Formulations of glucagon-like-peptide-2 (GLP-2) analogues

Abstract

To provide stable liquid formulations of glucagon-like-peptide-2 (GLP-2) analogues that are capable of long-term storage without undue levels of physical or chemical degradation of the active monomeric form of the peptide, and also to provide solid compositions comprising acetates of GLP-2 analogues useful for preparing the liquid formulations.SOLUTION: The liquid formulations are based on the findings that: acetates present in the formulations derived from the GLP-2 analogues affect the viscosity of the formulations; during long-term storage of GLP-2 analogues at 2-8°C, the concentration dependence for covalently linked oligomer formation is inversely dependent on increasing concentration of the GLP-2 analogues; and the GLP-2 analogues used in the formulations are incompatible with phosphate buffers commonly used in the prior art for reconstituted powder or lyophilized GLP-2 compositions.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to formulations of glucagon-like peptide-2 (GLP-2) analogs and their medical uses, such as in the treatment and / or prevention of gastrointestinal disorders and for improving the side effects of chemotherapy and radiotherapy.Furthermore, a solid composition comprising an acetate salt of a glucagon-like peptide-2 (GLP-2) analog that is useful for making a liquid formulation is also described. [Background technology]

[0002] Human GLP-2 is a 33 amino acid peptide with the following sequence: 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. Human GLP-2 is the result of specific post-translational processing of proglucagon in the enteroendocrine L-cells of the intestine and in specific regions of the brainstem. GLP-2 binds to a G protein-coupled receptor belonging to the class II glucagon secretin family.

[0003] GLP-2 has been reported to induce significant growth of the small intestinal mucosal epithelium through stimulation of stem cell proliferation in the crypts and by inhibition of 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 via upregulation of glucose transporters (Cheeseman, 1997, Am. J. Physiol. R 1965-71), and increases intestinal blood flow (Guan et al., 2003, Gastroenterology 125:136-147).

[0004] It has been 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 a clinical setting due to its extremely short half-life in humans, about 7 minutes for full-length GLP-2 [1-33] and 27 minutes for truncated GLP-2 [3-33]. This short half-life is mainly due to degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). Thus, there have been attempts in the art to develop GLP-2 receptor agonists with better pharmacokinetic properties, particularly to improve the half-life of the GLP-2 molecule. As an example, GLP-2 analogs with substitutions have been proposed, such as a Gly substitution at position 2 ([hGly2]GLP-2, teduglutide), which increases the half-life from 7 minutes (native GLP-2) to about 2 hours. Acylation of peptide drugs with fatty acid chains has also proven beneficial in extending systemic circulation and increasing enzyme stability without interfering with biological activity. However, while these efforts have improved the pharmacokinetics of GLP-2 analogs, which are sometimes described in the art as "long-acting," it must be kept in mind that this improvement is in comparison to native hGLP-2, which has a half-life of hours rather than minutes. This, in turn, means that GLP-2 analogs still need to be administered to patients one or more times per day.

[0005] US Patent No. 5,789,379 discloses GLP-2 analogs for administration by injection. The GLP-2 analogs are provided as powdered peptides and mixed with phosphate buffered saline (PBS) prior to injection at a pH of 7.3-7.4 with a GLP-2 concentration of 130 mg / ml. Optionally, the GLP-2 / PBS composition is mixed with gelatin to provide a depot formed from a solution of 130 mg / l GLP-2 in PBS / 15% gelatin. US Patent No. 5,789,379 does not disclose stable aqueous liquid formulations of GLP-2 analogs, and the GLP-2 analogs are generally reconstituted from a powder prior to injection.

[0006] WO97 / 39031 and U.S. Patent No. 6,184,201 disclose a GLP-2 analog, [Gly 2 ]GLP-2, in which alanine at position 2 was replaced with glycine to make the peptide resistant to DPP IV cleavage. As in U.S. Pat. No. 5,789,379, the GLP-2 analogs were provided as powdered peptides and mixed with saline, PBS, or 5% dextrose prior to injection, with optional acetic acid added as a solubility enhancer.

[0007] WO02 / 066511 describes GLP-2 analogues with increased half-life in vivo and their use as medicines in the treatment of gastrointestinal disorders such as inflammatory bowel disease. These GLP-2 analogues are stored in lyophilized form and reconstituted to be administered in a solvent, for example using saline or PBS.

[0008] WO01 / 41779 discloses the use of h[Gly] as a pretreatment to inhibit chemotherapy-induced apoptosis and promote cell survival. 2 ]GLP-2 is described. 2 ]GLP-2 is delivered by subcutaneous or intravenous injection or infusion after reconstitution of the analog in PBS.

[0009] WO2001 / 049314 is directed to formulations of GLP-2 peptides and analogues thereof that exhibit superior stability after storage at and / or exposure to elevated temperatures. The GLP-2 composition comprises a GLP-2 peptide or analogue thereof, a phosphate buffer, L-histidine, and mannitol.

[0010] WO2006 / 117565 discloses [hGly 2 ] describe GLP-2 analogs that contain one of a number of substitutions compared to GLP-2 and have improved biological activity in vivo and / or improved chemical stability, for example as assessed in an in vitro stability assay. In particular, GLP-2 analogs are described that 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 positions 2 and one or more of positions 3, 5, 7, 10, and 11, and / or deletions of one or more of amino acids 31-33. These substitutions may also be combined with the addition of N- or C-terminal stabilizing peptide sequences. Once or twice daily administration of these GLP-2 analogs is also described. Of the molecules disclosed in WO2006 / 117565, grepaglutide (ZP1848) is designed to be stable in liquid formulations and is typically administered by daily dosing using a pen-type injector. Summary of the Invention [Problem to be solved by the invention]

[0011] A challenge remains in this area to improve formulations of GLP-2 analogs, in particular to provide stable liquid formulations that can be stored for extended periods without excessive levels of physical or chemical degradation of the active monomeric form of the peptide. One of the chemical pathways that can result is the formation of covalently linked dimers and oligomers of the peptide, and the formation of these covalently linked high molecular weight oligomeric products reduces the amount of active monomeric form of the peptide. According to the law of mass action, the higher the concentration of the peptide drug in the formulation, the higher the probability of the formation of covalently linked oligomeric products is generally.

[0012] It is also believed to be a goal in the area of ​​GLP-2 analogue formulations to provide formulations in which the viscosity of the formulation is controlled within a range that renders the formulation suitable for use in delivery devices such as pre-filled syringes, infusion pumps, wearable injectors, or autoinjectors. [Means for solving the problem]

[0013] In general, the present invention is based on studies reported in the Examples, which have led to surprising findings regarding liquid formulations of GLP-2 analogues that, as liquids, are suitable for long-term storage and / or are particularly suitable for delivery by drug delivery devices.

[0014] In the first study, the inventors found that acetate present in formulations derived from GLP-2 analogs affects the viscosity of the formulation. This opens up the possibility of controlling the viscosity of the formulation by modifying and / or controlling the acetate concentration. Liquid formulations with low range viscosities are clinically useful because they provide advantages in the development and manufacture of drug delivery devices by potentially reducing breakage, dosing failures, dosing inaccuracies, and other defects during formulation manufacture and / or patient use. Furthermore, low viscosity may allow faster injection and / or the use of smaller bore (i.e., higher gauge) needles, resulting in less discomfort from injection. This opens up the possibility of providing formulations of GLP-2 analogs in the form of drug delivery devices, such as prefilled syringes, dose adjustable autoinjectors, disposable autoinjectors, wearable injectors, or infusion pumps, thereby providing patients with ready-to-use formulations using simpler, safer, and more patient-friendly devices. Higher viscosity controlled formulations may be advantageous in other drug delivery devices.

[0015] In a second study, the inventors found that the formation of covalently linked oligomers was concentration dependent during long-term storage of ZP1848 (grepaglutide) at 2-8°C. However, contrary to the usual situation where the law of mass action implies that covalent oligomer formation increases with increasing peptide drug concentration, the inventors found that the concentration dependence of oligomer formation was inversely dependent on increasing concentrations of the GLP-2 analogue. Without wishing to be bound by any particular theory, the inventors believe that the reduction in covalently linked oligomer formation with increasing concentrations of the GLP-2 analogue is a result of the lysine tail of the GLP-2 analogue promoting the formation of structural aggregates by self-association of the native peptide that prevent the formation of covalently linked oligomers in the formulation. This means that rather than causing some decrease in activity, as occurs when covalently linked oligomers are formed, the weakly self-associated species can dissociate after administration to a patient to release biologically active monomers.

[0016] In a third study, the inventors demonstrated that the GLP-2 analogs used in the formulations of the present invention were not compatible with phosphate buffers commonly used in the prior art for reconstituted powder or lyophilized GLP-2 compositions. This study demonstrated that only certain buffers were compatible with the formulation of these GLP-2 analogs, and as a result, these GLP-2 analogs were suitable for long-term storage in liquid dosage forms.

[0017] Thus, in a first aspect, the present invention is a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharma- ceutically acceptable salt or derivative thereof. and wherein the GLP-2 analog has the formula: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, and Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. is represented by (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL; (b) a buffer selected from the group consisting of histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, 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 at a concentration of about 90 mM to about 360 mM; (d) arginine in an amount sufficient to obtain a formulation having a pH of about 6.6 to about 7.4; and

[0018] In some embodiments, the formulation contains 5% or less of the GLP-2 analog in the form of covalently linked oligomer products.Alternatively or additionally, the total acetate concentration resulting from the GLP-2 analog in the formulation is 11% acetate or less per mg of GLP-2 analog.Alternatively or additionally, 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.

[0019] The formulation ingredients and their amounts are such that, upon storage at 2-8°C for at least 18 months, the formulation contains at least 90% GLP-2 analogue content and less than 10% chemical degradants.

[0020] In a further aspect, the invention provides an article of manufacture or kit comprising a container housing a stable pharmaceutical formulation of the invention. In a further embodiment, the present invention provides a delivery device containing a liquid formulation comprising a GLP-2 analogue of the present invention.

[0021] In a further aspect, the present invention provides formulations of the glucagon-like peptide 2 (GLP-2) analogues of the present invention for use in therapy. In a further aspect, the present invention provides a formulation of a glucagon-like peptide 2 (GLP-2) analogue of the present invention for use in a method for the treatment and / or prevention of a gastrointestinal-related disorder in a human patient.

[0022] In a further aspect, the present invention provides a method for preparing a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analog, or a pharma- ceutically acceptable salt or derivative thereof, wherein the GLP-2 analog has the formula: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr -Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. is represented by 1. A method for preparing a formulation comprising the steps of: (a) combining 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, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, 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, present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to obtain a formulation having a pH of about 6.6 to about 7.4; Methods are provided in which the formulation contains 5% or less of the GLP-2 analog in the form of a covalently attached oligomeric product.

[0023] In a further aspect, the present invention relates to the use of a formulation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharma- ceutically acceptable salt or derivative thereof to provide a liquid pharmaceutical formulation that is stable for 24 months when stored at 2-8° C., wherein the GLP-2 analogue has the formula R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. is represented by The formulation, (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL; (b) a buffer selected from the group consisting of histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, 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 at a concentration of about 90 mM to about 360 mM; (d) an appropriate amount of arginine to obtain a formulation having a pH of about 6.6 to about 7.4; and Provide use.

[0024] In a further aspect, the present invention provides a method for modulating the viscosity of a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analog or a pharma- ceutically acceptable salt or derivative thereof. The method, comprising the step of: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. is represented by The method includes the steps of: (a) combining 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, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, or MOPS buffer, present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity adjusting agent selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to obtain a formulation having a pH of about 6.6 to about 7.4; the total acetate concentration resulting from the GLP2 analog in the formulation is less than or equal to 11% acetate per mg of GLP-2 analog, and the formulation has a viscosity measured at 25° C. that is greater than 0.8 and less than or equal to 2.0 mPa / sec; A method is provided.

[0025] In a further aspect, the present invention provides a method of reducing the formation of covalently linked oligomeric products of a glucagon-like peptide 2 (GLP-2) analog in a stable liquid pharmaceutical formulation comprising a GLP-2 analog or a pharma- ceutically acceptable salt or derivative thereof, wherein the GLP-2 analog has the formula: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. is represented by The method includes the steps of: (a) combining 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, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, or MOPS buffer, present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity adjusting agent selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose, present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to obtain a formulation having a pH of about 6.6 to about 7.4; The method further comprises the step of: providing a formulation containing 5% or less of the GLP-2 analog in the form of a covalently linked oligomer product. Optionally, in this embodiment of the invention, 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.

[0026] In a further aspect, the present invention relates to the use of a formulation to reduce the formation of covalently linked oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue, or a pharma- ceutically acceptable salt or derivative thereof, in a liquid pharmaceutical formulation that is stable for 24 months when stored at 2-8°C, wherein the GLP-2 analogue has the formula: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. is represented by The formulation, (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL; (b) a buffer selected from the group consisting of histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, 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 at a concentration of about 90 mM to about 360 mM; (d) arginine in an amount sufficient to obtain a formulation having a pH of about 6.6 to about 7.4; and Contains 5% or less of a GLP-2 analog in the form of a covalently linked oligomeric product; Optionally, in this aspect of the invention, 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.

[0027] In a further aspect, the present invention relates to a use of a formulation for adjusting the viscosity of a liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharma- ceutically acceptable salt or derivative thereof, in a liquid pharmaceutical formulation stable for 24 months when stored at 2-8°C, wherein the GLP-2 analogue has the formula: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. is represented by The formulation, (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL; (b) a buffer selected from the group consisting of histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, 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 at a concentration of about 90 mM to about 360 mM; (d) an appropriate amount of arginine to obtain a formulation having a pH of about 6.6 to about 7.4; and the total acetate concentration resulting from the GLP2 analog in the formulation is less than or equal to 11% acetate per mg of GLP-2 analog, and the formulation has a viscosity between 0.8 and 2.0 mPa / sec measured at 25° C. Provide use.

[0028] In a further aspect, the present invention provides a compound of formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) (wherein x is 1.0 to 8.0) The present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula:

[0029] In a further aspect, the present invention provides a stable liquid pharmaceutical formulation comprising: (a) a solid composition of the present invention having a concentration of about 2 mg / mL to about 30 mg / mL; (b) a buffer selected from the group consisting of histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, 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 at a concentration of about 90 mM to about 360 mM; (d) an appropriate amount of arginine to obtain a formulation having a pH of about 6.6 to about 7.4; and Formulations are provided which contain up to 5% of a GLP-2 analogue in the form of a covalently linked oligomeric product and have a viscosity measured at 25°C between 0.8 and 2.0 mPa / sec.

[0030] In all of the aspects of the invention described herein, the buffer may be selected from the group consisting of a histidine buffer, a mesylate buffer, and an acetate buffer. In all of the aspects of the invention described herein, the non-ionic tonicity adjusting agent may be selected from the group consisting of mannitol, sucrose, glycerol, and sorbitol.

[0031] In some embodiments, the formulation contains 5% or less of the GLP-2 analog in the form of a covalently linked oligomer product. Alternatively or additionally, the total acetate concentration resulting from the GLP-2 analog in the formulation is 11% acetate / mg of GLP-2 analog or less. Alternatively or additionally, 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.

[0032] In a further aspect, the present invention provides a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue, or a pharma- ceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue has the formula: R1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Ly s-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4 alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, Z 2 is a peptide sequence of 6 amino acid units of Lys) and comprising the ingredients as described in any one of the aspects of the invention set forth herein.

[0033] In this aspect of the invention, formulations comprising a glucagon-like peptide 2 (GLP-2) analog or a salt thereof may be used for the treatment and / or prevention of gastrointestinal-related disorders such as ulcers, digestive disorders, malabsorption syndromes, short bowel syndrome, blind-tube syndrome, inflammatory bowel disease, celiac sprue (e.g. resulting from gluten-sensitive enteropathy or celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, small intestinal damage, or short bowel syndrome (SBS). Alternatively or additionally, the glucagon-like peptide 2 (GLP-2) analog may be used for the treatment and / or prevention of gastrointestinal-related disorders such as radiation enteritis, infectious or post-infectious enteritis, or small intestinal damage caused by toxic substances or other chemotherapeutic agents. In this case, treatment with the GLP-2 analogue may optionally be combined with one or more anti-cancer therapies and may thus include administering to the patient one or more chemotherapeutic agents or treating the patient with radiation therapy.

[0034] In some embodiments of the invention, in the above formula, X5 is Thr and / or X11 is Ala. Examples of these glucagon-like peptide 2 (GLP-2) analogs include: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1) ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO:2); ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3); ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4); ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 5); or ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6) In some embodiments of the invention, in the above formula, X5 is Ser and / or X11 is Ser. Examples of these glucagon-like peptide 2 (GLP-2) analogues include: ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7), ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 8), or ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (sequence number 9).

[0035] Embodiments of the invention will now be described, by way of non-limiting example, with reference to the accompanying drawings, in which: However, various further aspects and embodiments of the invention will be apparent to those skilled in the art in view of this disclosure. As used herein, "and / or" is used as a specific disclosure of each of two specified features or elements, with or without the other. For example, "A and / or B" is used as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually described herein.

[0036] Unless otherwise indicated, the above feature descriptions and definitions are not intended to be limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described. [Brief description of the drawings]

[0037] [Figure 1] FIG. 1 shows a typical chromatogram showing separation of oligomers from ZP1848 peptide. [Diagram 2]FIG. 1 shows how viscosity (squares) and hydrodynamic radius (z-average) (circles) changed as a function of acetate concentration after preparation of the formulations. The data shows that above 11% acetate, viscosity and hydrodynamic radius (z-average) begin to increase. [Diagram 3] FIG. 1 shows stability evaluation (normalized to starting 100%) at 20 mg / mL using various buffers at 40° C. for 0-3 weeks. [Figure 4] FIG. 1 shows stability (normalized to 100% starting) at 2 mg / mL using various buffers at 40° C. for 0-3 weeks. [Diagram 5] FIG. 1 shows stability evaluation (normalized to starting 100%) at 20 mg / mL with various tonicity agents at 40° C. for 0-3 weeks. [Figure 6] FIG. 1 shows stability evaluation (normalized to starting 100%) at 2 mg / mL with various tonicity agents at 40° C. for 0-3 weeks. [Figure 7] FIG. 1 shows the purity of formulations 1-5 using different concentrations of ZP1848 acetate, different salt forms, different tonicity agents and different buffers. [Figure 8] FIG. 1 shows the stability of peptides in combination with various preservatives at 25° C. for 13 weeks. [Figure 9] FIG. 1 shows the HPLC purity of the formulations studied at 25° C. (accelerated conditions). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] definition Unless otherwise stated, the following definitions apply to specific terms used in the above description.

[0039] Throughout the description and claims, the conventional one-letter and three-letter codes for natural amino acids are used. All amino acid residues in the peptides of the invention are preferably in the L-configuration, although D-configuration amino acids may be present.

[0040] Preferred compounds of the invention have at least one GLP-2 biological activity, particularly in intestinal growth. GLP-2 biological activity can be assessed, for example, in an in vivo assay measuring the whole intestine or a portion of the intestine after treating or exposing test animals to a GLP-2 analogue, for example, as described in the examples of WO2006 / 117565.

[0041] In some embodiments of the invention, the liquid formulation comprising a GLP-2 analogue has a total acetate concentration in the formulation of 11% or less acetate per mg of GLP-2 analogue, more preferably 10% or less acetate per mg of GLP-2 analogue, more preferably 9% or less acetate per mg of GLP-2 analogue, more preferably 8% or less acetate per mg of GLP-2 analogue, more preferably 10 ... Preferably, the total acetate concentration is 7% or less acetate per mg of GLP-2 analogue, more preferably 6% or less acetate per mg of GLP-2 analogue, more preferably 5% or less acetate per mg of GLP-2 analogue, more preferably 4% or less acetate per mg of GLP-2 analogue, more preferably 3% or less acetate per mg of GLP-2 analogue, and more preferably 2% or less acetate per mg of GLP-2 analogue. 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 with an acetate content of less than 11%. Thus, for example, for a formulation with 20 mg / mL of GLP-2 analogue, the total acetate concentration is 37 mM or less. For reference, 10% total acetate concentration corresponds to 34 mM, 9% corresponds to 30 mM, 8% corresponds to 27 mM, 7% corresponds to 24 mM, and 6% corresponds to 20 mM. Total acetate concentration may be determined using methods known in the art, for example, HPLC.

[0042] In the examples below, it is shown that the viscosity of the liquid formulations of the invention depends on the total acetate concentration. Preferably, the formulation has a viscosity between 0.8 and 2.0 mPa / sec when measured at 25°C. Conveniently, the viscosity may be measured by using a microVISC™. In parallel, the hydrodynamic radius may be measured using a dynamic light scattering (DLS) plate reader (Wyatt DynaPro II). Samples were prepared containing a drug substance (DS) of a GLP-2 analogue containing 6% acetate, and then acetate was added to mimic a DS with 7.8-15% acetate. Data obtained from producing formulations with various acetate concentrations from 6.7-15% are shown below in Figure 2. The effect of controlling the total acetate concentration is that it is possible to adjust the injectability of the formulations of the invention, for example by lowering the total acetate concentration, to provide a less viscous formulation that is more easily injectable.

[0043] 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 osmolality as body fluid. Preferably, the formulation of the present invention has an osmolality of about 300±60 mOsm as measured by an osmometer.

[0044] Additionally or alternatively, the present invention demonstrates that the formation of covalently linked oligomers of GLP-2 analogs is inversely dependent on the concentration of GLP-2 analogs in the formulation. As shown in the examples, this amount of covalently linked oligomers can be determined using size exclusion chromatography and determining the area under the peak for the monomeric GLP-2 analogs and the oligomers, respectively. This can be done using a Dionex Ultimate3000 HPLC system, and a linear gradient with a flow rate of 0.5 ml / min was used for the analysis. The mobile phase consisted of 45% acetonitrile and 0.1% TFA in 55% Milli-Q water. A wavelength of 215 nm was used for detection. This means that the formulations of the present invention generally contain GLP-2 analogs 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 some embodiments 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 more preferably 2% or less of the GLP-2 analogue in the form of covalently linked oligomeric products, preferably after 18 months of storage. By way of illustration, the amount of covalently linked oligomeric products may range between 2% and 5%, more preferably between 2% and 4%, and most preferably between 2% and 3%.

[0045] 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 analogue of the present invention may include multiple or course dosings separated by 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In a preferred embodiment, the dosings are separated by 3, 3.5, 4, 5, 6, 7, or 8 days. In a preferred embodiment, the dosings are separated by 3, 3.5, 4, or 7 days. As is understood in the art, the time between dosings may vary to some extent, so that each and every dosing is not separated by exactly the same time. This is often at the discretion of the physician. Thus, dosings may be separated by any clinically acceptable time period, for example, from about 2 days to about 10 days, or from about 3 or 4 days to about 7 or 8 days.

[0046] The formulation of the present invention is a stable liquid pharmaceutical formulation of GLP-2 analogue. A "stable" formulation is one in which the peptide therein substantially retains its physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially retains its physical and chemical stability and its biological activity during storage. The storage period is generally selected based on the intended shelf life of the formulation. The formulation of the present invention is provided as a stable liquid formulation, for example, a stable aqueous liquid formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, pp. 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Published (1991) and Jones, A. Adv. Drug Delivery Rev. 10: pp. 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 analogues in the formulation are active after storage at 2-8°C for at least 18 months.

[0047] Stability may be measured at a selected temperature for a selected period of time, e.g., the temperature may be increased to shorten the period for which the formulation is tested. Generally, storage at a temperature between 2 and 8°C refers to 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 qualitatively and / or quantitatively assessed in a wide variety of ways, including assessment of aggregate formation (e.g., using size-exclusion chromatography, by measuring turbidity, and / or by visual inspection); by assessing charge heterogeneity using cation-exchange chromatography, imaging capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide map (e.g., by trypsin or LYS-C) analysis; assessment of antibody biological activity or antigen-binding function, and the like. The instability may be associated with any one or more of 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 extensions, C-terminal processing, differences in glycosylation, and the like.

[0048] A peptide "retains its physical stability" in a pharmaceutical formulation if it shows no signs (or only minimal signs) of aggregation, precipitation, and / or denaturation as measured, for example, by visual inspection of color and / or clarity, or by UV light scattering, dynamic light scattering, circular dichroism, or by size exclusion chromatography, and is still considered to retain its biological activity.

[0049] A peptide "retains its chemical stability" in a pharmaceutical formulation if the chemical stability over a given period of time is such that the peptide is still considered to retain its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying chemically modified forms of the peptide. Chemical modifications can include isomerization, oxidation, size modification (e.g., clipping), which can be assessed, for example, using HPLC or size exclusion chromatography, SDS-PAGE, and / or mass spectrometry. Other types of chemical modifications include charge modification (e.g., resulting from deamidation), which can be assessed, for example, by HPLC or ion exchange chromatography or icIEF. GLP-2 analogue The GLP-2 analogs present in the formulations of the present invention have one or more amino acid substitutions, deletions, inversions, or additions as defined above, compared to native GLP-2. 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 of 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 lysine ε-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Preferably, herein, lower alkyl is a C1-C4 alkyl. Additionally, one or more side groups, or the terminal group, may be protected by a protecting group known to the skilled peptide chemist. The α-carbon of the amino acid may be mono- or di-methylated.

[0050] In some embodiments, the liquid formulations of the present invention have the formula: R 1 -Z 1 -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Z 2 -R 2 (In the formula, R 1 is hydrogen, C 1~4alkyl (e.g. methyl), acetyl, formyl, benzoyl, or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser, R 2 is NH2 or OH, and Z 1 and Z 2 are, independently, peptide sequences of 1 to 6 amino acid units, either absent or Lys. or a pharma- ceutically acceptable salt or derivative thereof is used.

[0051] In some embodiments of the invention, in the above formula, X5 is Thr and / or X11 is Ala. Examples of these glucagon-like peptide 2 (GLP-2) analogs include: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1) ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO:2); ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3); ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4); ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 5); or ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6) In an embodiment of the invention, the glucagon-like peptide 2 (GLP-2) analog is ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO:1).

[0052] In some embodiments of the invention, in the above formula, X5 is Ser and / or X11 is Ser. Examples of these glucagon-like peptide 2 (GLP-2) analogues include: ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7), ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 8), or ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9) In an embodiment of the invention, the glucagon-like peptide 2 (GLP-2) analog is ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7).

[0053] It is understood that the peptide (drug substance) of the present invention may also be provided in the form of a salt or other derivative. Salts include pharma- ceutically acceptable salts, such as acid addition salts and base salts. Examples of acid addition salts include hydrochloride, citrate, chloride, and acetate salts. Preferably, the salt is an acetate salt. In general, it is preferred that the salt is not a chloride salt. Examples of base salts include those in which the cation is an alkali metal, such as sodium and potassium, an alkaline earth metal, such as calcium, and an ammonium ion. + N(R 3 )3(R 4 )(wherein, R 3 and R 4 are independently optionally substituted C 1~6 -alkyl, optionally substituted C 2~6 -alkenyl, optionally substituted aryl, or optionally substituted heteroaryl). Other examples of pharma- ceutically acceptable salts include those salts selected from the group consisting of those described in "Remington's and Encyclopaedia of Pharmaceutical Technology, 17th ed., Edited by Alfonso R. Gennaro, Mark Publishing Company, Easton, PA, USA, 1985, and later editions.

[0054] In a preferred embodiment, the acetate salt of the GLP-2 analog of the present invention is selected from the group consisting of ZP1848-acetate, ZP2949-acetate, ZP2711-acetate, ZP2469-acetate, ZP1857-acetate, ZP2530-acetate, ZP1846-acetate, ZP1855-acetate, and ZP2242-acetate. In the present context, the term "ZP1848-acetate" refers to the ZP1848 molecule in the acetate form. The acetate salt of the GLP-2 analog has the following formula: (GLP-2 analog),x(CH3COOH) (wherein x is between 1.0 and 8.0, i.e., where x is 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0 In any composition of acetate salt of a GLP-2 analog, x is not necessarily an integer since there may be molecules with different numbers of acetate salt molecules. In some cases, x is 4.0 to 8.0, x is 6.0 to 8.0, or x is 4.0 to 6.5. In some cases, x is 4.0 to 6.0, x is 2.0 to 7.0, x is 3.0 to 6.0, x is 4.0 to 6.0, or x is 4.0 to 8.0.

[0055] 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), where x is between 1.0 and 8.0.

[0056] Thus, in a further aspect, the present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analogue. The solid composition is useful for formulation with excipients used to make liquid formulations of the present invention. In one embodiment, the present invention provides a compound having the formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) (wherein x is 1.0 to 8.0) The present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the formula:

[0057] The upper limit of 8.0 acetate molecules per GLP-2 analogue corresponds to an acetate content of less than 11% acetate, which can be formulated to have a viscosity between 0.8 and 2.0 mPa / sec measured at 25° C.

[0058] The range of the number of acetate molecules attached to each molecule of GLP-2 analogue defines the molecular weight range of this component of the formulation. For example, for the acetate salt of ZP1848, the range of the number of acetate molecules attached to each molecule of GLP-2 analogue defines the molecular weight range of ZP1848-acetate. As an example, each molecule of ZP1848 and one acetate equivalent has a molecular weight of 4316+60=4376 Da. Thus, the molecular weights of ZP1848 with increasing acetate equivalents are 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 then defines the molecular weight ranges as follows: 1-8 acetate equivalents=4376 Da to 4796 Da; 4-8 acetate equivalents=4556 Da to 4796 Da, and 6-8 acetate equivalents=4676 Da to 4796 Da.

[0059] Other derivatives of the GLP-2 analogues of the present invention include Mn 2+ and Zn 2+The compounds include coordination compounds with metal ions such as , esters such as in vivo hydrolyzable esters, free acids or bases, hydrates, prodrugs, or lipids. Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and suitable carboxylic acid or alcohol reaction partners using techniques known in the art. Derivatives as prodrugs of compounds can be converted into one of the parent compounds in vivo or in vitro. Typically, at least one of the biological activities 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 metabolites. Examples of prodrugs include the use of protective groups that can be removed in situ in vivo to release the active compound or act to inhibit the elimination of the drug in vivo.

[0060] Z 1 and Z 2 are independently present and / or absent or are peptide sequences of 1 to 6 amino acid units of Lys, i.e. 1, 2, 3, 4, 5 or 6 Lys residues. The Lys residues may have either the D- or L-configuration, but have the L-configuration. Particularly preferred sequences are those in which Z is a sequence of 4, 5 or 6 consecutive lysine residues, and in particular 6 consecutive lysine residues. Exemplary sequences Z are given in WO01 / 04156. In one embodiment, Z 1 does not exist. In such a case, Z 2 may or may not be present. Formulation of GLP-2 Analogues The formulation of the GLP-2 analogue 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 predetermined amount of diluent, such as water for injection or other suitable diluent, prior to use by a designated route of administration.

[0061] As described herein, the liquid formulation of the GLP-2 analogue of the present invention comprises a buffer, a non-ionic tonicity adjusting agent, and an appropriate amount of arginine to obtain the pH of the final formulation. In accordance with normal pharmaceutical practice, the formulation of the present invention is sterile and / or does not contain a reducing agent. In some cases, the liquid formulation of the present invention is an aqueous liquid formulation. In some cases, the liquid formulation of the present invention is a non-aqueous liquid formulation.

[0062] The term "buffer" as used herein means a pharma- ceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are known in the art and can be found in the literature. Screening experiments in the Examples show that the formulations of the invention preferably include a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, as these buffers dissolved the GLP-2 analog and resulted in a stable formulation that did not become viscous, turbid, or precipitate the peptide drug. In a preferred embodiment, the buffer is a histidine buffer, e.g., L-histidine. Generally, 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, it is preferred that the buffer is not a phosphate buffer, a citrate buffer, a citrate / Tris buffer, and / or a succinate buffer.

[0063] The term "tonicity modifier" as used herein means a pharma- ceutically acceptable isotonicity agent used to adjust the tonicity of a formulation. The formulation of the present invention is preferably isotonic, i.e., the formulation of the present invention has an osmolality that is substantially the same as human serum. The tonicity modifier used in the formulation is preferably a non-ionic tonicity modifier, preferably selected from the group consisting of mannitol, sucrose, glycerol, sorbitol, and trehalose. A preferred non-ionic tonicity modifier is mannitol, e.g., D-mannitol. The concentration of the tonicity modifier will depend on the concentration of the other formulation components, especially if the formulation is intended to be isotonic. Typically, the non-ionic tonicity modifier 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.

[0064] In general, the components and amounts of the liquid formulations of the present invention are selected to provide a formulation having a pH of about 6.6 to about 7.4, more preferably about 6.8 to about 7.2, and most preferably about 7.0. The pH may be adjusted by adding a qs amount of arginine to fall within the desired pH range. Based on the experiments shown in the examples, it is preferred that the pH adjustment is not performed using hydrochloric acid or sodium hydroxide.

[0065] In one embodiment, the liquid formulation of the present invention has a concentration of about 2 mg / mL to about 30 mg / mL. a buffer selected from the group consisting of histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, Tris buffer, Bis-Tris buffer, and MOPS buffer, 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, present at a concentration of about 90 mM to about 360 mM; and an appropriate amount of arginine to obtain a pH of about 6.6 to about 7.4.

[0066] In one embodiment, the liquid formulation of the present invention comprises 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, mesylate buffer, and acetate buffer 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, and sorbitol at a concentration of about 90 mM to about 360 mM, and an appropriate amount of arginine to obtain a pH of about 6.6 to about 7.4.

[0067] In a further embodiment, a 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, mannitol at a concentration of about 230 mM, and an appropriate amount of arginine to obtain a pH of about 7.0.

[0068] In a further embodiment, a liquid formulation of the invention comprises a GLP-2 analogue 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 has a pH of about 7.0.

[0069] In a further embodiment, a liquid formulation of the invention comprises ZP1848-acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (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 an appropriate amount of arginine to obtain a pH of about 7.0.

[0070] In a further embodiment, a liquid formulation of the invention comprises ZP1848-acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (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 has a pH of about 7.0.

[0071] In a further 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), where 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, mannitol at a concentration of about 230 mM, and a pH of about 7.0.

[0072] In a further embodiment, in a once or twice daily dosing regimen, the liquid formulation of the present invention comprises a compound having the following formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH) (wherein x is 1.0 to 8.0), acetate salt of a glucagon-like peptide 2 (GLP-2) analog, a histidine buffer at a concentration of about 15 mM, mannitol at a concentration of about 230 mM, and a pH of about 7.0.

[0073] In a further embodiment, in a once or twice weekly dosing regimen, the liquid formulation of the present invention comprises a compound having the following formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH) (wherein x is 1.0 to 8.0). The solution contains an acetate salt of a lucagon-like peptide 2 (GLP-2) analog, a histidine buffer at a concentration of about 15 mM, and mannitol at a concentration of about 230 mM, and has a pH of about 7.0.

[0074] In a further embodiment, a liquid formulation of the invention comprises ZP1846 H-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 an appropriate amount of arginine to obtain a pH of about 7.0.

[0075] In a further embodiment, a liquid formulation of the invention comprises ZP1846 H-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 has a pH of about 7.0.

[0076] Optionally, the liquid formulation of the present invention further comprises a preservative. Optionally, the preservative is a preservative selected from the group consisting of benzalkonium chloride, chlorobutanol, methylparaben and potassium sorbate. Typically, the preservative is present at a concentration of about 0.1% to about 1% of the final formulation volume.

[0077] In further embodiments, the liquid formulation is selected from the group consisting of aqueous liquid formulations, liquid formulations in various hydrophilic or hydrophobic solvents, emulsions, and liquid suspensions. In a preferred embodiment, the liquid formulation is an aqueous liquid formulation.

[0078] As an example, the liquid formulation of the present invention may be prepared by mixing a stock solution of GLP-2 analogue, a buffer, a non-ionic tonicity adjusting agent, and optionally a preservative with water, optionally diluting the resulting solution, and adjusting to the target pH. Conveniently, the buffer and non-ionic tonicity adjusting agent solutions may be mixed first to obtain the desired concentration of each excipient. The GLP-2 analogue solution may then be added, and the pH adjusted, if necessary, using, for example, acetic acid / 0.5M L-arginine. Water is added until the final volume is reached.

[0079] Preferably, the glucagon-like peptide 2 (GLP-2) analogue is administered to the patient parenterally, preferably by injection, most typically by subcutaneous, intramuscular, intravenous or intraperitoneal injection. Subcutaneous administration is preferred. The injection may be administered by a doctor, nurse or other medical personnel, or the patient may self-administer. As described herein, in some embodiments, the formulation of the present invention has a viscosity that facilitates the filling of the formulation into a pre-filled syringe, injection pen or other injection device. This may have the advantage that the dosage of the formulation to be administered to the patient is pre-determined, for example, without the need to measure from a multi-use vial. Thus, in other embodiments, the present invention provides a product or kit that includes a container that contains a stable, for example, aqueous stable pharmaceutical formulation of a GLP-2 analogue according to the present invention, or a pre-filled syringe or injection device or pen injection device that contains an aqueous liquid formulation comprising a GLP-2 analogue according to the present invention. Condition The GLP-2 analog formulations of the present invention are useful as pharmaceutical agents to prevent or treat individuals suffering from gastrointestinal disorders involving the upper gastrointestinal tract of the esophagus by administering an effective amount of a GLP-2 analog or a salt thereof as described herein. Gastrointestinal-related disorders include ulcers of any etiology (e.g., peptic ulcers, drug-induced ulcers, ulcers associated with infections or other pathogens), dyspepsia, malabsorption syndromes, short bowel syndrome, blind tube syndrome, inflammatory bowel disease, celiac sprue (e.g., resulting from gluten-sensitive enteropathy or celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, ulcerative colitis, small intestinal damage, and chemotherapy-induced These include chronic obstructive pulmonary disease (COPD), diarrhea, and mucositis (CID).

[0080] As mentioned above, generally, individuals who would benefit from increasing small intestinal mass and thus normalizing and / or maintaining normal small intestinal mucosal structure and function are candidates for treatment with the present GLP-2 analogues.Specific conditions that can be treated with GLP-2 analogues include various forms of sprue: celiac sprue, which is caused by a toxic reaction to α-gliadin by heating and can also be the result of gluten enteropathy or celiac disease, and is characterized by a significant loss of villi in the small intestine; tropical sprue, which is caused by infection and is characterized by partial flattening of villi; hypogammaglobulinemic sprue, which is generally found in patients with common variable immunodeficiency or hypogammaglobulinemia, and is characterized by a significant decrease in villus height.The therapeutic effectiveness of GLP-2 analogue treatment can be monitored by intestinal biopsy to examine villus morphology, by biochemical evaluation of nutrient absorption, by patient weight gain, or by improvement of symptoms associated with these conditions.

[0081] Another particular condition that may be treated with the GLP-2 analogs of the present invention, or for which a GLP-2 analog may be therapeutically and / or prophylactically useful, is short bowel syndrome (SBS), also known as short gut syndrome or simply short bowel syndrome. SBS is characterized by a "short gut" that results from poor intestinal absorption due to surgical resection, congenital defects, or disease, followed by an inability of patients to maintain fluid, electrolyte, and nutritional balance on a normal diet. Although typically adapted 2 years after resection, SBS patients experience reduced food intake and reduced fluid intake.

[0082] Other conditions that may be treated with the GLP-2 analogs of the present invention or for which they may be useful prophylactically include, in addition to the conditions mentioned above, radiation enteritis, infectious or post-infectious enteritis, and small intestinal damage caused by cancer chemotherapeutic or toxic agents.

[0083] GLP-2 analogues may also be used to treat malnutrition, such as cachexia and anorexia. A particular embodiment of the present invention relates to the use of the peptide for the prevention and / or treatment of intestinal damage and dysfunction. Such damage and dysfunction are well-known side effects of cancer chemotherapy treatment. The application of chemotherapy is frequently accompanied by unwanted side effects related to the gastrointestinal system, such as mucositis, diarrhea, bacterial translocation, malabsorption, abdominal cramps, gastrointestinal bleeding, and vomiting. These side effects are the clinical consequences of the structural and functional damage of intestinal epithelium, and often force the reduction of the dosage and frequency of chemotherapy.

[0084] The administration of this GLP-2 peptide analogue can enhance the trophic effect in intestinal crypts and rapidly bring about new cells to replace the intestinal epithelium damaged by chemotherapy.The ultimate goal achieved by the administration of this peptide is to create an optimal chemotherapy regimen for the treatment of cancer, while at the same time reducing the morbidity associated with gastrointestinal damage in patients undergoing chemotherapy treatment.Patients undergoing or about to undergo radiation therapy can also be provided with preventive or therapeutic treatment in accordance with the present invention.

[0085] Stem cells in the small intestinal mucosa have a high rate of proliferation and are therefore particularly susceptible to the cytotoxic effects of chemotherapy (Keefe et al., Gut, 47:632-7, 2000). Chemotherapy-induced damage to the small intestinal mucosa is often clinically referred to as gastrointestinal mucositis and is characterized by intestinal absorption and barrier dysfunction. For example, the widely used chemotherapeutic agents 5-FU, irinotecan, and methotrexate have been shown to increase apoptosis in the rodent small intestine, resulting in villous atrophy and crypt hypoplasia (Keefe et al., Gut, 47:632-7, 2000; Gibson et al., J Gastroenterol. Hepatol. September;18(9):1095-1100, 2003; Tamaki et al., J. Int. Med. Res. 31(1):6-16, 2003). In humans, chemotherapy agents have been shown to increase apoptosis in intestinal crypts 24 hours after administration, followed by a decrease in villus area, crypt length, number of mitoses per crypt, and enterocyte height 3 days after chemotherapy (Keefe et al., Gut, 47:632-7, 2000). Thus, structural changes in the small intestine directly result in intestinal dysfunction and, in some cases, diarrhea.

[0086] Gastrointestinal mucositis following cancer chemotherapy is an even greater problem, and although it gradually abates, once established it is essentially untreatable. Studies conducted with the commonly used cytostatic cancer drugs 5-FU and irinotecan have demonstrated that effective chemotherapy with these drugs primarily affects the structural integrity and function of the small intestine, whereas the colon is less affected, responding primarily with increased mucus formation (Gibson et al., J. Gastroenterol. Hepatol. September;18(9):1095-1100, 2003; Tamaki et al., J Int. Med. Res.31(1):6-16, 2003).

[0087] The formulations of the invention containing GLP-2 analogs may be useful in preventing and / or treating gastrointestinal damage and side effects of chemotherapy agents. This potentially important therapeutic application is indicative of the efficacy and safety of currently used chemotherapy agents, including, but not limited to, 5-FU, altretamine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydrochloride, cyclophosphamide ... Dicarbamide, Idarubicin, Ifosfamide, Irinotecan, Liposomal Doxorubicin, Leucovorin, Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin, Procarbazine, Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide, Thiotepa, Thioguanine / Thioguanine, Topotecan, Treosulfan, vinblastine, vincristine, vindesine, vinorelbine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan The present invention can be applied to the following drugs: cyclosporine, liposomal doxorubicin, leucovorin, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, streptozocin, tegafur-uracil, temozolomide, thiotepa, thioguanine / thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, and vinorelbine. Delivery of the formulation In some embodiments, the present invention relates to ready-to-use formulations of GLP-2 analogs intended for parenteral administration and suitable for use, for example, in vials, pre-filled syringes, infusion pumps, wearable injectors, disposable autoinjectors, or adjustable-dose autoinjectors. EXAMPLES

[0088] The following examples are intended to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. GLP-2 analogs administered according to the dosing regimen described herein may be administered in accordance with any of the methods described in WO2006 / 11331, the contents of which are expressly incorporated by reference in their entirety. They may also be prepared by methods such as solid phase peptide synthesis as described in US Pat. No. 17565.

[0089] Example 1 Synthesis of ZP1848-Acetate and Similar GLP-2 Analogues ZP1848-acetate peptide was synthesized using Fmoc solid phase peptide synthesis (SPPS) technique with standard coupling conditions. After synthesis was completed, the peptide sequence was deprotected, cleaved from the solid support, and the crude peptide was purified using preparative reversed-phase HPLC. The peptide was converted to the desired acetate form by subjecting the mobile phase to a final chromatography step containing an appropriate concentration of acetic acid, followed by lyophilization. The resulting drug substance product had less than 11% acetate content 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 protocol may be modified to produce other GLP-2 analogs used in the formulations of the present invention.

[0090] Example 2 Investigating the formation of covalently linked oligomers in pharmaceutical formulations of the GLP-2 analog ZP1848-acetate material and method Covalently linked oligomers were detected using a Dionex Ultimate3000 HPLC system with a linear gradient at a flow rate of 0.5 ml / min. The mobile phase consisted of 45% acetonitrile and 0.1% TFA in 55% Milli-Q water. A wavelength of 215 nm was used for detection. The injection amount was 4 μg peptide. The column used for the separation of covalently linked peptides was a TSKgel with 4 μm particle size and dimensions of 300*4.6 mm. The HPLC system was a SuperSW2000 (TSK BioScience). The total run time was 25 min. For the evaluation of the chemical stability of peptide monomers, a C18 column with an acidic mobile phase and an acetonitrile gradient was used.

[0091] Stock solutions of mannitol (700 mM), L-histidine (200 mM), and ZP1848 peptide (acetate salt; 60 mg / mL) were prepared in water (Milli-Q). The mannitol and histidine solutions were mixed in appropriate amounts to give 230 mM mannitol and 15 mM histidine. Peptide stock solutions were added to final concentrations of 0.2, 2, and 20 mg / mL, respectively. 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 reach the final volume. Results and Discussion It is known in the art that increasing the concentration of peptide or protein drugs in liquid formulations increases the concentration of dimers, trimers, and higher oligomers as a result of mass action effects that result in a higher probability of covalent reactions (see van Maarschalkerweerd et al., Intrinsically Disord. Proteins. 2015;3(1):e1071302). Thus, the formation of covalently linked high molecular weight degradants (cHMWDPs) increases with drug substance concentration and has the effect of reducing the amount of biologically active monomeric peptide available in the formulation. This was therefore investigated in a formulation of a GLP-2 analog, ZP1848-acetate.

[0092] A typical chromatogram for the separation of oligomers from ZP1848-acetate monomer is shown in Figure 1. The oligomers of ZP1848-acetate are well separated from the ZP1848-acetate monomer and all cluster together as one peak. The peak area percentages were used to quantitate the amount of oligomers, especially the covalently linked dimers and trimers.

[0093] The same formulations containing 0.2, 2, and 20 mg / mL ZP1848 were Analyzed after 24 months of storage at 8°C. Mainly dimers (two ZP1848-acetate molecules covalently linked) formed, but some trimers were also present (verified 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%. Initial oligomer content was less than 0.1%.

[0094] [Table 1]

[0095] Surprisingly, it was found that during long-term storage of ZP1848-acetate (grepaglutide) at 2-8°C, the formation of covalently linked oligomers was concentration dependent, but contrary to common expectations, the concentration dependence of oligomer formation was inversely dependent on increasing concentrations of GLP-2 analogues. Without wishing to be bound by any particular theory, the inventors believe that the decrease in the formation of covalently linked oligomers with increasing drug concentration is the result of the lysine tails of the GLP-2 analogues promoting competitive reactions that result in the formation of higher order species in which the GLP-2 analogue molecules are weakly associated together rather than covalently linked. This means that these weakly associated species can dissociate to release biologically active monomers, rather than causing some decrease in activity as occurs when covalently linked oligomers form.

[0096] Example 3 Screening of buffer solutions for formulation of GLP-2 analog ZP1848 acetate A study was conducted to investigate the effect of various buffer salts on the stability of ZP1848-acetate (4 mg / mL) formulations. The total buffer concentration in the formulation was 20 mM. material and method The buffers listed in Table 2 below were prepared. The pH of the buffers was adjusted with 1M HCl / 1M NaOH. ZP1848 peptide (acetate salt) was dissolved in the buffer at 80% of the final sample volume to give 4mg / mL in the final formulation. If necessary, the pH was then adjusted to the desired formulation pH using either 200mM acetic acid or 100mM L-arginine. Buffer was added to reach the final volume. Each formulation was filled into the appropriate vial (1ml / vial) for stability testing. Results and Discussion Appearance indicated that all formulations containing citrate, citrate / Tris, or succinate buffers were viscous and / or cloudy (see Table 2). Acetate buffer (20 mM, pH 5), mesylate buffer (20 mM, pH 6), histidine buffer (15 mM, pH 7), and histidine-arginine (15+5 mM, pH 7) yielded formulations that passed visual inspection as clear and non-viscous.

[0097] [Table 2]

[0098] Example 4 Incompatibility of phosphate buffers with the GLP-2 analog ZP1848-acetate material and method Stock solutions of mannitol (700 mM), phosphate buffer (200 mM), and ZP1848-acetate peptide (60.2 mg / mL) were prepared in water (Milli-Q). The stock solutions were mixed in appropriate amounts to obtain the formulations shown in Table 3 below. 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 until the final volume was reached. After 24 hours at room temperature, the sample containers were visually inspected for clarity and viscosity.

[0099] [Table 3]

[0100] Results and Discussion Visual inspection showed that formulations of 20 mg / mL ZP1848-acetate, pH 6.5-7.5, containing 20-50 mM phosphate buffer were cloudy and / or highly viscous after 24 hours at room temperature. Thus, phosphate buffer was determined to be incompatible with ZP1848-acetate in these formulations.

[0101] Example 5 Effect of acetate content on the viscosity of formulations of the GLP-2 analog ZP1848-acetate A study was conducted to determine the effect of acetate content on the viscosity of ZP1848-acetate formulations. material and method Samples were prepared using the drug substance (DS) of the GLP-2 analog ZP1848-acetate containing 6% acetate. Acetate was added to explore the effect of increasing acetate content ranging from 7.8 to 15% acetate (see Table 4).

[0102] Stock solutions of mannitol (700 mM), acetic acid (1000 mM), histidine (200 mM), and ZP1848-acetate peptide (60 mg / ml) were prepared in Milli-Q water. The stock solutions were mixed in appropriate amounts to obtain the formulations shown in Table 4 below. Water was added to 90% of the final volume. If necessary, the pH was then adjusted to the desired formulation pH using 250 mM arginine. Water was added to reach the final volume. Each formulation was filled into appropriate vials for stability testing.

[0103] Vials were visually inspected for clarity and viscosity. Viscosity was measured using a MicroVISC™ viscometer. Hydrodynamic radius was measured using a Wyatt DynaPro II dynamic light scattering (DLS) plate reader. Sample size on plate was 170 μl.

[0104] [Table 4]

[0105] Results and Discussion The viscosity and hydrodynamic radius of formulations with various acetate concentrations are shown in Figure 2. The results show that the viscosity of ZP1848-acetate formulations increases unexpectedly and nonlinearly with higher acetate concentrations. Thus, a total acetate concentration in the formulation of 11% acetate per mg of GLP-2 analogue or less opens up the possibility of providing a formulation of the GLP-2 analogue in the form of a drug delivery device, in which the total acetate concentration in the formulation is in this case favorable for controlling the viscosity at a low / unchanged level.

[0106] Example 6 Effect of buffer salts on the stability of formulations of the GLP-2 analog ZP1848-acetate at 2 and 20 mg / mL A study was conducted to determine the effect of various buffer salts on the stability of ZP1848-acetate (2 and 20 mg / mL) formulations. All buffers were at 15 mM concentration. material and method Mannitol (700 mM), L-histidine (200 mM), glycine (400 mM), lysine (200 mM), Tris (200 mM), Bis-Tris (200 mM), MOPS (100 mM), succinic acid (200 mM), MES (2-(N-morpholino)ethanesulfonic acid) (200 mM), mesylate (200 mM), phosphate (200 mM), and ZP1848 peptide (acetate salt; approximately 50 mg / ml) stock solutions were prepared in water (Milli-Q). Appropriate amounts of excipient solutions were mixed to obtain the formulations shown in Tables 5 and 6 below. All formulations contained 230 mM mannitol and 15 mM buffer. Peptide stock solution was added. Water was added to 90% of the final volume. If necessary, the pH was adjusted to pH 7 using 1M acetic acid / 0.5M L-arginine. Water was added to reach the final volume. The formulation was filled into vials and placed on stability testing at 40°C. Results and Discussion Based on the observed results, with regard to buffers, 15 mM concentrations of histidine, glycine, lysine, Tris, Bis-Tris, MOPS, mesylate, and MES were acceptable for use in the ZP1848-acetate formulations at 2 mg / mL and 20 mg / mL peptide and pH 7.0.

[0107] [Table 5]

[0108] [Table 6]

[0109] Covalent oligomer formation was evaluated in various buffers (Table 6). At 20 mg / mL, succinic acid formed a gel after 1 week, and stability could not be assessed at 2 and 3 weeks. In the same buffer, the rate of covalent oligomer formation was significantly higher at 2 mg / mL (2.1%). The overall trend was that after 3 weeks of accelerated storage at 40° C., the rate of covalent oligomer formation was higher in the 2 mg / mL formulation compared to the 20 mg / mL.

[0110] Phosphate and succinate buffers were incompatible with ZP1848-acetate at 2 and 20 mg / mL in these formulations. The stability of the peptide monomer was evaluated by determining the HPLC purity for a 3 week stability at 40° C. The results are shown in FIG. 3 and Table 7. At 20 mg / mL succinic acid, due to the gel formation mentioned above, only the first time point could be evaluated. At 2 mg / mL, results could be obtained for the 3 week study. Only minor, non-significant differences were detected between the buffers evaluated. Thus, the choice of buffer does not appear to affect the stability of the peptide monomer.

[0111] [Table 7]

[0112] Example 7 Effect of tonicity modifiers on the stability of formulations of the GLP-2 analog ZP1848-acetate at 2 and 20 mg / mL A study was conducted to investigate the effect of various tonicity modifiers on the stability of ZP1848-acetate (2 and 20 mg / mL) formulations. material and method Stock solutions of L-histidine (200 mM), sucrose (730 mM), glycerol (977 mM), D-sorbitol (801 mM), anhydrous D-(+) trehalose (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 obtain the formulations shown in Table 8 below. All formulations contained 15 mM histidine. Peptide stock solutions were added as necessary to obtain the peptide content shown in Table 8. Water was added to 90% of the final volume. The pH was adjusted to pH 7 using 1 M acetic acid / 0.5 M L-arginine as necessary. Water was added to reach the final volume. Each formulation was filled into a vial and placed in a stability study at 40° C. The sample vessels were visually inspected for clarity and viscosity, and analyzed for hydrodynamic radius by DLS (Dynamic Light Scattering) analysis. Results and Discussion According to the observed results shown in Table 8, mannitol, sucrose, glycerol, sorbitol, and trehalose were acceptable for use in these formulations containing ZP1848-acetate at 2 mg / mL and 20 mg / mL at pH 7.0.

[0113] [Table 8]

[0114] The formation of covalent oligomers was measured for formulations 1-10 at 40°C for up to 3 weeks. The results are shown in Table 9. Differences in the formation of covalent oligomers for the 10 formulations were already seen after 1 week of stability testing. Furthermore, the rate (slope) was almost constant throughout the test period. Formulation 3 (20 mg / mL-glycerol), formulation 7 (2 mg / mL-sucrose), formulation 8 (2 mg / mL-glycerol), and formulation 9 (2 mg / mL-sorbitol) had significantly higher rates of covalent oligomer formation than the other tablets. Mannitol showed the lowest rate of covalent oligomer formation. The overall trend was that for all isotonicity agents studied, the 2 mg / mL formulation had a higher rate of covalent oligomer formation compared to the 20 mg / mL formulation.

[0115] [Table 9]

[0116] The stability of the peptide monomers was evaluated by determining the HPLC purity for a 3 week stability at 40° C. Similar to the covalently linked oligomers, the chemical stability is poor with glycerol and deviates from other tonicity agents. The results are shown in Figures 5 and 6.

[0117] Example 8 Effect of the physical stability of acids and bases used to adjust the pH of formulations of the GLP-2 analog ZP1848-acetate material and method Stock solutions of mannitol, histidine, and ZP1848-acetate peptide in water were prepared. The mannitol and histidine stock solutions were added to water, mixed, and peptide solution was added to obtain a final peptide content of 10 mg / mL. Water was added to 90% of the final volume. The pH was then adjusted to pH 7 using 250 mM arginine / 1 M AcOH or 1 M NaOH / 1 M HCl (see Table 10). Water was added to reach the final volume. Each formulation was filled into vials for stability testing and placed in stability tests at 5° C., 25° C., and 40° C. Sample containers were visually inspected for clarity and viscosity. Results and Discussion The results shown in Table 10 indicate that the use of 1M NaOH / 1M HCl for pH adjustment has a detrimental effect on the physical stability of ZP1848-acetate formulations.

[0118] [Table 10]

[0119] Example 9 Use of ZP1848 peptide acetate and ZP1848 peptide chloride salts for the formulation of the GLP-2 analog ZP1848 peptide A study was conducted to examine the effect of salt using ZP1848 peptide acetate and ZP1848 peptide hydrochloride in selected ZP1848 formulations. The effect of salt type, concentration, buffer, and tonicity modifier was examined after accelerated storage at 40° C. Synthesis of ZP1848 peptide sodium salt was attempted but did not prove possible. material and method 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) were prepared in Milli-Q water. The excipient solutions were mixed in appropriate amounts to obtain the formulations shown in Tables 11 and 12 below. The peptide stock solutions were added to obtain the desired final peptide content. Water was added to 90% of the final volume. If necessary, the pH was adjusted to the desired formulation pH using 1 M acetic acid / 0.5 M L-arginine. Water was added until the final volume was reached. Each formulation was filled into vials and placed into a stability test at 40° C. Sample containers were visually inspected for clarity and viscosity and analyzed for hydrodynamic radius by DLS. Results and Discussion The results shown in Tables 11 and 12 indicate that the Z-average, viscosity, and appearance of Formulations 1, 2, 3, and 4 did not change in stability as assessed by appearance and DLS after 3 weeks at 40° C. Formulation 5 showed a change in stability over time as assessed by Z-average, viscosity, and appearance.

[0120] [Table 11]

[0121] [Table 12]

[0122] The chemical stability of formulations 1-5 was followed for up to 4 weeks at 40°C. 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 a slightly decreased but acceptable chemical stability after 4 weeks, which may be due to the low concentration of this formulation.

[0123] The formation of covalently linked oligomers was measured for formulations 1-5 at 40°C for up to 4 weeks. The results are shown in Table 13. Differences in the formation of covalently linked oligomers for the five formulations were observed even after 1 week of stability testing. Furthermore, the rate (slope) remained almost constant throughout the testing period. Formulation 1 (20 mg / mL, acetate salt of ZP1848, histidine as tonicity agent) was the most stable formulation, with covalently linked oligomer formation of approximately 1.1% after 4 weeks of stability at 40°C. Formulation 5 (20 mg / mL, chloride salt of ZP1848, histidine as tonicity agent) was the most stable formulation, with covalently linked oligomer formation of approximately 1.1% after 4 weeks of stability at 40°C. The second most stable formulation was formulation 3 (2 mg / mL, ZP1848 chloride salt, histidine as tonicity agent) with a covalent oligomer formation rate of approximately 2.1% after 4 weeks of stability at 40°C. The third most stable was formulation 2 (20 mg / mL, ZP1848 chloride salt, histidine as tonicity agent). The fourth most stable was formulation 2 (20 mg / mL, ZP1848 chloride salt, sorbitol as tonicity agent). The least stable formulation was formulation 4 (20 mg / mL, ZP1848 chloride salt, sorbitol as tonicity agent).

[0124] It was previously noted that the acetate salt of ZP1848 tends to be slightly less stable with sorbitol after 3 weeks at 40° C. compared to mannitol (0.9% for mannitol and 1.1% for sorbitol). See Example 7. The difference between the sorbitol- and mannitol-containing formulations is more pronounced when comparing the acetate salt with the chloride salt, where the rate of covalently linked oligomers formation for a 20 mg / mL formulation containing chloride and sorbitol is about 3.9%. When comparing 2 mg / mL and 20 mg / mL formulations of the chloride salt, after 4 weeks at 40° C., the formation of covalently linked oligomers was 2.4% for 2 mg / mL (increase of 0.53% per week) and 3.3% for 20 mg / mL (increase of 0.75% per week). This is surprising, since this is not observed with the acetate salt. At 20 mg / mL acetate, after 3 weeks at 40°C, the formation of covalent oligomers was 0.9% (an increase of 0.21% per week) compared to 1.2% at 2 mg / mL (an increase of 0.27% per week). This higher rate of covalent oligomer formation at lower acetate concentrations is consistent with what is observed during long-term stability. However, for the chloride salt, the situation was reversed, with the rate of covalent oligomer formation increasing with increasing ZP1848 concentration.

[0125] [Table 13]

[0126] Example 10 Use of ZP1848 peptide acetate and preservatives for formulation of GLP-2 analog ZP1848 peptide at 20 mg / mL A study was conducted to determine the compatibility of ZP1848 peptide acetate with commonly used preservatives. The effect of preservatives and temperature was investigated after accelerated storage. material and method Stock solutions of mannitol (700 mM), histidine (200 mM), and ZP1848 peptide solution (acetate salt; approximately 50 mg / mL) were prepared in Milli-Q water. The final peptide concentration was 20 mg / mL, with mannitol at 230 mM and histidine at 15 mM. The preservative solutions were mixed in appropriate amounts to obtain the formulations shown in Table 14 below. Peptide stock solutions were added to obtain the desired final peptide content. Water was added to 90% of the final volume. If necessary, the pH was then adjusted to the desired preparation using 1M acetic acid / 0.5M L-arginine. The pH of the formulation was adjusted to 0.05. Water was added to reach the final volume. Each formulation was filled into a vial. Sample containers were visually inspected for clarity and viscosity and analyzed by SEC for covalently attached oligomers and by HPLC for peptide monomer stability. Results and Discussion The results from this study are set forth in Table 14, Table 15, and Figure 8. The formulation appears to be unaffected by the addition of preservatives compared to Formulation 1, which has no added preservatives.

[0127] Chemical stability was evaluated by determining the stability (purity) of the covalently linked oligomers and peptide monomers. Formulation 4 (potassium sorbate) had a higher rate of covalently linked oligomer formation, but within acceptable limits. All other formulations have similar amounts of covalently linked oligomers. Normalized purity after 13 weeks at 25° C. shows that ZP1848-acetate also has similar stability, with formulation 4 having a slightly reduced but acceptable purity.

[0128] [Table 14]

[0129] [Table 15]

[0130] Example 11 Use of ZP1848 peptide acetate and preservatives for GLP-2 analog ZP1848 peptide formulations at 2 and 20 mg / mL material and method Stock solutions of mannitol (700 mM), histidine (200 mM), and ZP1848 peptide solution (acetate salt; approximately 50 mg / mL) were prepared in Milli-Q water. Final concentrations of peptides were 20 and 2 mg / mL, mannitol was 230 mM, and histidine was 15 mM. 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 using 1 M acetic acid / 0.5 M L-arginine. The viscosity was adjusted to (7.0). Water was added to reach the final volume. Each formulation was filled into a vial. Sample containers were visually inspected for clarity and viscosity and analyzed by HPLC for peptide monomer stability. Results and Discussion The results from this study are set forth below in Table 16. All formulations were tested for long-term stability for 52 weeks at 5° C. All test solutions remained clear and non-viscous throughout the study period.

[0131] [Table 16]

[0132] HPLC evaluation of ZP1848-acetate after accelerated stabilization at 25 °C is shown in Figure 9. Formulations containing phenol showed a slight decrease in chemical stability, while m-cresol gave similar chemical stability to the unpreserved formulation. For long-term stability at 5 °C, no obvious differences were observed between samples after 12 months of stability, with all samples having normalized ZP1848-acetate purity greater than 94% (data not shown). Thus, all formulations studied are stable for at least 52 weeks of long-term stability.

[0133] Although the present invention has been described with the above embodiments, numerous equivalent modifications and variations will become apparent to those skilled in the art given this disclosure. Accordingly, the described embodiments of the present invention are considered to be illustrative and not limiting. Various modifications to the described embodiments can be made without departing from the spirit and scope of the present invention. All documents cited herein are expressly incorporated by reference in their entirety for all purposes.

Claims

1. 1. A stable aqueous pharmaceutical formulation comprising a pharma- ceutically acceptable acetate salt of a glucagon-like peptide-2 (GLP-2) analog, the GLP-2 analog being: ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ２ (SEQ ID NO: 1) and The formulation comprises: a pharma- ceutically acceptable acetate salt of a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL; Histidine buffer at a concentration of 15 mM; Mannitol at a concentration of 90 mM to 360 mM; and Arginine in an amount sufficient to obtain a pH of about 7.0 Including, The total acetate concentration in the formulation is 11% acetate / mg GLP-2 analogue or less; The formulation.

2. 2. The formulation of claim 1, wherein the arginine is L-arginine / acetate.

3. 3. The formulation according to claim 1 or claim 2, wherein the mannitol is D-mannitol.

4. The formulation according to any one of claims 1 to 3, for administration to a subject by injection.

5. The formulation of claim 4, wherein the injection is a subcutaneous injection.

6. 6. The formulation of any one of claims 1 to 5, which is stable at 2-8°C for at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

7. 7. The formulation of claim 6, wherein the GLP-2 analogue in the formulation retains at least 90% of its biological activity after 18 months of storage at 2-8°C.

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