tonicity regulator being present at a concentration of 90 mM to 360 mM; and (d) an appropriate amount of arginine to provide a formulation having a pH of 6.6 to 7.4; wherein the total acetate concentration in the formulation resulting from the GLP2 analogue is less than or equal to 11% acetate / mg GLP-2 analogue, and wherein the viscosity of the formulation measured at 25 °C is greater than 0.8 and less than or equal to 2.0 mPa / s.

2. The method of claim 1, wherein (a) comprises formulating the pharmaceutically acceptable acetate of the GLP-2 analogue at a concentration of 2 mg / mL, 5 mg / mL, 10 mg / mL or 20 mg / mL.

3. A method for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue in a stable liquid pharmaceutical formulation, the liquid pharmaceutical formulation comprising a pharmaceutically acceptable acetate of the GLP-2 analogue, wherein the GLP-2 analogue is: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1) wherein the method comprises formulating (a) a pharmaceutically acceptable acetate of the GLP-2 analogue at a concentration of 2 mg / mL to 30 mg / mL together with: (b) a buffer selected from histidine buffer, methanesulfonate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer or MOPS buffer, the buffer being present at a concentration of 5 mM to 50 mM; (c) a non-ionic tonicity regulator selected from mannitol, sucrose, sorbitol and trehalose, the non-ionic tonicity regulator being present at a concentration of 90 mM to 360 mM; and (d) an appropriate amount of arginine to provide a formulation having a pH of 6.6 to 7.4; wherein the formulation contains 5% or less of the GLP-2 analogue in the form of covalently bonded oligomeric products; and wherein the total acetate concentration produced by the GLP2 analogue in the formulation is less than or equal to 11% acetate / mg GLP-2 analogue, and wherein the viscosity of the formulation measured at 25 °C is greater than 0.8 and less than or equal to 2.0 mPa / s.

4. The method of claim 3, wherein the formation of covalently linked oligomers of the GLP-2 analogue is negatively dependent on the concentration of the GLP-2 analogue in the formulation.

5. The method of claim 3, wherein (a) comprises formulating the pharmaceutically acceptable acetate of the GLP-2 analogue at a concentration of 2 mg / mL, 5 mg / mL, 10 mg / mL or 20 mg / mL.

6. Use of a formulation for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue in a stable liquid pharmaceutical composition, the liquid pharmaceutical composition comprising a pharmaceutically acceptable acetate of the GLP-2 analogue, the liquid pharmaceutical formulation being stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analogue is represented by the formula: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKUTDKKKKKK-NH2 (SEQ ID NO: 1) wherein the formulation comprises: (a) the pharmaceutically acceptable acetate of the GLP-2 analogue at a concentration of 2 mg / mL to 30 mg / mL; (b) a buffer selected from histidine buffer, mesylate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, the buffer being present at a concentration of 5 mM to 50 mM; (c) a non-ionic tonicity modifier selected from mannitol, sucrose, sorbitol and trehalose, at a concentration of 90 mM to 360 mM; and (d) an appropriate amount of arginine to provide a formulation with a pH of 6.6 to 7.4; wherein the formulation contains 5% or less of the GLP-2 analogue in the form of covalently bonded oligomeric products; and wherein the total acetate concentration produced by the GLP2 analogue in the formulation is less than or equal to 11% acetate / mg GLP-2 analogue, and wherein the viscosity of the formulation measured at 25 °C is greater than 0.8 and less than or equal to 2.0 mPa / s.

7. The use according to claim 6, wherein the formation of covalently linked oligomers of the GLP-2 analogue is negatively dependent on the concentration of the GLP-2 analogue in the formulation.

8. The use of claim 6, wherein the formulation comprises the pharmaceutically acceptable acetate of the GLP-2 analogue at a concentration of 2 mg / mL, 5 mg / mL, 10 mg / mL or 20 mg / mL.

9. Use of a formulation for reducing the viscosity of a liquid pharmaceutical formulation in a liquid pharmaceutical formulation comprising a pharmaceutically acceptable acetate of a glucagon-like peptide 2 (GLP-2) analogue, the liquid pharmaceutical formulation being stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analogue is: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1) Wherein the preparation comprises: (a) a pharmaceutically acceptable acetate of the GLP-2 analog, at a concentration of 2 mg / mL to 30 mg / mL; (b) a buffer selected from histidine buffer, mesylate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, present at a concentration of 5 mM to 50 mM; (c) a non-ionic tonicity regulator selected from mannitol, sucrose, sorbitol and trehalose, at a concentration of 90 mM to 360 mM; and (d) an appropriate amount of arginine to provide a preparation with a pH of 6.6 to 7.4; Wherein the total acetate concentration produced by the GLP2 analog in the preparation is less than or equal to 11% acetate / mg GLP-2 analog, and the viscosity of the preparation measured at 25 °C is 0.8 to 2.0 mPa / s.

10. The use according to claim 9, wherein the preparation comprises a pharmaceutically acceptable acetate of the GLP-2 analog at a concentration of 2 mg / mL, 5 mg / mL, 10 mg / mL or 20 mg / mL.

Images