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

Abstract

The present application relates to formulations of glucagon-like peptide 2 (GLP-2) analogs. A liquid formulation of a GLP-2 analogue is described that makes the GLP-2 analogue suitable for long term storage as a liquid and / or makes the GLP-2 analogue particularly suitable for delivery through a drug delivery device. Also described are solid compositions comprising an acetate of a glucagon-like peptide 2 (GLP-2) analogue useful in the preparation of a liquid formulation. The development of these liquid formulations is based on the discovery that acetate salts present in formulations derived from GLP-2 analogs have an effect on the viscosity of said formulations: during long-term storage of the GLP-2 analogs at 2 to 8 DEG C, the concentration dependency of covalent oligomer formation depends negatively on an increase in the concentration of the GLP-2 analogs; and the GLP-2 analogs used in the formulations are incompatible with phosphate buffers commonly used for reconstituted powdery or lyophilized GLP-2 compositions in the prior art.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 201980063594.1 and the invention name of "Formulations of Glucagon-like Peptide 2 (GLP-2) Analogs". The original application is the application that entered the Chinese national phase on March 26, 2021, of the PCT international application PCT / EP2019 / 076305 filed on September 27, 2019. Technical Field

[0002] The present invention relates to formulations of glucagon-like peptide 2 (GLP-2) analogs and their medical use, for example, in the treatment and / or prevention of stomach and intestine-related disorders and for improving the side effects of chemotherapy and radiotherapy. In addition, solid compositions comprising an acetate of a glucagon-like peptide 2 (GLP-2) analog that can be used to prepare liquid formulations are also described. Background Art

[0003] Human GLP-2 is a 33-amino acid peptide with the following sequence:

[0004] 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.

[0005] It is derived from the specific post-translational processing of proglucagon in intestinal enteroendocrine L cells of the intestine and specific regions of the brainstem. GLP-2 binds to a single G protein-coupled receptor belonging to the class II glucagon secretin family.

[0006] It has been reported that GLP-2 induces significant growth of the small intestinal mucosal epithelium by stimulating stem cell proliferation in the crypts and by inhibiting apoptosis in the villi (Drucker et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7911-7916). GLP-2 also has a growth effect on the colon. In addition, GLP-2 inhibits gastric emptying and gastric acid secretion (Wojdemann et al., 1999, J. Clin. Endocrinol. Metab. 84: 2513-2517), enhances intestinal barrier function (Benjamin et al., 2000, Gut 47: 112-119), stimulates intestinal hexose transport by upregulation of glucose transporters (Cheeseman, 1997, Am. J. Physiol. R1965-71), and increases intestinal blood flow (Guan et al., 2003, Gastroenterology, 125: 136-147).

[0007] In the art, it has been recognized that glucagon-like peptide-2 receptor analogs have therapeutic potential for treating enteropathy. However, native hGLP-2 (a 33-amino acid gastrointestinal peptide) is not available in a clinical setting due to its very short half-life in humans (about 7 minutes for full-length GLP-2 [1-33] and 27 minutes for truncated GLP-2 [3-33]). To a large extent, the short half-life is due to degradation by the enzyme dipeptidylpeptidase IV (DPP-IV). Thus, attempts have been made in the art to develop GLP-2 receptor agonists with better pharmacokinetic properties, particularly to improve the half-life of the GLP-2 molecule. For example, GLP-2 analogs with substitutions have been proposed, such as a GLP-2 analog containing 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. It has also been demonstrated that acylation of peptide drugs with fatty acid chains is beneficial for prolonging systemic circulation and increasing enzyme stability without disrupting biological potency. However, although these attempts have improved the pharmacokinetics of GLP-2 analogs, and they are sometimes described in the art as "long acting", it must be remembered that this is still in the order of hours rather than minutes compared to native hGLP-2. In turn, this means that GLP-2 analogs still need to be administered to patients once or more daily.

[0008] US 5,789,379 discloses GLP-2 analogs for administration by injection. The analogs are provided as powdered peptides and are mixed with phosphate buffered saline (PBS) at a GLP-2 concentration of 130 mg / ml at pH 7.3 to 7.4 prior to injection. In some cases, the GLP-2 / PBS composition is mixed with gelatin to provide a depot formed from a solution of 130 mg / 1 GLP-2 in PBS / 15% gelatin. US 5,789,379 does not disclose a stable aqueous liquid formulation of the GLP-2 analog, and the GLP-2 analog is generally reconstituted from powder prior to injection.

[0009] In WO 97 / 39031 and US 6,184,201, the GLP-2 analog [Gly 2 GLP-2 is disclosed. Here, the alanine at position 2 has been replaced by glycine such that the peptide is resistant to DPPIV cleavage. As with US 5,789,379, the GLP-2 analog is provided as a powdered peptide and is mixed with saline, PBS, or 5% dextrose prior to injection, optionally with the addition of acetic acid as a solubility enhancer.

[0010] WO 02 / 066511 describes GLP-2 analogs having an extended half-life in vivo and their use as a medicament in the treatment of gastrointestinal disorders such as inflammatory bowel disease. The GLP-2 analogs are stored in lyophilized form and reconstituted for administration in a medium such as using saline or PBS.

[0011] WO 01 / 41779 describes the use of h[Gly 2 GLP-2 as a pretreatment for inhibiting chemotherapy-induced apoptosis and promoting cell survival. h[Gly 2 GLP-2 is delivered by subcutaneous or intravenous injection or infusion after reconstituting the analog in PBS.

[0012] WO 2001 / 049314 relates to formulations of GLP-2 peptides and their analogs which exhibit excellent stability after storage and / or exposure to elevated temperatures. The GLP-2 composition comprises a GLP-2 peptide or its analog, a phosphate buffer, L-histidine, and mannitol.

[0013] WO 2006 / 117565 describes GLP-2 analogs which are 2The GLP-2 compared to contains one of multiple substitutions, and it has improved in vivo bioactivity and / or improved chemical stability, such as as evaluated in in vitro stability assays. In particular, GLP-2 analogs are described that have a substitution at one or more of positions 8, 16, 24, and / or 28 of the wild-type GLP-2 sequence, optionally in combination with one or more additional substitutions at positions 3, 5, 7, 10, and 11 and / or a deletion of one or more of amino acids 31 to 33 at position 2. These substitutions can also be combined with the addition of an N-terminal or C-terminal stabilizing peptide sequence. The administration of these GLP-2 analogs once daily or twice daily is also described. Among the molecules disclosed in WO 2006 / 117565, glepaglutide (ZP1848) has been designed to be stable in liquid formulations and is typically administered by once-daily dosing using an injection pen.

[0014] Improving the formulation of GLP-2 analogs, particularly providing a stable liquid formulation capable of long-term storage without excessive physical or chemical degradation of the active monomeric form of the peptide, remains a problem in the art. In liquid formulations of peptide drugs, the chemical pathways that can occur include the formation of covalently linked dimers and oligomers of the peptide, reducing the amount of the active monomeric form of the peptide by forming these covalently linked high-molecular-weight oligomeric products. The law of mass action means that generally, the higher the concentration of the peptide drug in the formulation, the higher the likelihood of forming covalently bonded oligomeric products.

[0015] It is also an objective in the field of GLP-2 analog formulation to provide a formulation in which the viscosity of the formulation is controlled within a range suitable for use in a delivery device (such as a prefilled syringe, infusion pump, wearable syringe, or autoinjector). Summary of the Invention

[0016] Broadly speaking, the present invention is based on studies that have led to some unexpected findings related to liquid formulations of GLP-2 analogs, the liquid formulations making the GLP-2 analogs suitable for long-term storage as a liquid and / or making the GLP-2 analogs particularly suitable for delivery by a drug delivery device.

[0017] In a first study, the inventors found that the acetate salt derived from the GLP-2 analogue present in the formulation has an effect on the viscosity of the formulation. This has opened up the possibility of controlling the viscosity of the formulation by varying and / or controlling the acetate concentration. Liquid formulations with a low range of viscosities can be used clinically as they offer advantages in the development and manufacture of drug delivery devices by potentially reducing breakage, dosing failures, dosing inaccuracies and other malfunctions during the manufacture of the drug product and / or patient use. In addition, the low viscosity may allow for faster injection and / or the use of needles with a narrower bore (i.e., higher gauge), which in turn can reduce injection discomfort. This has opened up the possibility of providing GLP-2 analogue formulations in the form of drug delivery devices (such as prefilled syringes, adjustable-dose autoinjectors, disposable autoinjectors, wearable syringes or infusion pumps), thereby providing ready-to-use formulations for patients in a simpler, safer and more patient-friendly device. Controlling the formulation to a higher viscosity may be applicable to other drug delivery devices.

[0018] In a second study, the inventors found that during the long-term storage of ZP1848 (glepaglutide) at 2 to 8 °C, the formation of covalently bound oligomers is concentration-dependent. However, contrary to the usual situation where the law of mass action implies that the formation of covalent oligomers increases with increasing peptide drug concentration, the inventors found that the concentration-dependence of oligomer formation negatively depends on the increasing GLP-2 analogue concentration. Without wishing to be bound by any particular theory, the inventors believe that the decrease in the formation of covalently linked oligomers with increasing GLP-2 analogue concentration is the result of the lysine tail of the GLP-2 analogue promoting the formation of self-association structural assemblies of the native peptide, which hinders the formation of covalently bound oligomers in the formulation. This means that the weakly self-associating material is able to dissociate after administration to the patient to release bioactive monomers rather than resulting in a loss of active substance, as occurs when covalently bound oligomers are formed.

[0019] In a third study, the inventors found that the GLP-2 analogues used in the formulations of the present invention are incompatible with the commonly used phosphate buffers for reconstituted powdered or lyophilized GLP-2 compositions in the prior art. This study found that only some buffers are compatible with formulating these GLP-2 analogues such that they are suitable for long-term storage in liquid form.

[0020] Accordingly, in a first aspect, the present invention provides a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the formula:

[0021] 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

[0022] Wherein:

[0023] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

[0024] X5 is Ser or Thr;

[0025] X11 is Ala or Ser;

[0026] R 2 is NH2 or OH; and

[0027] Z 1 and Z 2 independently do not exist, or are peptide sequences having 1 to 6 Lys amino acid units;

[0028] Wherein the preparation comprises:

[0029] (a) a GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL;

[0030] (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, the buffer being present at a concentration of about 5 mM to about 50 mM;

[0031] (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0032] (d) an appropriate amount of arginine to provide a preparation having a pH of about 6.6 to about 7.4.

[0033] In some embodiments, the formulation comprises 5% or less of the GLP-2 analog in the form of a covalently bonded oligomeric product. As an alternative or addition, the total acetate concentration produced by the GLP-2 analog in the formulation is less than or equal to 11% acetate / mg GLP-2 analog. As an alternative or addition, the formation of covalently linked oligomers of the GLP-2 analog is negatively dependent on the concentration of the GLP-2 analog in the formulation.

[0034] The components of the formulation and their amounts provide the formulation with at least 90% content of the GLP-2 analog and less than 10% of chemical degradation products when stored at 2 to 8 °C for at least 18 months.

[0035] In another aspect, the present invention provides an article or kit comprising a container containing the stable pharmaceutical formulation of the present invention.

[0036] In another aspect, the present invention provides a delivery device comprising a liquid formulation containing the GLP-2 analog of the present invention.

[0037] In another aspect, the present invention provides a formulation of the glucagon-like peptide 2 (GLP-2) analog of the present invention for therapeutic use.

[0038] In another aspect, the present invention provides a formulation of the glucagon-like peptide 2 (GLP-2) analog of the present invention for use in a method of treating and / or preventing stomach and intestine related disorders in a human patient.

[0039] In another aspect, the present invention provides a method for producing a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analog is represented by the following formula:

[0040] 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

[0041] Wherein:

[0042] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0043] X5 is Ser or Thr

[0044] X11 is Ala or Ser

[0045] R 2 is NH2 or OH;

[0046] Z 1 and Z 2 independently do not exist, or is a peptide sequence having 1 to 6 Lys amino acid units;

[0047] Wherein the method comprises formulating (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL with the following: (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, the buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol, and trehalose, the non-ionic tonicity regulator being present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0048] Wherein the formulation contains 5% or less of the GLP-2 analog in the form of a covalently bonded oligomeric product.

[0049] In another aspect, the present invention provides the use of a formulation comprising a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof for providing a liquid pharmaceutical formulation that is stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analog is represented by the following formula:

[0050] 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

[0051] Wherein:

[0052] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl, or trifluoroacetyl

[0053] X5 is Ser or Thr

[0054] X11 is Ala or Ser

[0055] R 2 is NH2 or OH;

[0056] Z 1 and Z 2 independently do not exist, or are a peptide sequence having 1 to 6 Lys amino acid units;

[0057] wherein the preparation comprises:

[0058] (a) a GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL;

[0059] (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, the buffer being present at a concentration of about 5 mM to about 50 mM;

[0060] (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0061] (d) an appropriate amount of arginine to provide a preparation having a pH of about 6.6 to about 7.4.

[0062] In another aspect, the present invention provides a method for regulating the viscosity of a stable liquid pharmaceutical preparation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the following formula:

[0063] 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

[0064] wherein:

[0065] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0066] X5 is Ser or Thr

[0067] X11 is A1a or Ser

[0068] R 2 is NH2 or OH;

[0069] Z 1 and Z 2 independently do not exist, or is a peptide sequence having 1 to 6 Lys amino acid units;

[0070] wherein the method comprises formulating (a) a GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL together with: (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer or MOPS buffer, said buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, said non-ionic tonicity regulator being present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0071] wherein the total acetate concentration in the formulation produced by the GLP2 analogue is less than or equal to 11% acetate / mg GLP-2 analogue, and wherein the viscosity of the formulation measured at 25 °C is greater than 0.8 and less than or equal to 2.0 mPa / sec.

[0072] In another aspect, the present invention provides a method for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue in a stable liquid pharmaceutical formulation, said liquid pharmaceutical formulation comprising a GLP-2 analogue represented by the following formula or a pharmaceutically acceptable salt or derivative thereof:

[0073] 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

[0074] wherein:

[0075] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0076] X5 is Ser or Thr

[0077] X11 is Ala or Ser

[0078] R 2 is NH2 or OH;

[0079] Z 1 and Z 2 independently do not exist, or are a peptide sequence having 1 to 6 Lys amino acid units;

[0080] wherein the method comprises formulating (a) a GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL with: (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer or MOPS buffer, the buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity modifier selected from mannitol, sucrose, glycerol, sorbitol and trehalose, the non-ionic tonicity modifier being present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0081] wherein the formulation contains 5% or less of the GLP-2 analogue in the form of a covalently bonded oligomeric product. In some cases, in this aspect of the invention, the formation of covalently linked oligomers of the GLP-2 analogue is negatively dependent on the concentration of the GLP-2 analogue in the formulation.

[0082] In another aspect, the present invention provides the use of a formulation for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof in a liquid pharmaceutical formulation, the liquid pharmaceutical formulation being stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analogue is represented by the formula:

[0083] 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

[0084] wherein:

[0085] R1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0086] X5 is Ser or Thr

[0087] X11 is Ala or Ser

[0088] R 2 is NH2 or OH;

[0089] Z 1 and Z 2 independently do not exist, or are peptide sequences having 1 to 6 Lys amino acid units;

[0090] wherein the preparation comprises:

[0091] (a) a GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL;

[0092] (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, and the buffer is present at a concentration of about 5 mM to about 50 mM;

[0093] (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0094] (d) an appropriate amount of arginine to provide a preparation with a pH of about 6.6 to about 7.4;

[0095] wherein the preparation contains 5% or less of the GLP-2 analog in the form of a covalently bonded oligomeric product. In some cases, in this aspect of the present invention, the formation of the covalently linked oligomer of the GLP-2 analog negatively depends on the concentration of the GLP-2 analog in the preparation.

[0096] In another aspect, the present invention provides the use of a preparation for regulating the viscosity of a liquid pharmaceutical preparation in a liquid pharmaceutical preparation comprising a glucagon-like peptide 2 (GLP-2) analog or a pharmaceutically acceptable salt or derivative thereof, and the liquid pharmaceutical preparation is stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analog is represented by the following formula:

[0097] 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

[0098] Wherein:

[0099] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0100] X5 is Ser or Thr

[0101] X11 is Ala or Ser

[0102] R 2 is NH2 or OH;

[0103] Z 1 and Z 2 independently do not exist, or are peptide sequences having 1 to 6 Lys amino acid units;

[0104] Wherein the preparation comprises:

[0105] (a) A GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL;

[0106] (b) A buffer selected from 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;

[0107] (c) A non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0108] (d) An appropriate amount of arginine to provide a preparation with a pH of about 6.6 to about 7.4;

[0109] Wherein the total acetate concentration produced by the GLP2 analogue in the preparation is less than or equal to 11% acetate / mg GLP-2 analogue, and the viscosity of the preparation measured at 25 °C is 0.8 to 2.0 mPa / s.

[0110] In another aspect, the present invention provides a solid composition comprising an acetate of a glucagon-like peptide 2 (GLP-2) analogue having the following formula:

[0111] (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH), where x is from 1.0 to 8.0.

[0112] In another aspect, the present invention provides a stable aqueous pharmaceutical formulation comprising:

[0113] (a) The solid composition of the present invention at a concentration of about 2 mg / mL to about 30 mg / mL;

[0114] (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;

[0115] (c) A non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0116] (d) An appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0117] wherein the formulation comprises 5% or less of the GLP-2 analogue in the form of covalently bonded oligomeric products, and wherein the viscosity of the formulation measured at 25 °C is 0.8 to 2.0 mPa / s.

[0118] In all aspects of the present invention described herein, the buffer can be selected from histidine buffer, mesylate buffer, and acetate buffer.

[0119] In all aspects of the present invention described herein, the non-ionic tonicity regulator can be selected from mannitol, sucrose, glycerol, and sorbitol.

[0120] In some embodiments, the formulation comprises 5% or less of the GLP-2 analogue in the form of covalently bonded oligomeric products. As an alternative or in addition, the total acetate concentration in the formulation produced by the GLP-2 analogue is less than or equal to 11% acetate / mg GLP-2 analogue. As an alternative or in addition, the formation of covalently linked oligomers of the GLP-2 analogue is negatively dependent on the concentration of the GLP-2 analogue in the formulation.

[0121] In another aspect, the present invention relates to a stable liquid pharmaceutical formulation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the following formula:

[0122] R 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

[0123] Wherein:

[0124] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

[0125] X5 is Ser or Thr;

[0126] X11 is Ala or Ser;

[0127] R 2 is NH2 or OH; and

[0128] Z 2 is a peptide sequence having 6 Lys amino acid units;

[0129] The formulation comprises the components listed in any aspect of the present invention as listed herein.

[0130] In this aspect of the invention, a preparation comprising a glucagon-like peptide 2 (GLP-2) analogue or a salt thereof can be used for treating and / or preventing stomach- and intestine-related disorders, such as ulcers, digestive disorders, malabsorption syndromes, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue (e.g., caused by gluten-induced enteropathy or celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, small intestine injury or short bowel syndrome (SBS). As an alternative or in addition, the glucagon-like peptide 2 (GLP-2) analogue can be used for treating and / or preventing stomach- and intestine-related disorders, such as radiation enteritis, infectious or post-infectious enteritis, or small intestine injury caused by a toxic agent or other chemotherapeutic agent. In this case, the treatment with the GLP-2 analogue can optionally be combined with one or more anticancer treatments and can thus include administering to the patient one or more chemotherapeutic agents or treating the patient with radiotherapy.

[0131] In some embodiments of the invention, in the above formula, X5 is Thr and / or X11 is Ala. Some examples of these glucagon-like peptide 2 (GLP-2) analogues include:

[0132] ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 1)

[0133] ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO: 2);

[0134] ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3);

[0135] ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4);

[0136] ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 5); or

[0137] ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6).

[0138] In some embodiments of the present invention, in the above formula, X5 is Ser and / or X11 is Ser. Some examples of these glucagon-like peptide 2 (GLP-2) analogs include:

[0139] ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7);

[0140] ZP1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO: 8); or

[0141] ZP2242 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9).

[0142] Some embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying drawings. However, in light of the present disclosure, many other aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0143] As used herein, "and / or" is considered to be a specific disclosure of each of two particular features or components with or without the other. For example, "A and / or B" is considered to be a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were listed separately herein.

[0144] Unless otherwise indicated in the context, the descriptions and definitions of the features listed above are not limited to any specific aspect or embodiment of the present invention and equally apply to all aspects and embodiments described. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1 A typical chromatogram showing the separation of the oligomer from the ZP1848 peptide is shown.

[0147] Figure 2Shows how viscosity (squares) and hydrodynamic radius (z-average) (circles) vary as a function of acetate concentration after formulation manufacture. The data indicate that above 11% acetate, the viscosity and hydrodynamic radius (z-average) start to increase.

[0148] Figure 3 Shows the stability evaluation at 20 mg / mL (normalized to 100% relative to the start) using different buffers over 0 to 3 weeks at 40 °C.

[0149] Figure 4 Shows the stability at 2 mg / mL (normalized to 100% relative to the start) using different buffers over 0 to 3 weeks at 40 °C.

[0150] Figure 5 Shows the stability evaluation at 20 mg / mL (normalized to 100% relative to the start) using different tonicity agents over 0 to 3 weeks at 40 °C.

[0151] Figure 6 Shows the stability evaluation at 2 mg / mL (normalized to 100% relative to the start) using different tonicity agents over 0 to 3 weeks at 40 °C.

[0152] Figure 7 Shows the purity of Formulations 1 to 5 using different concentrations of ZP1848 acetate, different salt forms, different tonicity agents, and different buffers.

[0153] Figure 8 Shows the stability of peptides in combination with different preservatives over 13 weeks at 25 °C.

[0154] Figure 9 Shows the HPLC purity of the studied formulations at 25 °C (accelerated conditions). Detailed Description

[0155] Definition

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

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

[0158] The preferred compounds of the present invention have at least one GLP-2 biological activity, particularly in terms of causing intestinal growth. This can be evaluated in in vivo assays, such as those described in the examples of (for example) WO 2006 / 117565, where the mass of the intestine or a part thereof is determined after the test animals have been treated or exposed to a GLP-2 analogue.

[0159] In some aspects of the present invention, the total acetate concentration in the liquid formulation containing a GLP-2 analogue is less than or equal to 11% acetate / mg GLP-2 analogue, and more preferably less than or equal to 10% acetate / mg GLP-2 analogue, more preferably less than or equal to 9% acetate / mg GLP-2 analogue, more preferably less than or equal to 8% acetate / mg GLP-2 analogue, more preferably less than or equal to 7% acetate / mg GLP-2 analogue, more preferably less than or equal to 6% acetate / mg GLP-2 analogue, more preferably less than or equal to 5% acetate / mg GLP-2 analogue, more preferably less than or equal to 4% acetate / mg GLP-2 analogue, more preferably less than or equal to 3% acetate / mg GLP-2 analogue, and more preferably less than or equal to 2% acetate / mg 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 chromatographic step. This will result in a drug substance with an acetate content below 11%. Thus, for example, for a formulation with a GLP-2 analogue at 20 mg / mL, the total acetate concentration will be less than or equal to 37 mM. For reference, a 10% total acetate concentration is equal to 34 mM, 9% is equal to 30 mM, 8% is equal to 27 mM, 7% is equal to 24 mM, and 6% is equal to 20 nM. The total acetate concentration can be determined using methods known in the art, such as HPLC.

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

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

[0162] As a supplement or alternative, the present invention shows that the formation of covalently linked oligomers of the GLP-2 analog depends negatively on the concentration of the GLP-2 analog in the preparation. As shown in the examples, the amount of the covalently bonded oligomer can be determined using size exclusion chromatography and separately determining the peak areas under the curves of the monomeric GLP-2 analog and the oligomer. This can be carried out using a Dionex Ultimate3000 HPLC system giving a linear gradient, at a flow rate of 0.5 mL / minute for analysis. The mobile phase consists of 45% acetonitrile and 55% Milli-Q water with 0.1% TFA. Detection is carried out using a wavelength of 215 nm. This means that the preparation of the present invention generally contains the GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL, more preferably at a concentration of about 15 mg / mL to about 25 mg / mL, and most preferably at a concentration of about 20 mg / mL. In some other embodiments, the present invention generally contains the GLP-2 analog at a concentration of about 2 mg / mL, 5 mg / mL, 10 mg / mL or 20 mg / mL. In some aspects of the present invention, preferably, the concentration of the GLP-2 analog is selected such that the preparation 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 analog in the form of a covalently bonded oligomeric product, preferably after 18 months of storage. By way of illustration, the amount of the covalently bonded oligomeric product can be in the range of 2% to 5%, more preferably in the range of 2% to 4%, and most preferably in the range of 2% to 3%.

[0163] In some cases, the formulations of the present invention can be used in a once-daily or twice-daily dosing regimen. In some cases, the formulations of the present invention can be used in a once-weekly or twice-weekly dosing regimen. As an alternative or supplement, the dosing regimen of the GLP-2 analog of the present invention can comprise several doses or courses of doses spaced 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days apart in time. In a preferred embodiment, the doses are spaced 3 days, 3.5 days, 4 days, 5 days, 6 days, 7 days, or 8 days apart in time. In a preferred embodiment, the doses are spaced 3 days, 3.5 days, 4 days, or 7 days apart in time. As would be understood in the art, the time between doses may vary somewhat such that each and every dose is not spaced exactly the same time apart. This will generally be guided by the judgment of the physician. Thus, the doses can be spaced apart by a clinically acceptable time range, such as from about 2 days to about 10 days, or from about 3 or 4 days to about 7 or 8 days.

[0164] The formulations of the present invention are stable liquid pharmaceutical formulations of a GLP-2 analog. A "stable" formulation is one in which the peptide therein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability as well as its biological activity after storage. The storage period is typically selected based on the intended shelf life of the formulation. The formulations of the present invention are provided as stable liquid formulations, such as stable aqueous liquid formulations. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). In the present invention, a "stable" formulation includes one 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 analog has activity after the formulation has been stored at 2 to 8 °C for at least 18 months.

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

[0166] If a peptide shows no signs (or very few signs) of aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or as measured by UV light scattering, dynamic light scattering, circular dichroism, or size exclusion chromatography, then the peptide "maintains its physical stability" in the pharmaceutical formulation and is considered to still retain its biological activity.

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

[0168] GLP-2 analog

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

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

[0171] 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

[0172] Wherein:

[0173] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

[0174] X5 is Ser or Thr;

[0175] X11 is Ala or Ser;

[0176] R 2 is NH2 or OH; and

[0177] Z 1 and Z 2Independently does not exist, or is a peptide sequence having 1 to 6 Lys amino acid units.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0191] It should be understood that the peptides (drug substances) of the present invention may also be provided in the form of salts or other derivatives. Salts include pharmaceutically acceptable salts, such as acid addition salts and basic salts. Some examples of acid addition salts include hydrochloride, citrate, chloride salt, and acetate. Preferably, the salt is acetate. Generally, preferably the salt is not a chloride salt. Some examples of basic salts include salts in which the cation is selected from the following: alkali metals (such as sodium and potassium); alkaline earth metals (such as calcium); and ammonium ions + N(R 3 )3(R 4 ), where R 3 and R 4 independently represent an optionally substituted C 1-6 alkyl, an optionally substituted C 2-6 alkenyl, an optionally substituted aryl, or an optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th Edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S.A., 1985 and more recent editions, and in Encyclopaedia of Pharmaceutical Technology.

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

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

[0194] Thus, in another aspect, the present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog. The solid composition can be used to formulate with excipients for preparing the liquid formulations of the present invention. In one embodiment, the present invention provides a solid composition comprising an acetate salt of a glucagon-like peptide 2 (GLP-2) analog having the following formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2), x(CH3COOH), where x is from 1.0 to 8.0.

[0195] The upper limit of 8.0 acetate molecules / GLP-2 analog is equal to an acetate content of less than 11% acetate and can be formulated to have a viscosity of 0.8 to 2.0 mPa / sec measured at 25°C.

[0196] The number of acetate molecules associated with each molecule of the GLP-2 analog defines a range of molecular weights for the formulation component. For example, for the acetate of ZP1848, the number of acetate molecules associated with each molecule of the GLP-2 analog defines the molecular weight range of ZP1848-acetate. By way of example, 1 acetate equivalent per molecule of ZP1848 provides a molecular weight = 4316 + 60 = 4376 Da. Thus, for increasing acetate equivalents of ZP1848, the molecular weights are as follows: 1 acetate equivalent = 4376 Da; 2 acetate equivalents = 4436 Da; 3 acetate equivalents = 4496 Da; 4 acetate equivalents = 4556 Da; 5 acetate equivalents = 4616 Da; 6 acetate equivalents = 4676 Da; 7 acetate equivalents = 4736 Da and 8 acetate equivalents = 4796 Da. This in turn defines the following molecular weight ranges: 1 to 8 acetate equivalents = 4376 Da to 4796 Da; 4 to 8 acetate equivalents = 4556 Da to 4796 Da and 6 to 8 acetate equivalents = 4676 Da to 4796 Da.

[0197] Other derivatives of the GLP-2 analogs of the present invention include coordination complexes having metal ions (such as Mn 2+ and Zn 2+ ), esters (such as in vivo hydrolysable esters), free acids or bases, hydrates, prodrugs or lipids. Using techniques well known in the art, esters can be formed between hydroxyl or carboxylic acid groups present in the compound and suitable carboxylic acid or alcohol reaction partners. Derivatives that are prodrugs of the compound can be converted in vivo or in vitro to one of the parent compounds. Generally, at least one biological activity of the compound 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. Some examples of prodrugs include the use of protecting groups that can be removed in situ to release the active compound or to inhibit the clearance of the drug in vivo.

[0198] Z 1 and Z 2 independently are present and / or absent, or are peptide sequences having 1 to 6 Lys amino acid units (i.e., 1, 2, 3, 4, 5 or 6 Lys residues). The Lys residues can have a D-configuration or an L-configuration, but have one L-configuration. Particularly preferred sequences for Z are sequences having 4, 5 or 6 consecutive lysine residues, and especially 6 consecutive lysine residues. Exemplary sequences for Z are shown in WO 01 / 04156. In certain embodiments, Z 1 is absent. In such cases, Z 2 can be present or absent.

[0199] Formulation of GLP-2 analog

[0200] The formulations of GLP-2 analogs are ready-to-use formulations. The term "ready-to-use" as used herein refers to a formulation that does not require constitution or dilution with a specified amount of diluent (such as water for injection or other suitable diluents) prior to use by the designated route of administration.

[0201] As described herein, the liquid formulations of the GLP-2 analogs of the present invention comprise a buffer, a non-ionic tonicity modifier, and a suitable amount of arginine to provide the pH of the final formulation. In accordance with conventional pharmaceutical practice, the formulations of the present invention are sterile and / or free of reducing agents. In some cases, the liquid formulations of the present invention are aqueous liquid formulations. In some cases, the liquid formulations of the present invention are non-aqueous liquid formulations.

[0202] The term "buffer" as used herein refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. The screening experiments in the examples show that the formulations of the present invention preferably comprise a buffer selected from the group consisting of histidine buffers, mesylate buffers, acetate buffers, glycine buffers, lysine buffers, TRIS buffers, Bis-Tris buffers, and MOPS buffers, because these buffers provide a stable formulation in which the GLP-2 analog is dissolved and does not become viscous, turbid, or precipitate the peptide drug. In some preferred embodiments, the buffer is a histidine buffer, such as 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, preferably, the buffer is not a phosphate buffer, a citrate buffer, a citrate / Tris buffer, and / or a succinate buffer.

[0203] The term "tonicity modifier" as used herein refers to a pharmaceutically acceptable tonicity agent for adjusting the tonicity of a formulation. The formulations of the present invention are preferably isotonic, i.e., they have substantially the same osmotic pressure as human serum. The tonicity modifier used in the formulation is preferably a non-ionic tonicity modifier and is preferably selected from mannitol, sucrose, glycerol, sorbitol, and trehalose. The preferred non-ionic tonicity modifier is mannitol, such as D-mannitol. The concentration of the tonicity modifier will depend on the concentration of the other components of the formulation, especially in the case where the formulation is intended to be isotonic. Generally speaking, 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.

[0204] Typically, the components and amounts of the liquid formulation of the present invention are selected to provide a formulation having a pH of from about 6.6 to about 7.4, more preferably a pH of from about 6.8 to about 7.2, and most preferably a pH of about 7.0. Arginine may be added in an appropriate amount (quantum sufficit, q.s.) to adjust the pH such that it is within the desired pH range. According to the experiments shown in the examples, it is preferred not to use hydrochloric acid or sodium hydroxide for pH adjustment.

[0205] In one embodiment, the liquid formulation of the present invention consists of: a GLP-2 analogue at a concentration of from about 2 mg / mL to about 30 mg / mL; a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, said buffer being present at a concentration of from about 5 mM to about 50 mM; a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose at a concentration of from about 90 mM to about 360 mM; and an appropriate amount of arginine to provide a pH of from about 6.6 to about 7.4.

[0206] In one embodiment, the liquid formulation of the present invention consists of: a GLP-2 analogue at a concentration of from about 2 mg / mL to about 30 mg / mL; a buffer selected from histidine buffer, mesylate buffer and acetate buffer, said buffer being present at a concentration of from about 5 mM to about 50 mM; a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol and sorbitol at a concentration of from about 90 mM to about 360 mM; and an appropriate amount of arginine to provide a pH of from about 6.6 to about 7.4.

[0207] In another embodiment, the liquid formulation of the present 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 an appropriate amount of arginine to provide a pH of about 7.0.

[0208] In another embodiment, the liquid formulation of the present 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 a pH of about 7.0.

[0209] In another embodiment, the liquid formulation of the present invention comprises ZP1848-acetate or H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 acetate (SEQ ID NO: 1) at a concentration of about 20 mg / mL, a histidine buffer at a concentration of about 15 mM, mannitol at a concentration of about 230 mM and an appropriate amount of arginine to provide a pH of about 7.0.

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

[0211] In another embodiment, the liquid formulation of the present invention comprises an acetate of a glucagon-like peptide 2 (GLP-2) analogue having the formula (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) (where x is from 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.

[0212] In another embodiment, the liquid formulation of the present invention comprises an acetate of a glucagon-like peptide 2 (GLP-2) analogue having the formula (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) (where x is from 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, and is for a once-daily or twice-daily dosing regimen.

[0213] In another embodiment, the liquid formulation of the present invention comprises an acetate of a glucagon-like peptide 2 (GLP-2) analogue having the formula (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2),x(CH3COOH) (where x is from 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, and is for a once-weekly or twice-weekly dosing regimen.

[0214] In another embodiment, the liquid formulation of the present invention comprises ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO: 7) 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 suitable amount of arginine to provide a pH of about 7.0.

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

[0216] In some cases, the liquid formulation of the present invention further comprises a preservative. In some cases, the preservative is a preservative selected from the following: benzalkonium chloride, chlorobutanol, methylparaben, and potassium sorbate. Generally, the preservative is present at a concentration of about 0.1% to about 1% of the final formulation volume.

[0217] In another embodiment, the liquid formulation is selected from 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.

[0218] For example, the liquid formulation of the present invention can be prepared by mixing a stock solution of a GLP-2 analogue, a buffer, a non-ionic tonicity modifier, and optionally a preservative in water, optionally diluting the resulting solution and adjusting to the target pH. Conveniently, a solution of the buffer and the non-ionic tonicity modifier can be first mixed to provide the desired concentration of each excipient. Then a solution of the GLP-2 analogue can be added and the pH adjusted if necessary, for example using acetic acid / 0.5 M L-arginine. Water is added up to the final volume.

[0219] Preferably, the glucagon-like peptide 2 (GLP-2) analogue is administered parenterally to a patient, preferably by injection, most typically by subcutaneous injection, intramuscular injection, intravenous injection, or intraperitoneal injection. Administration is preferably by subcutaneous injection. The injection can be performed by a physician, nurse, or other healthcare professional, or can be self-administered by the patient. As listed herein, in some aspects, the formulations of the present invention have a viscosity that aids in loading the formulation into a pre-filled syringe, injection pen, or other syringe device. This can have the advantage of pre-determining the dose of the formulation to be administered to the patient, for example without the need for measurement via a multi-use vial. Thus, in other aspects, the present invention provides an article or kit that comprises a container containing a stable (e.g., aqueous-stable) pharmaceutical formulation containing a GLP-2 analogue according to the present invention; or a pre-filled syringe or syringe device or injection pen that contains an aqueous liquid formulation containing a GLP-2 analogue according to the present invention.

[0220] Medical condition

[0221] By administering an effective amount of a GLP-2 analogue or a salt thereof as described herein, the GLP-2 analogue formulation of the present invention can be used as a pharmaceutical agent for preventing or treating an individual suffering from a gastrointestinal disorder (including the upper gastrointestinal tract of the esophagus). Gastric and intestinal related disorders include: ulcers of any etiology (e.g., peptic ulcer, drug-induced ulcer, ulcer associated with infection or other pathogens), digestive disorders, malabsorption syndromes, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, celiac sprue diarrhea (e.g., caused by gluten-induced enteropathy or celiac disease), tropical sprue diarrhea, hypogammaglobulinemic sprue diarrhea, enteritis, ulcerative colitis, small intestine injury, and chemotherapy-induced diarrhea / mucositis (CID).

[0222] As described above, generally, individuals who would benefit from an increased small intestine mass and subsequent maintenance of normal small intestine mucosal structure and function and / or normal small intestine mucosal structure and function are candidates for treatment with the GLP-2 analogue of the present invention. Specific disorders treatable with the GLP-2 analogue include various forms of sprue diarrhea, including: celiac sprue diarrhea, which is caused by a toxic reaction to α-gliadin from wheat and may be a result of gluten-induced enteropathy or celiac disease and is marked by a significant loss of small intestine villi; tropical sprue diarrhea, which is caused by infection and is marked by partial flattening of the villi; hypogammaglobulinemic sprue diarrhea, which is typically observed in patients with common variable immunodeficiency or hypogammaglobulinemia and is marked by a significant decrease in villus height. The therapeutic efficacy of GLP-2 analogue treatment can be monitored by intestinal biopsy examining villus morphology, by biochemical assessment of nutrient absorption, by patient weight gain, or by improvement of symptoms associated with these disorders.

[0223] Another specific disorder treatable with the GLP-2 analogue of the present invention or for which the GLP-2 analogue may be therapeutically and / or prophylactically useful is short bowel syndrome (SBS), also known as short gut syndrome or simply short gut, which is caused by surgical resection, congenital defect, or disease-related intestinal absorption loss, where the patient is subsequently unable to maintain fluid, electrolyte, and nutrient balance on a regular diet. Although adaptation typically occurs within two years after resection, dietary intake and fluid losses in SBS patients are reduced.

[0224] Other disorders treatable with the GLP-2 analogue of the present invention or for which the GLP-2 analogue may be prophylactically useful include infectious or post-infectious enteritis and small intestine injury caused by cancer chemotherapeutic agents or toxins in addition to the aforementioned radiation enteritis.

[0225] GLP-2 analogs can also be used to treat malnutrition (such as cachexia and anorexia).

[0226] One specific embodiment of the invention relates to the use of the peptides of the invention for the prevention and / or treatment of intestinal injury and dysfunction. Such injury and dysfunction are well-known side effects of cancer chemotherapy. Chemotherapy administration is often associated with undesirable side effects related to the gastrointestinal system, such as mucositis, diarrhea, bacterial translocation, malabsorption, abdominal cramps, gastrointestinal bleeding, and vomiting. These side effects are the clinical consequences of structural and functional damage to the intestinal epithelium and often necessitate a reduction in the dose and frequency of chemotherapy.

[0227] Administration of the GLP-2 peptide analogs of the invention can enhance the trophic effects of the intestinal crypts and rapidly provide new cells to replace the damaged intestinal epithelium after chemotherapy. The ultimate goal achieved by administering the peptides of the invention is to reduce the morbidity associated with gastrointestinal injury in patients undergoing chemotherapy while generating an optimal chemotherapy regimen for the treatment of cancer. Prophylactic or therapeutic treatment can be provided concomitantly to patients undergoing or about to undergo radiotherapy according to the invention.

[0228] Stem cells of the small intestinal mucosa are particularly vulnerable to the cytotoxic effects of chemotherapy due to their high proliferation rate (Keefe et al., Gut, 47: 632-7, 2000). Chemotherapy-induced injury to the small intestinal mucosa is commonly referred to clinically as gastrointestinal mucositis and is characterized by absorptive and barrier damage to the small intestine. For example, it has been shown that the widely used chemotherapeutic agents 5-FU, irinotecan, and methotrexate increase apoptosis in the small intestine of rodents, resulting in villous atrophy and crypt hypoplasia (Keefe et al., Gut 47: 632-7, 2000; Gibson et al., J Gastroenterol. Hepatol. Sep; 18(9): 1095-1100, 2003; Tamaki et al., J.Int.Med.Res. 31(1): 6-16, 2003). In humans, chemotherapeutic agents have been shown to increase apoptosis in intestinal crypts at 24 hours after administration and subsequently reduce the villous area, crypt length, mitotic count per crypt, and intestinal epithelial cell height three days after chemotherapy (Keefe et al., Gut, 47: 632-7, 2000). Thus, structural changes in the small intestine directly lead to intestinal dysfunction and, in some cases, diarrhea.

[0229] Gastrointestinal mucositis following cancer chemotherapy is an increasingly serious problem that, once established, is essentially incurable despite its gradual remission. Studies with the commonly used cytostatic cancer drugs 5-FU and irinotecan have shown that effective chemotherapy with these drugs mainly affects the structural integrity and function of the small intestine, while the colon is less sensitive and mainly responds with increased mucus formation (Gibson et al., J. Gastroenterol. Hepatol. Sep; 18(9): 1095-1100, 2003; Tamaki et al., J Int. Med. Res. 31(1): 6-16, 2003).

[0230] The pharmaceutical preparation of the present invention containing GLP-2 analogs can be used for preventing and / or treating gastrointestinal injuries and side effects of chemotherapeutic agents. This potentially important therapeutic application can be applied to currently used chemotherapeutic agents, such as but not limited to: 5-FU, Altretamine, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxycarbamide, Idarubicin, Ifosfamide, Irinotecan, Liposomaldoxorubicin, Leucovorin, Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin, Procarbazine, Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide, Thiotepa, Tioguanine / Thioguanine, Topotecan, Treosulfan, Vinblastine, Vincristine, Vindesine, Vinorelbine, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Asparaginase, Cyclophosphamide, Cytarabine, Dacarbazine, Actinomycin D, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Liposomal Doxorubicin, Leucovorin, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin, Procarbazine, Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide, Thiotepa, Tioguanine / Thioguanine, Topotecan, Treosulfan, Vinblastine, Vincristine, Vindesine and Vinorelbine.

[0231] Delivery of formulation

[0232] In some aspects, the present invention relates to ready-to-use formulations of GLP-2 analogs, which are intended for parenteral administration and are suitable for use in, for example, vials, pre-filled syringes, infusion pumps, wearable syringes, disposable auto-injectors or adjustable-dose auto-injectors.

[0233] Example

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

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

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

[0237] Example 2. Study of the formation of covalently bound oligomers in a pharmaceutical formulation of the GLP-2 analog ZP1848-acetate Formation

[0238] Materials and Methods

[0239] For the detection of covalently linked oligomers, analysis was performed 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 55% Milli-Q water with 0.1% TFA. Detection was carried out at a wavelength of 215 nm. The injection volume was 4 μg of peptide. The column used for the separation of covalently formed peptides was a TSKgel SuperSW2000 (TSK BioScience) with a particle size of 4 μm and dimensions of 300 * 4.6 mm. The total run time was 25 minutes. For the evaluation of the chemical stability of the peptide monomer, a C18 column with an acidic mobile phase and an acetonitrile gradient was used.

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

[0241] Results and Discussion

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

[0243] A typical chromatogram showing the separation of oligomers from the ZP1848-acetate monomer is shown in Figure 1 . The oligomers of ZP1848-acetate are well separated from the ZP1848-acetate monomer and all integrate into one peak. The percentage of the peak area is used to quantify the oligomers, particularly the amount of covalently linked dimers and trimers.

[0244] After storage at 2 to 8 °C for 24 months, formulations containing 0.2, 2, and 20 mg / mL ZP1848 in the formulation were analyzed. It was mainly dimer (two covalently linked ZP1848-acetate molecules) formation, but to some extent also trimer formation (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%. The initial value of the amount of oligomers was less than 0.1%.

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

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

[0247] During the long-term storage of ZP1848-acetate (glepaglutide) at 2 to 8 °C, it was unexpectedly found that the formation of covalently bound oligomers is concentration-dependent. Contrary to general expectations, however, the concentration-dependence of oligomer formation negatively depends on the increasing concentration of the GLP-2 analogue. 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 tail of the GLP-2 analogue promoting a competitive reaction that leads to the formation of higher-order species in which GLP-2 analogue molecules are weakly associated rather than covalently linked. This means that these weakly associated species are able to dissociate to release bioactive monomers, rather than resulting in loss of active substance, as occurs when covalently bound oligomers are formed.

[0248] Example 3: Buffer screening for a formulation of the GLP-2 analog ZP1848 acetate

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

[0250] Materials and Methods

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

[0252] Results and Discussion

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

[0254] Table 2: Formulations screening the effect of different buffers

[0255]

[0256] Example 4: Incompatibility of phosphate buffer with the GLP-2 analog ZP1848-acetate

[0257] Materials and Methods

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

[0259] Table 3: Formulations for Testing the Effect of Phosphate Buffer

[0260]

[0261] Results and Discussion

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

[0263] Example 5: Effect of acetate content on the viscosity of a formulation of the GLP-2 analog ZP1848-acetate Do With

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

[0265] Materials and Methods

[0266] Prepare samples using the drug substance (DS) of GLP-2 analogue ZP1848-acetate containing 6% acetate. Add acetate to explore the effect of increased acetate content in the range of 7.8% to 15% acetate (see Table 4).

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

[0268] Visually inspect the clarity and viscosity of the vials. Use microVISC TMThe viscometer measures viscosity. The hydrodynamic radius was measured using a Wyatt DynaPro II dynamic light scattering (DLS) plate reader. The sample loaded on the plate was 170 μl in size.

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

[0270]

[0271] Results and Discussion

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

[0273] Example 6: Effect of buffer salts on the stability of a formulation of the GLP-2 analog ZP1848-acetate at 2 and 20 mg / mL Effect on stability

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

[0275] Materials and Methods

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

[0277] Results and Discussion

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

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

[0280]

[0281]

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

[0283]

[0284]

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

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

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

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

[0289]

[0290] Example 7: Effect of tonicity modifiers on the stability of a formulation of the GLP-2 analog ZP1848-acetate at 2 and 20 m g / mL Effect on stability

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

[0292] Materials and Methods

[0293] Prepare stock solutions of L - histidine (200 mM), sucrose (730 mM), glycerol (977 mM), D - sorbitol (801 mM), D-(+)-trehalose dihydrate (500 mM), D - mannitol (700 mM) and ZP1848 - acetate peptide (acetate; approximately 50 mg / ml) in water (Milli - Q). Mix the excipient solutions in appropriate amounts to obtain the formulations shown in Table 8 below. All formulations contain 15 mM histidine. Add the peptide stock solution as needed to obtain the peptide content shown in Table 8. Add water up to 90% of the final volume. Adjust the pH to pH 7 using 1 M acetic acid / 0.5 M L - arginine if necessary. Add water up to the final volume. Fill each formulation in vials and place them in a stability study at 40 °C. Visually inspect the clarity and viscosity of the sample containers and analyze the hydrodynamic radius using DLS (Dynamic Light Scattering).

[0294] Results and Discussion

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

[0296] Table 8: Formulations Prepared Using Different Tonicity Modifiers

[0297]

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

[0299] Table 9: Formation of Covalently Linked Oligomers of Formulations 1 to 10 at 40 °C from 0 to 3 Weeks

[0300]

[0301] The stability of the peptide monomer was evaluated by determining the HPLC purity of the stability study for 3 weeks at 40 °C. Similar to the covalently linked oligomers, the chemical stability was very poor when using glycerol and was different from other tonicity agents. The results are presented in Figure 5 and Figure 6 .

[0302] Example 8: Physical stability impact of acids and bases for pH adjustment in a formulation of the GLP-2 analog ZP1848-acetate Impact on stability

[0303] Materials and Methods

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

[0305] Results and Discussion

[0306] The results shown in Table 10 indicate that pH adjustment using 1 M NaOH / 1 M HCl has an adverse effect on the physical stability of the ZP1848-acetate preparation.

[0307] Table 10: Effects of Acid / Base on Physical Stability

[0308]

[0309] Example 9: Use of ZP1848 peptide acetate and ZP1848 peptide chloride salts in a formulation of the GLP-2 analog ZP1848 peptide Use

[0310] A study was conducted to examine the effects of the salts of ZP1848 peptide acetate and ZP1848 peptide hydrochloride in the selected ZP1848 preparations. After accelerated storage at 40 °C, the effects of salt type, concentration, buffer, and tonicity modifier were examined. An attempt was made to synthesize the sodium salt of ZP1848 peptide, but it was found to be not possible.

[0311] Materials and Methods

[0312] Prepare stock solutions of mannitol (700 mM), histidine (200 mM), sorbitol (700 mM), mesylate (200 mM), and ZP1848 peptide solution (chloride salt; approximately 50 mg / mL) in Milli-Q water. Mix the excipient solutions in appropriate amounts to obtain the formulations shown in Tables 11 and 12 below. Add the peptide stock solution to obtain the desired final peptide content. Add water up to 90% of the final volume. If necessary, then adjust the pH to the desired formulation pH using 1 M acetic acid / 0.5 M L-arginine. Add water up to the final volume. Fill each formulation in vials and place them in a stability study at 40 °C. Visually inspect the clarity and viscosity of the sample containers and analyze the hydrodynamic radius using DLS.

[0313] Results and Discussion

[0314] The results shown in Tables 11 and 12 indicate that after 3 weeks at 40 °C, the Z-average, viscosity, and visual appearance of Formulations 1, 2, 3, and 4 have invariant stability, as evaluated by visual appearance and DLS. Formulation 5 shows a change in stability over time, as evaluated by the Z-average, viscosity, and visual appearance.

[0315] Table 11: Effect of Peptide Salt Type

[0316]

[0317] Table 12: Effect of Peptide Salt Type

[0318]

[0319]

[0320] Track the chemical stability of Formulations 1 to 5 at 40 °C for up to 4 weeks. The purity obtained is normalized relative to 100% at release. The results are shown in the figure. For Formulations 1, 2, 4, and 5, no significant differences in peptide monomer chemical stability are seen. After 4 weeks, Formulation 3 shows slightly lower but acceptable chemical stability, and this is most likely due to the lower concentration of this formulation.

[0321] Measure the formation of covalent oligomers of Formulations 1 to 5 up to 4 weeks at 40°C. The results are shown in Table 13. Even after 1 week of the stability test, differences in the formation of covalent oligomers of the 5 formulations can still be seen. Additionally, the rate (slope) is quite consistent throughout the test period. Formulation 1 (20 mg / mL, acetate of ZP1848, histidine as a tonicity agent) is the most stable formulation, with approximately 1.1% of covalent oligomers formed after 4 weeks of the stability study at 40°C. Formulation 5 (20 mg / mL, chloride salt of ZP1848, mesylate as a tonicity agent) is the second most stable formulation, with approximately 2.1% of covalent oligomer formation after 4 weeks of the stability study at 40°C. The third most stable is Formulation 3 (2 mg / mL, chloride salt of ZP1848, histidine as a tonicity agent). The fourth most stable is Formulation 2 (20 mg / mL, chloride salt of ZP1848, histidine as a tonicity agent). The least stable formulation is Formulation 4 (20 mg / mL, chloride salt of ZP1848, sorbitol as a tonicity agent).

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

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

[0324]

[0325] Example 10: Use of ZP1848 peptide acetate and preservatives in a 20 mg / mL formulation of the GLP-2 analog ZP1848 peptide Use of

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

[0327] Materials and Methods

[0328] Prepare stock solutions of mannitol (700 mM), histidine (200 mM), and ZP1848 peptide solution (acetate; approximately 50 mg / mL) in Milli-Q water. The final concentration of the peptide was 20 mg / mL, mannitol was 230 mM, and histidine was 15 mM. Mix the preservative solutions in appropriate amounts to obtain the formulations shown in Table 14 below. Add the peptide stock solution to obtain the desired final peptide content. Add water up to 90% of the final volume. If necessary, then adjust the pH to the desired formulation pH using 1 M acetic acid / 0.5 M L-arginine. Add water up to the final volume. Fill each formulation in vials. Visually inspect the clarity and viscosity of the sample containers, analyze covalently linked oligomers by SEC, and analyze peptide monomer stability by HPLC.

[0329] Results and Discussion

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

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

[0332] Table 14: Effects of Preservative Screening at 25 °C for 26 Weeks

[0333]

[0334] Table 15: Formation of Covalently Linked Oligomers of Different Preservatives at 25 °C for 13 Weeks

[0335]

[0336] Example 11: Use of ZP1848 peptide acetate and preservatives in 2 and 20 mg / mL formulations of the GLP-2 analog ZP1848 peptide Use of formulation

[0337] Materials and Methods

[0338] Prepare stock solutions of mannitol (700 mM), histidine (200 mM), and ZP1848 peptide solution (acetate; approximately 50 mg / mL) in Milli-Q water. The final concentrations of the peptide are 20 and 2 mg / mL, mannitol is 230 mM, and histidine is 15 mM. Mix the preservative solutions (m-cresol and phenol) in appropriate amounts to obtain the formulations shown in Table 14 below. Add water up to 90% of the final volume. If necessary, then adjust the pH to the desired formulation pH (7.0) using 1 M acetic acid / 0.5 M L-arginine. Add water up to the final volume. Fill each formulation in vials. Visually inspect the clarity and viscosity of the sample containers and analyze the peptide monomer stability by HPLC.

[0339] Results and Discussion

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

[0341] Table 16: Physical appearance of the tested formulations after a 52-week stability study at 5 °C

[0342]

[0343]

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

[0345] Although the invention has been described in connection with the above embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when the present disclosure is given. Accordingly, some embodiments of the invention set forth herein are considered illustrative rather than restrictive. Various changes can be made to the described embodiments without departing from the spirit and scope of the invention. All documents cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0346] The original claims of the parent case of this application are hereby incorporated into this specification:

[0347] 1. A stable liquid pharmaceutical preparation, which contains a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the following formula:

[0348] 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

[0349] Wherein:

[0350] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

[0351] X5 is Ser or Thr;

[0352] X11 is Ala or Ser;

[0353] R 2 is NH2 or OH; and

[0354] Z 1 and Z 2 independently do not exist, or are peptide sequences having 1 to 6 Lys amino acid units;

[0355] Wherein the preparation contains:

[0356] (a) The GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL;

[0357] (b) A buffer selected from histidine buffer, methanesulfonate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, and the buffer is present at a concentration of about 5 mM to about 50 mM;

[0358] (c) A non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, with a concentration of about 90 mM to about 360 mM; and

[0359] (d) An appropriate amount of arginine to provide a preparation with a pH of about 6.6 to about 7.4;

[0360] wherein the formulation contains 5% or less of the GLP-2 analog in the form of a covalently bonded oligomeric product.

[0361] 2. The formulation according to item 1, wherein the total acetate concentration produced by the GLP2 analog in the formulation is less than or equal to 11% acetate / mg GLP-2 analog.

[0362] 3. The formulation according to item 1 or item 2, wherein the formation of the covalently linked oligomer of the GLP-2 analog negatively depends on the concentration of the GLP-2 analog in the formulation.

[0363] 4. The formulation according to any one of items 1 to 3, wherein the formulation is an aqueous formulation.

[0364] 5. The formulation according to any one of items 1 to 4, wherein the formulation is stable for at least 18 months when stored at 2 to 8 °C.

[0365] 6. The formulation according to any one of the foregoing items, wherein the viscosity of the formulation measured at 25 °C is 0.8 to 2.0 mPa / s.

[0366] 7. The formulation according to any one of the foregoing items, wherein the GLP-2 analog is present in the formulation at a concentration of about 15 mg / mL to about 25 mg / mL.

[0367] 8. The formulation according to any one of the foregoing items, wherein the formulation is a ready-to-use formulation.

[0368] 9. The formulation according to any one of the foregoing items, wherein the GLP-2 analog is present in the formulation at a concentration of about 20 mg / mL.

[0369] 10. The formulation according to any one of the foregoing items, wherein the buffer is present in the formulation at a concentration of about 5 mM to about 25 mM.

[0370] 11. The formulation according to any one of the foregoing items, wherein the buffer is a histidine buffer.

[0371] 12. The formulation according to item 11, wherein the histidine buffer is present in the formulation at a concentration of about 15 mM.

[0372] 13. The formulation according to any one of the foregoing items, wherein the non-ionic tonicity regulator is present in the formulation at a concentration of about 150 mM to about 250 mM.

[0373] 14. The formulation according to any one of the foregoing items, wherein the non-ionic tonicity regulator is mannitol.

[0374] 15. The preparation according to item 14, wherein the mannitol is present in the preparation at a concentration of about 230 mM.

[0375] 16. The preparation according to any one of the preceding items, wherein the pH of the preparation is from about 6.8 to about 7.2.

[0376] 17. The preparation according to any one of the preceding items, wherein the pH of the preparation is about 7.0.

[0377] 18. The preparation according to any one of the preceding items, wherein the preparation consists of: the GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / ml; a buffer selected from histidine buffer, mesylate buffer and acetate buffer, the buffer being present at a concentration of about 5 mM to about 50 mM; a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol and sorbitol, at a concentration of about 90 mM to about 360 mM; an appropriate amount of arginine to provide a pH of about 6.6 to about 7.4.

[0378] 19. The preparation according to any one of the preceding items, wherein the preparation contains the 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 an appropriate amount of arginine to provide a pH of about 7.0.

[0379] 20. The preparation according to any one of the preceding items, wherein the histidine buffer is L-histidine.

[0380] 21. The preparation according to any one of the preceding items, wherein the mannitol is D-mannitol.

[0381] 22. The preparation according to any one of the preceding items, wherein the arginine is L-arginine / acetate.

[0382] 23. The preparation according to any one of the preceding items, wherein the preparation does not contain a reducing agent.

[0383] 24. The preparation according to any one of the preceding items, wherein the preparation is stable at 2 to 8 °C for at least 6 months, at least 12 months, at least 18 months or at least 24 months.

[0384] 25. The preparation according to item 24, wherein after storage at 2 to 8 °C for 18 months, the GLP-2 analogue in the preparation retains at least about 90% of its biological activity.

[0385] 26. The preparation according to any one of the preceding items, which is sterile.

[0386] 27. The preparation according to any one of the preceding items, wherein the preparation is administered to a subject by injection.

[0387] 28. The preparation according to item 27, wherein the injection is a subcutaneous injection.

[0388] 29. The preparation according to any one of the preceding items, wherein the GLP-2 analogue is provided as an acetate.

[0389] 30. The preparation according to any one of the preceding items, wherein the GLP-2 analogue is ZP1848 or ZP1848-acetate.

[0390] 31. The preparation according to any one of the preceding items, wherein the preparation consists of: ZP1848-acetate 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 suitable amount of arginine to provide a pH of about 7.0.

[0391] 32. A product or kit, which comprises a container containing the stable pharmaceutical preparation according to any one of items 1 to 31.

[0392] 33. A delivery device, which comprises a liquid preparation containing the GLP-2 analogue according to any one of items 1 to 31.

[0393] 34. The delivery device according to item 33, wherein the delivery device is a pre-filled syringe, a syringe device, an injection pen, an adjustable-dose auto-injector, a disposable auto-injector, a wearable syringe, an infusion pump.

[0394] 35. A preparation of a glucagon-like peptide 2 (GLP-2) analogue according to any one of items 1 to 31, for treatment.

[0395] 36. A preparation of a glucagon-like peptide 2 (GLP-2) analogue according to any one of items 1 to 31, for use in a method for treating and / or preventing stomach and intestine related disorders in a human patient.

[0396] 37. The preparation of a glucagon-like peptide 2 (GLP-2) analogue for the treatment and / or prevention method according to item 36, wherein the stomach and intestine related disorders are ulcers, digestive disorders, malabsorption syndromes, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, celiac diarrhea (e.g., caused by gluten-induced enteropathy or celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, small intestine injury or short bowel syndrome (SBS).

[0397] 38. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment and / or prevention method according to item 37, wherein the gastric and intestinal related disorder is short bowel syndrome.

[0398] 39. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to item 36, wherein the gastric and intestinal related disorder is radiation enteritis, infectious or post-infectious enteritis, or small intestine injury caused by a toxic agent or other chemotherapeutic agent.

[0399] 40. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to item 39, wherein the treatment with the GLP-2 analogue is combined with one or more anti-cancer treatments.

[0400] 41. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to item 40, wherein the treatment of the anti-cancer treatment includes administering one or more chemotherapeutic agents to the patient or treating the patient with radiotherapy.

[0401] 42. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to item 40 or item 41, wherein the preparation is for treating and / or preventing side effects of chemotherapy or radiotherapy.

[0402] 43. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to item 42, wherein the side effects of the chemotherapy are diarrhea, abdominal cramps, vomiting, or structural and functional damage of the intestinal epithelium caused by chemotherapy.

[0403] 44. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to any one of items 40 to 43, wherein the human patient is a patient with SBS intestinal failure.

[0404] 45. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to any one of items 40 to 44, wherein the human patient is a patient on the borderline between a patient with SBS intestinal insufficiency and a patient with SBS intestinal failure.

[0405] 46. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a treatment method according to any one of items 35 to 45, wherein the method includes administering the GLP-2 analogue to the patient once a week.

[0406] 47. A preparation of a glucagon-like peptide 2 (GLP-2) analogue for a therapeutic method according to any one of items 29 to 43, wherein the method comprises administering the GLP-2 analogue to the patient twice a week.

[0407] 48. A method for producing a stable liquid pharmaceutical preparation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the following formula:

[0408] 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

[0409] Wherein:

[0410] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0411] X5 is Ser or Thr

[0412] X11 is Ala or Ser

[0413] R 2 is NH2 or OH;

[0414] Z 1 and Z 2 independently do not exist, or are peptide sequences having 1 to 6 Lys amino acid units;

[0415] Wherein the method comprises formulating (a) the GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL with the following: (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, the buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, the non-ionic tonicity regulator being present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to provide a preparation with a pH of about 6.6 to about 7.4;

[0416] Wherein the preparation contains 5% or less of the GLP-2 analogue in the form of a covalently bonded oligomeric product.

[0417] 49. The method according to item 48, wherein the formation of the covalently linked oligomer of the GLP-2 analogue is negatively dependent on the concentration of the GLP-2 analogue in the preparation.

[0418] 50. The method according to item 48 or item 49, wherein the preparation is stable for at least 18 months when stored at 2 to 8 °C.

[0419] 51. The method according to any one of items 48 to 50, wherein the total acetate concentration produced by the GLP2 analogue in the preparation is less than or equal to 11% acetate / mg GLP-2 analogue.

[0420] 52. The method according to any one of items 48 to 51, wherein the viscosity of the preparation measured at 25 °C is greater than 0.8 and less than or equal to 2.0 mPa / s.

[0421] 53. Use of a preparation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof for providing a liquid pharmaceutical preparation that is stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analogue is represented by the following formula:

[0422] 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

[0423] Wherein:

[0424] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0425] X5 is Ser or Thr

[0426] X11 is Ala or Ser

[0427] R 2 is NH2 or OH;

[0428] Z 1 and Z2 either independently does not exist, or is a peptide sequence having 1 to 6 Lys amino acid units;

[0429] wherein the preparation comprises:

[0430] (a) the GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL;

[0431] (b) a buffer selected from 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;

[0432] (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0433] (d) an appropriate amount of arginine to provide a preparation having a pH of about 6.6 to about 7.4;

[0434] wherein the preparation contains 5% or less of the GLP-2 analogue in the form of a covalently bonded oligomeric product.

[0435] 54. The use according to item 53, wherein the formation of the covalently linked oligomer of the GLP-2 analogue is negatively dependent on the concentration of the GLP-2 analogue in the preparation.

[0436] 55. The use according to item 53 or item 54, wherein the total acetate concentration produced by the GLP2 analogue in the preparation is less than or equal to 11% acetate / mg GLP-2 analogue.

[0437] 56. The use according to any one of items 53 to 54, wherein the viscosity of the preparation measured at 25 °C is 0.8 to 2.0 mPa / s.

[0438] 57. A method for regulating the viscosity of a stable liquid pharmaceutical preparation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, wherein the GLP-2 analogue is represented by the following formula:

[0439] 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

[0440] wherein:

[0441] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0442] X5 is Ser or Thr

[0443] X11 is Ala or Ser

[0444] R 2 is NH2 or OH;

[0445] Z 1 and Z 2 independently are absent, or are a peptide sequence having 1 to 6 Lys amino acid units;

[0446] wherein the method comprises formulating (a) the GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL with: (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer or MOPS buffer, the buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, the non-ionic tonicity regulator being present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0447] wherein the total acetate concentration in the formulation produced by the GLP2 analogue is less than or equal to 11% acetate / mg GLP-2 analogue, and wherein the viscosity of the formulation measured at 25 °C is greater than 0.8 and less than or equal to 2.0 mPa / sec.

[0448] 58. A method for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue in a stable liquid pharmaceutical formulation, the liquid pharmaceutical formulation comprising a GLP-2 analogue represented by the following formula or a pharmaceutically acceptable salt or derivative thereof:

[0449] 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

[0450] Wherein:

[0451] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0452] X5 is Ser or Thr

[0453] X11 is Ala or Ser

[0454] R 2 is NH2 or OH;

[0455] Z 1 and Z 2 independently do not exist, or are peptide sequences having 1 to 6 Lys amino acid units;

[0456] Wherein the method comprises formulating (a) the GLP-2 analog at a concentration of about 2 mg / mL to about 30 mg / mL with the following: (b) a buffer selected from histidine buffer, mesylate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer or MOPS buffer, the buffer being present at a concentration of about 5 mM to about 50 mM; (c) a non-ionic tonicity modifier selected from mannitol, sucrose, glycerol, sorbitol and trehalose, the non-ionic tonicity modifier being present at a concentration of about 90 mM to about 360 mM; and (d) an appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0457] Wherein the formulation contains 5% or less of the GLP-2 analog in the form of covalently bonded oligomeric products.

[0458] The method according to item 58, wherein the formation of covalently linked oligomers of the GLP-2 analog is negatively dependent on the concentration of the GLP-2 analog in the formulation.

[0459] 60. Use of a formulation for reducing the formation of covalently bonded oligomeric products of a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof in a liquid pharmaceutical formulation, the liquid pharmaceutical formulation being stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analogue is represented by the following formula:

[0460] 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

[0461] Wherein:

[0462] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0463] X5 is Ser or Thr

[0464] X11 is Ala or Ser

[0465] R 2 is NH2 or OH;

[0466] Z 1 and Z 2 independently do not exist, or are peptide sequences having 1 to 6 Lys amino acid units;

[0467] Wherein the formulation comprises:

[0468] (a) the GLP-2 analogue at a concentration of about 2 mg / mL to about 30 mg / mL;

[0469] (b) a buffer selected from histidine buffer, methanesulfonate buffer, acetate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, the buffer being present at a concentration of about 5 mM to about 50 mM;

[0470] (c) a non-ionic tonicity regulator selected from mannitol, sucrose, glycerol, sorbitol and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0471] (d) an appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0472] wherein the preparation contains 5% or less of the GLP-2 analogue in the form of a covalently bonded oligomeric product.

[0473] 61. Use according to item 60, wherein the formation of the covalently linked oligomer of the GLP-2 analogue is negatively dependent on the concentration of the GLP-2 analogue in the preparation.

[0474] 62. Use of a preparation for regulating the viscosity of a liquid pharmaceutical preparation comprising a glucagon-like peptide 2 (GLP-2) analogue or a pharmaceutically acceptable salt or derivative thereof, the liquid pharmaceutical preparation being stable for 24 months when stored at 2 to 8 °C, wherein the GLP-2 analogue is represented by the following formula:

[0475] 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

[0476] wherein:

[0477] R 1 is hydrogen, C 1-4 alkyl (such as methyl), acetyl, formyl, benzoyl or trifluoroacetyl

[0478] X5 is Ser or Thr

[0479] X11 is Ala or Ser

[0480] R 2 is NH2 or OH;

[0481] Z 1 and Z 2 independently are absent, or are a peptide sequence having 1 to 6 Lys amino acid units;

[0482] wherein the preparation comprises:

[0483] (a) the GLP-2 analogue at a concentration of from about 2 mg / mL to about 30 mg / mL;

[0484] (b) A buffering agent 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;

[0485] (c) A non-ionic tensioactive agent selected from mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0486] (d) An appropriate amount of arginine to provide a formulation with a pH of about 6.6 to about 7.4;

[0487] wherein the total acetate concentration in the formulation produced by the GLP2 analogue is less than or equal to 11% acetate / mg GLP-2 analogue, and the viscosity of the formulation measured at 25 °C is 0.8 to 2.0 mPa·s.

[0488] 63. A solid composition comprising an acetate of a glucagon-like peptide 2 (GLP-2) analogue having the following formula:

[0489] (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2)x(CH3COOH), where x is from 1.0 to 8.0.

[0490] 64. The solid composition according to item 63, wherein x is from 4.0 to 6.0.

[0491] 65. The solid composition according to item 63, wherein x is from 2.0 to 7.0.

[0492] 66. The solid composition according to item 63, wherein x is from 3.0 to 6.0.

[0493] 67. The solid composition according to item 63, wherein x is from 4.0 to 6.0.

[0494] 68. The solid composition according to item 63, wherein x is from 4.0 to 8.0.

[0495] 69. A stable aqueous pharmaceutical formulation, the formulation comprising

[0496] (a) The solid composition according to items 63 to 67, at a concentration of about 2 mg / mL to about 30 mg / mL;

[0497] (b) A buffering agent 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;

[0498] (c) A nonionic tensioactive agent selected from mannitol, sucrose, glycerol, sorbitol, and trehalose, at a concentration of about 90 mM to about 360 mM; and

[0499] (d) An appropriate amount of arginine to provide a formulation having a pH of about 6.6 to about 7.4;

[0500] wherein the formulation contains 5% or less of the GLP-2 analog in the form of covalently bonded oligomeric products, and wherein the viscosity of the formulation measured at 25 °C is 0.8 to 2.0 mPa / s.

[0501] 70. The formulation according to item 69, wherein the formulation is stable for at least 18 months when stored at 2 to 8 °C.

[0502] 71. The formulation according to item 69 or item 70, wherein after storage at 2 to 8 °C for 18 months, the GLP-2 analog in the formulation retains at least about 90% of its biological activity.

Claims

1. A solid composition comprising an acetate of a glucagon-like peptide 2 (GLP-2) analogue having the following formula: (H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2)×(CH3COOH), where x is from 1.0 to 8.

0.

2. The solid composition according to claim 1, wherein x is from 2.0 to 7.

0.

3. The solid composition according to claim 1, wherein x is from 3.0 to 6.

0.

4. The solid composition according to claim 1, wherein x is from 4.0 to 6.

0.

5. The solid composition according to claim 1, wherein x is from 4.0 to 8.

0.

6. A stable aqueous liquid pharmaceutical formulation comprising a pharmaceutically acceptable acetate of a glucagon-like peptide 2 (GLP-2) analogue formulated by mixing: (a) The solid composition according to claim 1, wherein the pharmaceutically acceptable acetate of the GLP-2 analogue is present in the formulation at a concentration of 2 mg / mL to 30 mg / mL; (b) A buffer selected from histidine buffer, mesylate buffer, glycine buffer, lysine buffer, TRIS buffer, Bis-Tris buffer and MOPS buffer, the buffer being present at a concentration of 5 mM to 50 mM; (c) A non-ionic tonicity regulator selected from mannitol, sucrose, sorbitol and trehalose, at a concentration of 90 mM to 360 mM; and (d) An appropriate amount of arginine to provide a formulation having a pH of 6.6 to 7.4; wherein the total acetate concentration in the formulation produced by the GLP2 analogue is less than or equal to 11% acetate / mg GLP-2 analogue, and wherein the viscosity of the formulation measured at 25 °C is greater than 0.8 and less than or equal to 2.0 mPa / s.

7. The formulation according to claim 6, wherein the formulation contains 5% or less of the GLP-2 analogue in the form of covalently bonded oligomeric products, and wherein the viscosity of the formulation measured at 25 °C is 0.8 to 2.0 mPa / s.

8. The formulation according to claim 6, wherein the formulation is stable for at least 18 months when stored at 2 to 8 °C.

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

10. The formulation according to claim 6, wherein the viscosity of the formulation measured at 25 °C is 0.8 to 2.0 mPa / s.

11. The formulation according to claim 6, wherein the pharmaceutically acceptable acetate of the GLP-2 analogue is present in the formulation at a concentration of 15 mg / mL to 25 mg / mL.

12. The formulation according to claim 6, wherein the formulation is a ready-to-use formulation.

13. The formulation according to claim 6, wherein the pharmaceutically acceptable acetate of the GLP-2 analogue is present in the formulation at a concentration of 20 mg / mL.

14. The preparation according to claim 6, wherein the pharmaceutically acceptable acetate of the GLP-2 analogue is present in the preparation at a concentration of 10 mg / mL.

15. The preparation according to claim 6, wherein the pharmaceutically acceptable acetate of the GLP-2 analogue is present in the preparation at a concentration of 5 mg / mL.

16. The preparation according to claim 6, wherein the pharmaceutically acceptable acetate of the GLP-2 analogue is present in the preparation at a concentration of 2 mg / mL.

17. The preparation according to claim 6, wherein the buffer is present in the preparation at a concentration of 5 mM to 25 mM.

18. The preparation according to claim 6, wherein the buffer is a histidine buffer.

19. The preparation according to claim 18, wherein the histidine buffer is present in the preparation at a concentration of 15 mM.

20. The preparation according to claim 6, wherein the non-ionic tonicity regulator is present in the preparation at a concentration of 150 mM to 250 mM.

21. The preparation according to claim 6, wherein the non-ionic tonicity regulator is mannitol.

22. The preparation according to claim 21, wherein the mannitol is present in the preparation at a concentration of 230 mM.

23. The preparation according to claim 6, wherein the pH of the preparation is 6.8 to 7.

2.

24. The preparation according to claim 6, wherein the pH of the preparation is 7.

0.

25. The preparation according to claim 6, wherein the preparation consists of the following: The pharmaceutically acceptable acetate of the GLP-2 analogue, at a concentration of 2 mg / mL to 30 mg / ml; a buffer selected from histidine buffer and mesylate buffer, the buffer being present at a concentration of 5 mM to 50 mM; a non-ionic tonicity regulator selected from mannitol, sucrose and sorbitol, at a concentration of 90 mM to 360 mM; an appropriate amount of arginine to provide a pH of 6.6 to 7.

4.

26. The preparation according to claim 6, wherein the preparation comprises the pharmaceutically acceptable acetate of the GLP-2 analogue at a concentration of 20 mg / mL, a histidine buffer at a concentration of 15 mM, mannitol at a concentration of 230 mM and an appropriate amount of arginine to provide a pH of 7.

0.

27. The preparation according to claim 6, wherein the histidine buffer is L-histidine.

28. The preparation according to claim 6, wherein the mannitol is D-mannitol.

29. The preparation according to claim 6, wherein the arginine is L-arginine / acetate.

30. The preparation according to claim 6, wherein the preparation does not contain a reducing agent.

31. The preparation according to claim 6, which is sterile.

32. The preparation according to claim 6, wherein the preparation is formulated for administration to a subject by injection.

33. The preparation according to claim 32, wherein the injection is subcutaneous injection.

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