Manufacture, formulation and dosing of apraglutide

Abstract

To provide methods of making, formulating and administering GLP-2 analogs.SOLUTION: The present invention provides a sodium salt of apraglutide having the following structure, with a purity of 95% or more, as measured by high performance liquid chromatography, for use in methods to treat short bowel syndrome-associated intestinal insufficiency or short bowel syndrome-associated intestinal dysfunction.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 036,507, filed June 9, 2020, U.S. Provisional Application No. 63 / 074,119, filed September 3, 2020, and U.S. Provisional Application No. 63 / 157,083, filed March 5, 2021. The contents of each of the foregoing patent applications are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains an ASCII formatted Sequence Listing submitted via EFS-Web, which is hereby incorporated by reference in its entirety. This ASCII copy, created on June 7, 2021, is titled "VECT-002_001WO_SeqList.txt" and is approximately 2KB in size.

[0003] The present disclosure relates to the manufacture, formulation, and dosing of apraglutide. [Background technology]

[0004] Glucagon-like peptide 2 (GLP-2) is a hormone that regulates the intestinal enteroendocrine system. It is a 33 amino acid peptide released from post-translational processing of proglucagon in L cells.

[0005] The inventors contemplate using apraglutide to treat intestinal disorders or dysfunction. In one embodiment, the inventors contemplate treating short bowel syndrome. Short bowel syndrome is a malabsorptive condition characterized by an extreme reduction in functional bowel length, most commonly as a result of mesenteric ischemia or surgical resection due to Inflammatory Bowel Disease (IBD), although other etiologies exist. SBS with intestinal failure (SBS-IF) is a condition characterized by the inability to tolerate the intestinal tract in order to maintain health and / or growth. SBS-IF is defined as a decline in intestinal function below the minimum required for absorption of macronutrients and / or fluids and electrolytes such that intravenous supplementation is required to maintain adequate fluid, energy, electrolytes, trace elements, vitamins, and nutritional balance. SBS-IF patients range from those with stomas and colon-in-continuity (CIC) requiring large PS volumes, to those with lower PS volumes. There is a range of patients with CIC who are able to tolerate PS for up to 10 days. As a result of this range, a decrease in dependency on PS can be demonstrated in various outcomes depending on the anatomy, including a decrease in PS volume, days off PS, or achievement of enteral autonomy. After surgical resection, the remaining intestine undergoes a process called intestinal adaptation, which increases its absorptive capacity and compensates for its reduced length. It has been demonstrated that this process of intestinal adaptation can be enhanced by administering glucagon-like peptide 2 (GLP-2) or more stable analogs with extended half-lives (e.g., teduglutide and apraglutide). Due to the physiological role of the colon, patients with CIC are better able to manage fluid balance than patients with a non-functioning colon and require less parenteral support at baseline compared to stoma patients. PS is defined as any intravenous infusion containing fluids and electrolytes, which may or may not include parenteral nutrition (PN). Parenteral nutrition is defined as PS that contains protein, carbohydrates, fats, vitamins, and / or trace elements. Patients with chronic intestinal failure due to benign conditions are more likely to survive long-term with PS.

[0006] Since its identification as a potent stimulator of mucosal epithelial proliferation, GLP-2 has been considered a potential therapeutic agent for intestinal injury. It has been noted as a potential therapeutic agent for the treatment of chronic bronchitis. Preliminary studies in patients with short bowel syndrome have shown improvements in intestinal absorption of both fluids and nutrients. GLP-2 induces significant proliferation of the small intestinal mucosal epithelium. It also slows intestinal transit. However, naturally occurring GLP-2 is not a suitable drug candidate because it is rapidly degraded by peptidases (e.g., DPP IV). As a result, GLP-2 has a very short half-life (t1 / 2=10 min in humans) and rapid clearance (CL).

[0007] There is a need for methods of producing substantially pure GLP-2 analog peptides in high yields with improved pharmacokinetic properties. Additionally, there is a need for substantially pure, e.g., substantially free of impurities, GLP-2 analog peptide compositions. The present disclosure is directed to meeting this need.

[0008] Summary of the Invention In one aspect, a novel synthesis of a highly pure GLP-2 analog is disclosed. In another aspect, a novel sodium salt of a GLP-2 analog is disclosed. The method comprises performing solid phase peptide synthesis (SPPS) on a resin and eluting the synthesized GLP-2 analog with 1,000 ng / mL of ... The P-2 analog peptide was cleaved from the resin, the side chains of the peptide were deprotected by treating the resin with a solution containing trifluoroacetic acid (TFA), water, and anisole, and purified twice using reversed-phase high performance liquid chromatography (RP-HPLC). and lyophilizing and packaging the purified peptide powder comprising the substantially pure GLP-2 analog peptide. In some embodiments, the apraglutide has a purity of 95% or greater. In some embodiments, the apraglutide has a purity of 97% or greater.

[0009] Another aspect of the present disclosure relates to a GLP-2 analog peptide having a purity of at least 97%. In some embodiments, the GLP-2 analog peptide, such as apraglutide, contains less than 1% Des-Gly. 4 Apraglutide impurities and / or aspartimide less than 3% 3 Apraglutide, Asp 33 -OH Apraglutide and Des-Ser 7 Apraglutide impurities and / or less than 1% [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity.

[0010] Another aspect of the present disclosure relates to a GLP-2 analog peptide having a purity of at least 97%. In some embodiments, the GLP-2 analog peptide, such as apraglutide, has 1% or less of Des-Gly. 4 Apraglutide impurities and / or aspartimide less than 3% 3 Apraglutide, Asp 33 -OH Apraglutide and Des-Ser 7 Apraglutide impurities and / or less than 1% [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity.

[0011] Another aspect of the present disclosure relates to a GLP-2 analog peptide having a purity of at least 97%. In some embodiments, the GLP-2 analog peptide, such as apraglutide, is selected from the group consisting of Des-Gly. 4 Apraglutide and Aspartimide 3 Less than 1% of total apraglutide impurities, D-aspartimide 3 Apraglutide impurity less than 1%, Asp 33 -OH apraglutide impurity less than 1%, Des-Ser 7 Less than 1% of apraglutide impurity and / or [Trp 25 ,2-(2,4,6-trimethoxyphenyl)] apraglutide contains less than 1% of the impurity.

[0012] Another aspect of the present disclosure relates to a GLP-2 analog peptide having a purity of at least 97%. In some embodiments, the GLP-2 analog peptide, such as apraglutide, has 1% or less of Des-Gly. 4 Apraglutide and Aspartimide 3 Apraglutide impurities, Less than 1% D-aspartimide 3 Apraglutide impurity, less than 1% Asp 33 -OH apraglutide impurity, Des-Ser less than 1% 7 Apraglutide impurities and / or less than 1% [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity.

[0013] The inventors have recognized the following. i) Fmoc-protected amino acids, namely Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Gly-OH, during Fmoc deprotection and coupling cycles, Fmoc-Gln(Trt)-Thr(Ψ Me,Me Substitution with Fmoc-Tmb-Gly-OH and Fmoc-Tmb-Gly-OH improves coupling efficiency and reduces aspartimide formation. ii) Using oxymer instead of HOBt as a coupling additive during the Fmoc deprotection and coupling cycles reduced peptide oxidation and [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity formation is minimized. iii) The use of acetonitrile (ACN) for the mobile phase composition, the introduction of a pH adjustment step, and the elimination of the titration step during the sodium salt conversion by preparative RP-HPLC improves the sodium salt conversion of apraglutide and the quality of the purified apraglutide. In another aspect, novel formulations and salts of apraglutide are disclosed.

[0014] According to one embodiment, the GLP-2 analog peptide of the present disclosure has the following chemical structure:

[0015] [ka]

[0016] In another aspect, a novel method of administering an effective amount of apraglutide to a patient in need thereof is disclosed. In some embodiments, the effective amount comprises 1-10 mg of apraglutide or a pharma- ceutically acceptable salt thereof. In a preferred embodiment, apraglutide or a pharma- ceutically acceptable salt thereof is administered at 2.5 mg once a week to a patient weighing less than 50 kg. In another preferred embodiment, apraglutide or a pharma- ceutically acceptable salt thereof is administered at 5.0 mg once a week to a patient weighing 50 kg or more. In some embodiments, the method comprises administering apraglutide or a pharma- ceutically acceptable salt thereof intravenously. In other embodiments, the method comprises administering apraglutide or a pharma- ceutically acceptable salt thereof subcutaneously.

[0017] In some embodiments, the methods comprise administering the compound or a pharma- ceutically acceptable salt thereof at a frequency ranging from twice daily to twice monthly, preferably once weekly.

[0018] There is a need for GLP-2 analogs with longer half-lives, such as apraglutide, that can be dosed less than once daily, preferably once weekly. [Brief description of the drawings]

[0019] [Figure 1] 1 shows the peptide sequence and structure of an exemplary GLP-2 agonist (apraglutide) that may be used in the methods of the present disclosure. The sequences shown in FIG. 1 correspond to SEQ ID NOs: 1-4. [Figure 2A] FIG. 1 is a schematic diagram of a flow diagram of steps 1-2 in a first embodiment of the present disclosure directed to an improved method for producing GLP-2 analog peptides such as apraglutide (hereinafter referred to as Process B). [Figure 2B]FIG. 1 is a schematic diagram of a flow diagram of steps 3 to 6 in a first embodiment of the present disclosure directed to an improved method for making GLP-2 analog peptides such as apraglutide (hereinafter referred to as Process B). [Diagram 3] FIG. 1 is a schematic flow diagram of the manufacturing process of the present disclosure directed to the production of a pharmaceutical composition comprising apraglutide. [Figure 4] FIG. 1 is a schematic diagram of the study design of the present disclosure. [Diagram 5] Graph showing individual and mean change from baseline to end of treatment in urine output (mL / day). Dashed lines indicate mean, Δ indicates mean change from baseline (standard deviation). Greyscale difference indicates individual patients. [Figure 6] Graph showing change from baseline in urine output (mL / day) with adjusted means (dots) and 95% CI (black line) from analysis Parts A+B. [Figure 7] Graph showing individual and mean change from baseline to end of treatment in urinary sodium excretion (mmol / day). Dashed line indicates mean, B indicates baseline, T indicates treatment, and Δ indicates mean change from baseline (standard deviation). Greyscale difference indicates individual patients. [Figure 8] Graph showing individual and mean change in urine output from baseline to immediately after first injection. Dashed line indicates mean, B indicates baseline, T indicates treatment, and Δ indicates mean change from baseline (standard deviation). Greyscale difference indicates individual patients. [Figure 9] Graph showing individual and mean change in urinary sodium excretion from baseline to immediately after the first treatment. Dashed line indicates mean, B indicates baseline, T indicates treatment, and Δ indicates mean change from baseline (standard deviation). Greyscale difference indicates individual patients. [Figure 10A]Graph showing individual and mean changes in the combined fluid effect in placebo-treated patients, defined as the sum of increased urine production (days 27-29), decreased PS volume, and decreased spontaneous oral fluid intake (days 20-22). [Figure 10B] Graph showing individual and mean changes in combined fluid effect in patients receiving apraglutide 5 mg, defined as the sum of increased urine production (days 27-29), decreased PS volume, and decreased spontaneous oral fluid intake (days 20-22). [Figure 10C] Graph showing individual and mean changes in combined fluid effect in patients receiving 10 mg apraglutide, defined as the sum of increased urine production (days 27-29), decreased PS volume, and decreased spontaneous oral fluid intake (days 20-22). [Figure 11] Graph showing individual and mean changes from baseline to end of treatment in absolute plasma L-citrulline concentrations. Dashed line indicates mean, B indicates baseline, T indicates treatment, and Δ indicates mean change from baseline (standard deviation). Greyscale difference indicates individual patients. [Figure 12] Schematic diagram of the Consolidated Standards of Reporting Trials (CONSORT). [Figure 13] Schematic diagram of the Consolidated Standards of Reporting Clinical Trials (CONSORT). [Figure 14] Graphs showing individual and mean changes in wet weight food intake, fecal output, intestinal absorption, and urine production from baseline to end of treatment. Bold line indicates mean, B indicates baseline, T indicates treatment, and Δ indicates mean change from baseline (standard deviation). Dashed line indicates patients with SBS-II. Grey scale difference indicates individual patients. [Figure 15]Graph showing individual and mean changes from baseline to end of treatment in potassium, sodium, magnesium and calcium absorption. Bold line indicates mean, B indicates baseline, T indicates treatment, Δ indicates mean change from baseline (standard deviation). Dashed line indicates patients with SBS-II. Greyscale difference indicates individual patients. [Figure 16] Graphs showing individual and mean changes from baseline to end of treatment in energy dietary intake, fecal output, and absorption. Bold line indicates mean, B indicates baseline, T indicates treatment, and Δ indicates mean change from baseline (standard deviation). Dashed line indicates patients with SBS-II. Grey scale difference indicates individual patients. [Figure 17] Graph showing individual and mean changes in macronutrient absorption from baseline to end of treatment. Bold line indicates mean, B indicates baseline, T indicates treatment, and Δ indicates mean change from baseline (standard deviation). Dashed line indicates patients with SBS-II. Greyscale difference indicates individual patients. [Figure 18] Graph showing plasma citrulline concentration (ug / mL) as a function of time (days) after the first dose of apraglutide. SD = standard deviation, SC = subcutaneous. Data are arithmetic mean (±SD). Weekly SC administration of 1, 5 or 10 mg apraglutide was scheduled on days 1, 8, 15, 22, 29 and 36. [Figure 19] FIG. 1 shows the corresponding least squares (LS) mean plot of the change from baseline in plasma citrulline concentration (ug / mL) as a function of time (days) after the first dose of apraglutide. CI = confidence interval, SC = subcutaneous. Data are estimated least squares means (95% CI). Weekly SC administration of 1, 5 or 10 mg apraglutide was scheduled on days 1, 8, 15, 22, 29 and 36. [Figure 20A]Graph showing plasma apraglutide concentrations (ng / mL) as a function of time (days) after the first dose of apraglutide. SC = subcutaneous, SD = standard deviation. Weekly SC administration of 1, 5 or 10 mg apraglutide was scheduled on days 1, 8, 15, 22, 29 and 36. Values ​​below the limit of quantification (<1 ng / mL) were set to 0. Data per dose level are presented as arithmetic mean ± SD on a linear scale. [Figure 20B] Graph showing plasma apraglutide concentrations (ng / mL) as a function of time (days) after the first dose of apraglutide. SC = subcutaneous, SD = standard deviation. Weekly SC administration of 1, 5 or 10 mg apraglutide was scheduled on days 1, 8, 15, 22, 29 and 36. Values ​​below the limit of quantification (<1 ng / mL) were set to 0. Data per dose level are presented as arithmetic mean ± SD on a logarithmic scale. [Figure 21] Graph showing individual apraglutide Cmax values ​​and corresponding citrulline Rmax values ​​(ug / mL) by dose level at week 6. Cmax apraglutide (ng / mL), Cmax=maximum concentration, Rmax=maximum response. For regression, the regression line, equation, p-value for slope and R-squared value are displayed. [Figure 22] Graph showing individual apraglutide AUCtau values ​​and corresponding citrulline Rmax values ​​(ug / mL) by dose level at week 6. AUCtau = area under the plasma concentration-time curve until the end of the treatment period for the corresponding dose, Rmax = maximum response. For regression, the regression line, equation, p-value for slope and R-squared value are displayed. [Figure 23] Correlation plot of individual apraglutide AUCtau values ​​and corresponding citrulline Rmax values ​​by dose level at week 6. Black dots represent assessments before the last dose, and lines connect subsequent assessments up to 2 weeks after the last dose. [Figure 24] Correlation plot of all apraglutide concentrations and corresponding citrulline concentrations for each subject dosed with 1 mg at week 6. Black dots represent assessments before the last dose, and lines connect subsequent assessments up to 2 weeks after the last dose. [Diagram 25] Correlation plot of all apraglutide concentrations and corresponding citrulline concentrations for each subject dosed with 5 mg at week 6. Black dots represent assessments before the last dose, and lines connect subsequent assessments up to 2 weeks after the last dose. [Figure 26A] 1 is a graph showing predicted plasma apraglutide concentrations (ng / mL) for a 70 kg individual administered 2.5, 5, or 10 mg of apraglutide subcutaneously weekly. [Figure 26B] 1 is a graph showing predicted plasma citrulline concentrations (ug / mL) for a 70 kg individual administered 2.5, 5 or 10 mg of subcutaneous apraglutide weekly. [Figure 27] Graph showing individual and mean changes from baseline to post-treatment in plasma L-citrulline concentrations. Mean changes from baseline were analyzed using paired t-test. *p<0.05 Solid black lines represent mean changes, colored lines represent individual patients. Dotted lines=SBS-II (intestinal dysfunction), solid lines=SBS-IF (intestinal dysfunction). Δ indicates mean change from baseline. [Figure 28] FIG. 1 is a graph showing citrulline plasma concentrations (ug / mL) after three weekly doses of apraglutide. Mean±standard error of the mean (SEM) is shown. [Figure 29] 1 is a graph showing plasma apraglutide concentration (ng / mL) as a function of time (hours) following a single subcutaneous dose of 5 mg apraglutide.

[0020] Detailed Description Short bowel syndrome (SBS) is a disabling malabsorption disorder caused by extensive surgical intestinal resection. Patients who have undergone extensive intestinal resection may suffer from disorders in the gastrointestinal neuroendocrine feedback that regulates fluid and nutrient absorption. This disorder includes impaired postprandial secretion of GLP-2, which is normally produced by L-cells in the terminal ileum and colon. Lack of GLP-2 can lead to accelerated gastrointestinal emptying, gastrointestinal hypersecretion, reduced intestinal blood flow, disruption of immunological and barrier function, and inhibition of mucosal growth. The resulting lack of intestinal adaptation leads to the pathophysiological characteristics of SBS, including frequent diarrhea or stoma emptying, malnutrition, dehydration, electrolyte imbalance, and weight loss. SBS is also associated with significant morbidity and mortality, and reduced quality of life. In patients with SBS and intestinal failure (SBS-IF), parenteral support (PS), including any combination of intravenous fluids and / or parenteral nutrition, is required to maintain health and / or growth.

[0021] Providing long-term PS can result in severe complications such as catheter-related blood stream infection (CRBSI) and intestinal failure-associated liver disease (IFALD). In contrast to patients with SBS-IF, patients with SBS intestinal insufficiency (SBS-II) are managed without PS due to their ability to compensate for malabsorption with hyperphagia, metabolic regulation, and / or pharmacological treatment. However, patients with SBS-II may be at risk for fluid and electrolyte imbalance, necessitating repeated hospitalizations for PS administration. Overall, patients with SBS suffer from impaired quality of life, high morbidity and mortality, and high healthcare costs. Globally, SBS-IF is a neglected organ disorder with limited therapeutic possibilities. Therefore, identifying new therapies could improve disease awareness, morbidity and mortality, reduce debilitating symptoms, and ease the treatment burden on patients.

[0022] After surgical resection, the remaining intestine may undergo structural and functional changes to enhance its absorptive capacity (generally referred to as intestinal adaptation). Secretion of neuroendocrine peptides throughout the gastrointestinal tract contributes to this adaptation. The pathophysiological traits of SBS are often caused by impaired neuroendocrine feedback mechanisms and lack of intestinal adaptation. This includes impaired postprandial secretion of glucagon-like peptide-2 (GLP-2), which is produced by intestinal L-cells, primarily located in the terminal ileum and proximal colon.

[0023] Natural GLP-2 is produced by dipeptidyl peptidase-IV (DIPEPTIDYL PEPTIDASE-IV, It has a short circulating half-life due to cleavage by DPP4. Apraglutide is a next-generation synthetically produced GLP-2 analogue with a molecular structure designed to provide long-lasting consistent exposure and an increased half-life of at least 30 hours compared to human GLP-2 and other GLP-2 analogues. Apraglutide differs from human GLP-2 by four amino acid substitutions, identified through chemical structure-activity relationship studies of lipophilic amino acid substitutions at positions 11 and 16 of [Gly2]hGLP-2(1-33). In animal models, apraglutide promoted increases in intestinal length and body weight, villus height, and crypt depth. Pharmacokinetic (PK) and pharmacodynamic studies in animals have demonstrated that apraglutide is an effective anti-inflammatory drug. This suggests that glutide may have low clearance, long elimination half-life and high plasma protein binding compared to other GLP-2 analogues.Therefore, apraglutide may be a candidate for weekly dosing regimen.Apraglutide treatment may potentially help patients to restore intestinal autonomy or reduce PS requirement, improve malabsorption symptoms, reduce organ failure secondary to II and IF, and prevent patients with SBS-II from deteriorating into intermittent or chronic IF.

[0024] As used herein, the term "amino acid" includes both naturally occurring and non-naturally occurring amino acids. Unless otherwise specified, amino acids are L-amino acids. Unless otherwise specified, amino acid sequences are presented from the N-terminus to the C-terminus.

[0025] As used herein, the term "control" when used alone or in reference to an amount or level can refer to a level observed in a subject prior to administration of a treatment (e.g., an effective dose of a GLP-2 agonist), a level observed in a control subject or population of subjects (including levels observed over time). When used in reference to a subject, "control" can refer to the same subject prior to receiving a given treatment (e.g., an effective dose of a GLP-2 agonist), a similar subject not receiving any treatment for a given condition, or a similar subject receiving a treatment (e.g., surgery, wound care, and / or nutritional support) that does not include the given treatment (e.g., administration of an effective dose of a GLP-2 agonist).

[0026] As used herein, the term "GLP-2 agonist" collectively refers to analogs of naturally occurring GLP-2 in vertebrates that elicit activity similar or comparable to naturally occurring GLP-2, but that are structurally altered relative to a given vertebrate GLP-2 by the addition, deletion, substitution, modification of at least one amino acid, and / or by the incorporation of one or more amino acids bearing blocking groups. Such agonists preferably have an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of either GLP-2 or a fragment of GLP-2 having the same number of amino acid residues.

[0027] As used herein, the terms "patient" and "subject" are used interchangeably and refer to any subject for whom treatment is desired. In some embodiments, the subject is a mammal, e.g., a human or a non-human mammal. In some embodiments, the subject is a human.

[0028] As used herein, the term "treatment" (also referred to as "treat" or "treating") refers to any administration of a therapeutic entity (e.g., a therapeutic compound or composition described herein) that partially or completely reduces the need for parenteral support.

[0029] As used herein, the term "purity" is used to refer to purity as determined by chromatographic methods, more specifically, by Ultra Performance Liquid Chromatography (UPLC) and / or High Performance Liquid Chromatography (HPLC) methods.

[0030] As used herein, the term "equivalent" of a substance (e.g., an oxymer) is used to refer to the molar ratio of the substances, more specifically, the number of moles of a substance that will react with one mole of another substance.

[0031] Any one of the embodiments and / or aspects described herein may be combined with any other embodiment and / or aspect described herein, and any number of the embodiments and / or aspects may be combined.

[0032] GLP-2 agonists that can be used in the methods of the present invention include, for example, those disclosed in International Patent Application No. 2006 / 117565 and U.S. Patent No. 8,589,918, the entire contents of each of which are incorporated herein by reference. The GLP-agonist apraglutide and related compounds (see, for example, U.S. Patent No. 8,589,918) have excellent pharmacokinetic properties and are preferred for use in the methods of the present invention.

[0033] SEQ ID NO: Amino acid sequence

[0034] [Table 1]

[0035] In a preferred embodiment, the GLP-2 agonist is apraglutide, a peptide having the amino acid sequence of SEQ ID NO:2, where Nle is norleucine and D-Phe is the D-amino acid phenylalanine.

[0036] synthesis Methods for preparing apraglutide have been described in the art, including solid-phase peptide synthesis methods. For example, see U.S. Patent No. 8,580,918, which describes a method for preparing GLP-2 agonists, including apraglutide. In contrast to previous methods for synthesizing apraglutide (e.g., the method disclosed in U.S. Patent No. 8,580,918), which could only achieve a purity of 93%, the present disclosure provides a method for making apraglutide that is substantially, for example, ≧95% or ≧97% pure, which also provides a high enough yield to be used in a commercial manufacturing process. Furthermore, U.S. Patent No. 8,580,918 discloses an ammonia salt of apraglutide. A preferred salt of apraglutide is The synthesis of a new salt, the sodium salt, is disclosed herein.

[0037] The apraglutide production process is based on solid-phase peptide synthesis (SPPS) using the Fmoc (9-fluorenylmethyloxycarbonyl) strategy. The amino acid sequence is built stepwise from the C-terminus by successive cycles of Fmoc deprotection and coupling of the next Fmoc amino acid. The novel synthesis method of apraglutides has many improvements described below. Fmoc-Rink-amide-MBHA (MethylBenzHydril Amine) resin is used, and the link between the peptide and the resin is performed via a Knorr linker. After Fmoc deprotection, piperidine / DMF (N,N-dimethylformamide) washing is performed using oxyma (ethyl [hydroxyamino] cyanoacetate) to reduce the level of oxidized impurities. Two DIC (Diisopropylcarbodiimide) additions are performed to reduce the coupling time. After each amino acid coupling reaction, the completeness is controlled by a semi-quantitative Kaiser test based on revealing unreacted amines. After assembly and final Fmoc group deprotection, the peptide resin is washed with IPA (Isopropanol) and dried under vacuum. Using the novel synthesis process, the overall coupling time is reduced by about 25% compared to the initial conditions without affecting the crude peptide HPLC (High Performance Liquid Chromatography) purity and process yield.

[0038] After completion of peptide synthesis, cleavage of the peptide from the resin is carried out by treatment with a TFA (trifluoroacetic acid) / HO / anisole mixture at room temperature under nitrogen. The cleaved peptide is separated from the resin by filtration and further processed in improved downstream purification steps to produce the sodium salt of apraglutide with an HPLC purity of ≧97%.

[0039] The improved version of the apraglutide synthesis process (Process B; shown in FIG. 2A and FIG. 2B) includes a first purification by RP-HPLC (C18) chromatography in a TFA-based mobile phase (HO / acetonitrile) (adjusting the pH of the fractions using sodium bicarbonate (NaHCO3) to ≧90% purity), followed by a second purification by RP-HPLC (C18) in a NaHCO3 mobile phase (HO / acetonitrile) (to ≧97% purity), followed by desalting / buffer exchange by RP-HPLC (C18) in a sodium acetate (NaOAc) / HO / acetonitrile mobile phase. The fractions containing the product are pooled and lyophilized to produce the sodium salt of apraglutide with a purity of ≧97%. The improved version of the apraglutide synthesis process described herein can provide high purity apraglutide (e.g., ≧95% or ≧97% purity) while still maintaining a yield suitable for large-scale commercial production of apraglutide. As will be appreciated by those skilled in the art, methods for producing ultra-high purity compounds typically suffer from low yields, making their use in commercial manufacturing impractical.Surprisingly, the apraglutide synthesis method described herein not only provides apraglutide of high purity, but also exhibits yields suitable for use in commercial manufacturing situations.

[0040] The present disclosure provides a method for making a GLP-2 analog peptide, the method comprising: a) carrying out solid phase peptide synthesis (SPPS) to synthesize a GLP-2 analog peptide on a resin; b) cleaving the synthesized GLP-2 analog peptide from the resin and deprotecting the side chains of the synthesized GLP-2 analog peptide by treating the resin with a solution containing trifluoroacetic acid (TFA), water, and anisole; c) purifying the synthesized GLP-2 analog peptide using preparative reversed-phase high performance liquid chromatography (RP-HPLC); d) further purifying the product from step (c) using a second preparative RP-HPLC (and optionally a third preparative RP-HPLC with desalting and buffer exchange) to produce a purified peptide solution; e) lyophilizing the purified peptide solution to produce a purified peptide powder; f) packaging the purified peptide powder under argon.

[0041] The present disclosure provides a method for making a GLP-2 analog peptide, comprising performing SPPS on a resin to synthesize the GLP-2 analog peptide, where the resin is a 4-Methylbenzhydrylamine (MBHA) resin.

[0042] The present disclosure provides a method for making a GLP-2 analog peptide, comprising performing SPPS to synthesize the GLP-2 analog peptide on a resin, the SPPS comprising washing the resin with a solution comprising DMF and oxyma.

[0043] The present disclosure provides a method for making a GLP-2 analog peptide, comprising synthesizing a GLP-2 analog peptide by performing SPPS on a resin, the SPPS comprising a coupling step performed by contacting the resin with two volumes of a solution comprising DIC and oxyma, the volumes being contacted 30 minutes apart.

[0044] The present disclosure provides a method for making a GLP-2 analog peptide comprising purifying a synthesized GLP-2 analog peptide by contacting the peptide with a solution containing water, acetonitrile (ACN), and ammonium hydroxide (NHOH) to extract the crude peptide from the cleaved peptide-resin mixture, and omitting the subsequent acidification step.

[0045] The present disclosure provides a method for making a GLP-2 analog peptide, comprising purifying a synthesized GLP-2 analog peptide by RP-HPLC using a solution containing 0.05% TFA, water, and acetonitrile as an eluent, and adjusting the pH of the purified peptide fraction to about pH 7.9 using 0.1% (acetic acid) AcOH in water.

[0046] The present disclosure provides a method for producing a GLP-2 analog peptide, comprising purifying the synthesized GLP-2 analog peptide with a second purification by RP-HPLC using a solution comprising 0.05 M sodium bicarbonate (NaHCO3), water, and acetonitrile as the eluent.

[0047] The present disclosure provides a method for making a GLP-2 analog peptide, comprising performing HPLC purification using a 1.5 mM NaOAc / H2O / ACN solution as the mobile phase.

[0048] In some aspects of the methods of the present disclosure, step (a) comprises: i) preparing a resin on which SPPS is performed; ii) performing an initial Fmoc deprotection followed by a coupling reaction to add a first Fmoc-protected amino acid to the resin, thereby forming a protected peptide on the resin; iii) performing an Fmoc deprotection reaction followed by a coupling reaction to add at least one Fmoc-protected amino acid to the protected peptide; iv) repeating step iii until the GLP-2 analog peptide is synthesized on the resin, wherein the Fmoc-protected and side-chain-protected GLP-2 analog peptide is coupled to the resin. and repeating the peptides comprising L-histidyl-glycyl-L-aspartyl-glycyl-L-seryl-L-phenylalanyl-L-seryl-L-aspartyl-L-glutamyl-L-norleucyl-D-phenylalanyl-L-threonyl-lysoleucyl-L-leucyl-L-aspartyl-L-leucyl-L-leucyl-L-alanyl-L-alanyl-L-arginyl-L-aspartyl-L-phenylalanyl-L-isoleucyl-L-asparaginyl-L tryptophanyl-L-leucyl-L-isoleucyl-L-glutaminyl-L-threonyl-L-lysyl-L-isoleucyl-L-threonyl-laspartamide; v) performing an Fmoc deprotection reaction to produce a resin-linked side-chain protected GLP-2 analog peptide; vi) drying the resin-linked side-chain protected GLP-2 analog peptide.

[0049] In some aspects of the methods of the present disclosure, step (a)(i) comprises: (a1) The resin is dissolved in a solution containing dimethylformamide (DMF) and N,N-diisopropylethylamine (DIEA) under a N2 atmosphere, and 1 g of the resin is dissolved in the solution. Wash with 5 mL of solution per tube and (b1) Coupling of the Rink amide linker to the resin in a solution containing 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), DIEA, and hydroxybenzotriazole (HOBt) in DMF. And (c1) washing the product formed in step (b1) with DMF; (d1) carrying out a reduction reaction by contacting the resin with a solution containing acetic anhydride (AcO) and DIEA in DMF; (e1) washing the product formed in step (d1) with DMF.

[0050] In some aspects of the methods of the present disclosure, the resin is a methylbenzhydrylamine resin.

[0051] In some aspects of the disclosed methods, the product of steps (a)(i)(a1) through (a)(i)(e1) is Fmoc-Rink-amide-MBHA resin.

[0052] In some aspects of the disclosed methods, step (c1) and / or step (e1) include washing the product three times with a ratio of 5 milliliters of DMF per gram of resin.

[0053] In some aspects of the methods of the present disclosure, a coupling test may be performed between steps (d1) and (e1).

[0054] In some aspects of the methods of the present disclosure, a Kaiser test is performed to determine whether coupling completion has been achieved.

[0055] In some aspects of the disclosed methods, upon completion of step (a)(i)(e1), the product is Fmoc-His(Trt)-Gly-Asp(OtBu)-TmbGly-Ser(tBu)-Phe-Ser(tBu)-Asp(OtBu)-Glu(OtBu)-Nle-DPhe-Thr(tBu)-Ile-Leu-Asp(OtBu)-Leu-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Asn(Trt)-Trp(Boc)-Leu-Ile-Gln(Trt)-Thr(ψ Me,Me pro)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Rink-amide-MBHA resin.

[0056] In some embodiments of the methods of the present disclosure, performing an Fmoc deprotection reaction followed by a coupling reaction comprises: (a2) treating the resin with a solution of piperidine in DMF; (b2) washing the resin with DMF; (c2) The resin is reacted with at least one Fmoc-protected amino acid and diisopropylcarbodiimide (DIC) and cyanohydroxyiminoacetic acid in DMF. a solution containing ethyl acetate (oxyma), thereby coupling at least one Fmoc-protected amino acid; (d2) washing the product formed in step (c2) with DMF.

[0057] In some embodiments of the disclosed methods, the solution comprising piperidine in DMF is a 35% solution of piperidine in DMF.

[0058] In some embodiments of the disclosed methods, treating the resin with a solution of piperidine in DMF is carried out by washing the resin with a solution of piperidine in DMF for 3 minutes, followed by washing the resin with a solution of piperidine in DMF a second time for 3 minutes, followed by washing the resin with a solution of piperidine in DMF for 10 minutes.

[0059] In some embodiments of the disclosed methods, the solution comprising piperidine in DMF is a 20% solution of piperidine in DMF.

[0060] In some embodiments of the disclosed methods, the treatment of the resin with a solution of piperidine in DMF is carried out by washing the resin with a solution of piperidine in DMF for 15 minutes, followed by washing the resin with a solution of piperidine in DMF for a second 15 minutes.

[0061] In some embodiments of the disclosed methods, washing is performed at a ratio of 5 milliliters of a solution comprising piperidine in DMF to 1 gram of resin.

[0062] In some embodiments of the disclosed methods, washing the resin in step (b2) comprises performing a first DMF wash at a ratio of 15 milliliters of DMF per gram of resin, followed by a second DMF wash at a ratio of 5 milliliters of DMF per gram of resin.

[0063] In some embodiments of the disclosed method, washing the resin in step (b2) comprises: i) washing the resin with DMF; ii) washing the resin with a solution containing DMF and Oxyma.

[0064] In some embodiments of the disclosed methods, the solution comprising DMF and oxyma comprises two equivalents of oxyma.

[0065] In some embodiments of the disclosed methods, the residue Asp 3 Carrying out the Fmoc deprotection reaction involves treating the resin with a solution of piperidine and oxymer in DMF.

[0066] In some embodiments of the disclosed methods, the solution comprising piperidine and oxyma in DMF is a 10% piperidine and 2% oxyma solution in DMF.

[0067] In some embodiments of the disclosed methods, the solution comprising piperidine and oxyma in DMF is a 5% piperidine and 1% oxyma solution in DMF.

[0068] In some embodiments of the disclosed method, the solution containing piperidine and oxymer in DMF is 15% piperidine and 3% oxyma solution in MF.

[0069] In some embodiments of the disclosed methods, the solution comprising piperidine and oxyma in DMF is a 20% piperidine and 4% oxyma solution in DMF.

[0070] In some embodiments of the disclosed methods, the solution comprising piperidine and oxyma in DMF is a 25% piperidine and 5% oxyma solution in DMF.

[0071] In some embodiments of the disclosed methods, the solution comprising piperidine and oxyma in DMF is a 30% piperidine and 6% oxyma solution in DMF.

[0072] In some embodiments of the disclosed method, the treatment of the resin with a solution containing piperidine and oxyma in DMF is carried out by washing the resin with a solution containing piperidine and oxyma in DMF for 15 minutes, followed by a second wash of the resin with a solution containing piperidine and oxyma in DMF for 30 minutes.

[0073] In some aspects of the disclosed method, step (c2) comprises: i) contacting the resin with a solution comprising at least one Fmoc-protected amino acid and DIC and oxymer in DMF; ii) contacting the resin with a second amount of a solution comprising DIC.

[0074] In some embodiments of the disclosed methods, after contacting the resin with at least one Fmoc-protected amino acid, the resin is contacted with a second amount of a solution comprising DIC for about 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes.

[0075] In some embodiments, at least one protected amino acid is a dipeptide comprising Boc-His(Trt)-Gly-OH.

[0076] In some embodiments, at least one protected amino acid is a dipeptide comprising Fmoc-His(Trt)-Gly-OH.

[0077] In some embodiments, the dipeptide is pre-activated for 10 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, or 4 hours prior to coupling the dipeptide.

[0078] In some aspects, the dipeptide is preactivated at 5°C ± 2°C, 10°C ± 2°C, 15°C ± 2°C, 20°C ± 2°C, 25°C ± 2°C, 30°C ± 2°C, or 35°C ± 2°C prior to coupling the dipeptide.

[0079] In some embodiments, the dipeptide is pre-activated in a solution containing Boc-His(Trt)-Gly-OH / Oxima / DIC 2.5 mmol / 2.5 mmol / 2.5 mmol in 7 mL of DMF prior to coupling the dipeptide.

[0080] In some aspects of the disclosed method, step (c2) comprises: i) contacting the resin with at least one Fmoc-protected amino acid and a solution comprising DIC and oxymer in DMF; ii) contacting the resin with a second amount of a solution comprising DIC and Oxyma.

[0081] In some embodiments of the disclosed methods, a second amount of a solution comprising DIC and oxyma is contacted with the resin about 30 minutes after contacting the resin with the at least one Fmoc-protected amino acid.

[0082] In some aspects of the methods of the disclosure, in step (c2), the at least one Fmoc-protected amino acid is provided in a concentration of 1, 2, 3, 4, or 5 equivalents.

[0083] In some aspects of the disclosed methods, in step (c2), DIC is provided at a concentration of 1, 2, 3, 4, or 5 equivalents and oxyma is provided at a concentration of 1, 2, 3, 4, or 5 equivalents.

[0084] In some embodiments of the disclosed method, in step (c2), the at least one Fmoc-protected amino acid is Fmoc-Gln(Trt)-Thr(Ψ Me,Me pro)-OH.

[0085] In some embodiments of the method of the present disclosure, in step (c2), Fmoc-Gln(Trt)-Thr(Ψ Me,Me pro)-OH is provided in a concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 equivalents.

[0086] In some aspects of the methods of the present disclosure, the method further comprises between step (b2) and step (c2), performing a test for residual piperidine by measuring the amount of piperidine.

[0087] In some embodiments of the disclosed method, if the amount of piperidine measured is greater than 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ppm, the resin is washed again with DMF and / or a solution comprising DMF and oxymer.

[0088] In some aspects of the methods of the present disclosure, a coupling test is performed between step (c) and step (d).

[0089] In some aspects of the disclosed methods, the coupling test is a ninhydrin assay.

[0090] In some aspects of the disclosed methods, step a(v) comprises: (a3) treating the resin with a solution of piperidine in DMF; (b3) washing the resin with DMF; (c3) washing the resin with isopropanol.

[0091] In some embodiments of the disclosed method, washing the resin in step (b3) comprises: i) washing the resin with DMF; ii) washing the resin with a solution containing DMF and Oxyma.

[0092] In some aspects of the disclosed methods, washing the resin with isopropanol in step (c3) comprises washing the resin five times with isopropanol, each wash being performed at a ratio of 5 milliliters of isopropanol per gram of resin.

[0093] In some aspects of the disclosed methods, the product upon completion of step (a)(v) is H-His(Trt)-Gly-Asp(OtBu)-TmbGly-Ser(tBu)-Phe-Ser(tBu)-Asp(OtBu)-Glu(OtBu)-Nle-DPhe-Thr(tBu)-Ile-Leu-Asp(OtBu)-Leu-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Asn(Trt)-Trp(Boc)-Leu-Ile-Gln(Trt)-Thr(ψ Me,Me The protected peptide on resin is comprised of (pro)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Rink-amide-MBHA resin.

[0094] In some embodiments of the disclosed methods, the solution comprising TFA, water, and anisole is a solution comprising TFA, water, and anisole in a ratio of 95:2.5:2.5 TFA:water:anisole.

[0095] In some embodiments of the disclosed methods, the solution comprising TFA, water, and anisole is a solution comprising TFA, water, and anisole in a ratio of 90:5:5 TFA:water:anisole.

[0096] In some embodiments of the disclosed methods, the solution comprising TFA, water, and anisole is a solution comprising TFA, water, and anisole in a ratio of 80:10:10 TFA:water:anisole.

[0097] In some embodiments of the disclosed methods, the solution comprising TFA, water, and anisole is a solution comprising TFA, water, and anisole in a ratio of 70:15:15 TFA:water:anisole.

[0098] In some aspects of the disclosed method, step (b) comprises cleaving and deprotecting the cleaved and deprotected GLP-2 using tert-butyl methyl ether (MTBE). It further comprises precipitating and washing the analog peptide.

[0099] In some aspects of the disclosed methods, the product upon completion of step (b) is H-His-Gly-Asp-Gly-Ser. 5 -Phe-Ser-Asp-Glu-NLe 10 -DPhe-Thr-Ile-Leu-Asp 15 -Leu-Leu-Ala-Ala-Arg 20 -Asp-Phe-Ile-Asn-Trp 25 -Leu-Ile-Gln-Thr-Lys 30 -Ile-Thr-Asp-NH2.

[0100] In some aspects of the disclosed methods, step (c) comprises: (i) contacting the product of step (b) with a solution comprising water, acetonitrile and NH4OH; (ii) purifying the synthesized GLP-2 analog peptide using preparative reversed-phase high performance liquid chromatography (RP-HPLC).

[0101] In some embodiments, the solution of step (c)(i) comprising water and acetonitrile comprises a mixture of HO / ACN in an ammonia buffer in a ratio of 80:20.

[0102] In some embodiments, the solution of step (c)(i) comprising water and acetonitrile comprises a mixture of HO / ACN in an ammonia buffer in a ratio of 70:30.

[0103] In some embodiments, the solution of step (c)(i) comprising water and acetonitrile comprises a mixture of HO / ACN in an ammonia buffer in a ratio of 90:10, 80:20, 70:30, 60:40, 50:50, or 40:60.

[0104] In some aspects, the solution of step (c)(i) comprising water and acetonitrile is adjusted to a target pH of 7, pH 8, pH 9, pH 10, or pH 11.

[0105] In some embodiments, the solution of step (c)(i) comprising water and acetonitrile is adjusted to a target pH > 7.

[0106] In some embodiments, the solution of step (c)(i) comprising water and acetonitrile is adjusted to a target pH of 8.0±0.1.

[0107] In some aspects, the solution of step (c)(i) comprising water and acetonitrile is adjusted to a target pH of 8.0±0.1, ±0.2, ±0.3, ±0.4, ±0.5, ±0.6, ±0.7, ±0.8, or ±0.9.

[0108] In some embodiments, the solution of step (c)(i) comprising water and acetonitrile is adjusted to about pH 10.

[0109] In some embodiments of the disclosed methods, the pH is adjusted using 25% acetic acid.

[0110] In some embodiments of the disclosed methods, the pH is adjusted using 25% NH4OH in H2O.

[0111] In some aspects, the solution in step (c)(i) is maintained at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, or 80°C.

[0112] In some embodiments, the solution in step (c)(i) is maintained at 50°C.

[0113] In some aspects, the solution of step (c)(i) is maintained at room temperature.

[0114] In some aspects, the solution of step (c)(i) is maintained at a temperature disclosed herein for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 20 hours, 24 hours, 30 hours, 40 hours, 50 hours, 60 hours, or 72 hours.

[0115] In some embodiments, the solution of step (c)(i) is maintained at a temperature disclosed herein for 65 minutes.

[0116] In some embodiments, the solution of step (c)(i) is maintained at a temperature disclosed herein for 24 hours.

[0117] In some embodiments, the solution of step (c)(i) is maintained at 50° C. for 65 minutes.

[0118] In some embodiments, the solution of step (c)(i) is maintained at room temperature °C for 24 hours.

[0119] In some embodiments of the methods of the present disclosure, RP-HPLC is performed using a C18 column.

[0120] In some embodiments of the disclosed methods, RP-HPLC is performed using a solution comprising NaHCO3, water, and acetonitrile as the eluent.

[0121] In some aspects of the disclosed methods, the solution comprising NaHCO3, water, and acetonitrile comprises 0.01M, 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, or 0.5M NaHCO3.

[0122] In some embodiments of the disclosed methods, RP-HPLC is performed using a solution comprising TFA, water, and acetonitrile as the eluent.

[0123] In some embodiments of the disclosed methods, the solution comprising TFA, water, and acetonitrile is 0. Contains 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% TFA.

[0124] In some embodiments of the methods of the present disclosure, the RP-HPLC comprises adjusting the pH of the purified peptide fraction.

[0125] In some aspects of the disclosed methods, in step (d), the synthesized GLP-2 analog peptide is converted to its sodium cation form.

[0126] In some aspects of the disclosed methods, step (d) comprises adjusting the pH of the purified peptide solution to about pH 7.9 using 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, or 2% AcOH in water.

[0127] In some aspects of the disclosed methods, step (d) comprises adjusting the pH of the purified peptide solution to about pH 7.9 using 0.1% AcOH in water.

[0128] In some aspects of the disclosed methods, the product from step (c) is purified using HPLC to produce a further purified peptide solution.

[0129] In some aspects of the disclosed methods, HPLC is performed using a solution comprising AcOH, water, and acetonitrile.

[0130] In some aspects of the disclosed methods, the solution comprising AcOH, water, and acetonitrile is a 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% AcOH solution.

[0131] In some aspects of the disclosed methods, HPLC is performed using a solution comprising sodium acetate (NaOAc), water, and acetonitrile.

[0132] In some aspects of the disclosed methods, the solution comprising NaOAc, water, and acetonitrile is a 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, or 3 mM NaOAc solution.

[0133] In some embodiments of the disclosed methods, after HPLC purification, the pH of the purified peptide solution is further adjusted to about pH 7.9 using 0.1% AcOH in water.

[0134] In some embodiments of the disclosed methods, after HPLC purification, the pH of the purified peptide solution is further adjusted to about pH 5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 7.9, about pH 8, about pH 8.5, or about pH 9 using 0.1% AcOH in water.

[0135] In some embodiments, the starting materials shown in Table 1 can be used in the methods of the present disclosure. In some embodiments, the starting materials shown in Table 1 can have the purity shown in the "Purity" column of Table 1.

[0136] [Table 2]

[0137] In some embodiments of the methods of the present disclosure, the apraglutide has a purity of 95% or greater.

[0138] In some embodiments of the methods of the present disclosure, the apraglutide has a purity of 97% or greater.

[0139] In some embodiments, Des-Gly 4 Apraglutide, Aspartimide 3 , Asp 33 -OH, Des-Ser 7 Apraglutide and [Trp 25The concentration and / or presence of 2-(2,4,6-trimethoxyphenyl) can be determined using RP-HPLC. Table 2a shows the purity and major impurities in two variations of the synthesis. Table 2b shows the purity and yield in three variations of the apraglutide synthesis process described herein.

[0140] [Table 3]

[0141] [Table 4]

[0142] In some embodiments, the purified peptide pool does not contain any unspecified impurities at a concentration greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%. In some embodiments, the purified peptide pool does not contain any unspecified impurities at a concentration greater than 1%.

[0143] In some embodiments, the purified peptide pool comprises Des-Gly 4 In some embodiments, the purified peptide pool does not contain impurities, including GLP-2 analogs, at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the purified peptide pool does not contain Des-Gly 4 Does not contain GLP-2 analogues at concentrations greater than 3%.

[0144] In some embodiments, the purified peptide pool is 3 , Asp 33 -OH and Des-Ser 7 - Contains no impurities, including total GLP-2 analog impurities, at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the purified peptide pool contains no aspartimide, 3 , Asp 33-OH and Des-Ser 7 - Contains no total GLP-2 analogue impurities at a concentration greater than 2%.

[0145] In some embodiments, the purified peptide pool comprises a peptide pool of [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analogs at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the purified peptide pool does not contain impurities, including [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analog impurity at a concentration greater than 2%.

[0146] In some embodiments, the purity of the GLP-2 analog peptide sodium salt is greater than 95%, 97% or 99% as measured by RP-HPLC. In some embodiments, the purity of the purified peptide pool is greater than 95% as measured by RP-HPLC.

[0147] In some embodiments, the purity of the GLP-2 analog peptide sodium salt is such that it does not contain unspecified impurities at a concentration of more than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%. In some embodiments, the GLP-2 analog peptide sodium salt is such that it does not contain unspecified impurities at a concentration of more than 1%.

[0148] In some embodiments, the GLP-2 analog peptide sodium salt is Des-Gly. 4 In some embodiments, the GLP-2 analog peptide sodium salt does not contain impurities, including GLP-2 analogs, at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2 analog peptide sodium salt is Des-Gly. 4 Contains no GLP-2 analog impurities at concentrations greater than 3%.

[0149] In some embodiments, the GLP-2 analog peptide sodium salt is aspartimide.3 , Asp 33 -OH and Des-Ser 7 - Contains no impurities, including total GLP-2 analog impurities, at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2 analog peptide sodium salt is aspartimide. 3 , Asp 33 -OH and Des-Ser 7 -Contains no total GLP-2 analog impurities at concentrations greater than 2%.

[0150] In some embodiments, the GLP-2 analog peptide sodium salt is 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analogues at concentrations of more than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2 analogue peptide sodium salt contains no impurities, including [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analog impurity at a concentration greater than 2%.

[0151] In some aspects of the disclosed method, the method can further include an in-process control after conversion of the peptide to a sodium salt form, the in-process control being performed by RP-HPLC, and the acceptance criteria is that the peptide has a purity of ≧95.0% and is free of the following impurities: Des-Gly 4 Apraglutide ≦3.0%, aspartimide 3 , Asp 33 -OH and Des-Ser 7 Sum of apraglutides ≦2.0%, [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide≦2.0%, and any other unspecified impurities≦1.0%.

[0152] Without wishing to be bound by theory, in some embodiments, Fmoc-Gln(Trt)-Thr(Ψ Me , Me The use of Fmoc-Pro)-OH and Fmoc-Tmb-Gly-OH improves coupling efficiency and reduces aspartimide formation.

[0153] Without wishing to be bound by theory, in some embodiments, the use of oxime as a coupling additive during the Fmoc deprotection and coupling cycles reduces the oxidation and [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analog impurity formation is minimized.

[0154] Without wishing to be bound by theory, in some embodiments, the use of ACN for the mobile phase composition during sodium salt conversion by preparative RP-HPLC improves the sodium salt conversion of the GLP-2 analog peptide composition. In some embodiments, the elimination of the titration step during sodium salt conversion by preparative RP-HPLC improves the sodium salt conversion of the GLP-2 analog peptide composition. In some embodiments, the introduction of a pH adjustment during sodium salt conversion by preparative RP-HPLC improves the sodium salt conversion of the GLP-2 analog peptide composition.

[0155] Des-Gly 4 The GLP-2 analog impurity is a Gly that is not present in the peptide. 4 Contains aspartimide 3 The GLP-2 analog impurity is Asp 3 Including aspartimide formation during coupling. Asp 33 -OH GLP-2 analog impurities include C-terminal amide hydrolysis of the peptide. 7 GLP-2 analog impurities include Ser in the peptide. 7 Contains deletions. [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analog impurities include Fmoc-Gly(Tmb)-OH formation during the cleavage process.

[0156] In some embodiments, the GLP-2 analog peptide composition does not contain any individual impurity at a concentration greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4%. In some embodiments, the GLP-2 analog peptide composition does not contain any individual impurity at a concentration greater than 1.5%.

[0157] In some embodiments, the GLP-2 analog peptide composition comprises Des-Gly 4 Contaminants, including GLP-2 analogs, do not exceed 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2 analog peptide composition comprises Des-Gly. 4 Contains no GLP-2 analog impurities at concentrations greater than 3%.

[0158] In some embodiments, the GLP-2 analog peptide composition comprises β-Asp 3 In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including GLP-2 analogs, at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. 3 In some embodiments, the GLP-2 analog peptide composition does not contain GLP-2 analog impurities at a concentration of more than 1.5%. 3 Contains no GLP-2 analog impurities at concentrations greater than 1%.

[0159] In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including D-His GLP-2 analogs, at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2 analog peptide composition does not contain D-His GLP-2 analog impurities at concentrations greater than 1.5%. In some embodiments, the GLP-2 analog peptide composition does not contain D-His GLP-2 analog impurities at concentrations greater than 1%.

[0160] In some embodiments, the GLP-2 analog peptide composition comprises aspartimide. 3 , Asp 33 -OH and Des-Ser 7 - Contains no impurities, including total GLP-2 analog impurities, at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2 analog peptide composition contains aspartimide. 3 , Asp 33 -OH and Des-Ser 7 -Contains no total GLP-2 analog impurities at concentrations greater than 2%.

[0161] In some embodiments, the GLP-2 analog peptide composition comprises a peptide selected from the group consisting of [Trp 25 In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analog impurity at a concentration greater than 2%.

[0162] In some embodiments, the GLP-2 analog peptide is Des-Gly. 4 GLP-2 analogue, aspartimide 3 GLP-2 analogue, Asp33 -OH GLP-2 analogue, Des-Ser 7 GLP-2 analogues or [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analogs at concentrations greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%, 2.5%, 3%, 3.5%, 4%, or 5%.

[0163] In some embodiments, the GLP-2 analog peptide is Des-Gly. 4 GLP-2 analog peptide, aspartimide 3 GLP-2 analog peptide, Asp 33 -OH GLP-2 analog peptide, Des-Ser 7 GLP-2 analog peptide, or [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]GLP-2 analog peptide at concentrations greater than 0.5%.

[0164] In some embodiments, the GLP-2 analog peptide composition does not contain a total amount of impurities at a concentration of more than 1%, 3%, 5%, or 7%. In some embodiments, the GLP-2 analog peptide composition does not contain a total amount of impurities at a concentration of more than 5%.

[0165] In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including acetate, at concentrations greater than 1%, 2%, 3%, 4%, or 5%. In some embodiments, the GLP-2 analog peptide composition does not contain acetate at concentrations greater than 5%.

[0166] In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including trifluoroacetic acid, at concentrations greater than 0.01%, 0.03%, 0.05%, 0.07%, 0.1%, 0.2%, 0.5%, or 1%. In some embodiments, the GLP-2 analog peptide composition does not contain trifluoroacetic acid at concentrations greater than 0.1%.

[0167] In some embodiments, the GLP-2 analog peptide composition contains less than 5,000, 4,000, 3,000, 2,000, 1,000, 500, 400, 300, 200, or 100 ppm of impurities, including acetonitrile. In some embodiments, the GLP-2 analog peptide composition contains less than 450 ppm of acetonitrile.

[0168] In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including bacterial endotoxins, at concentrations greater than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg. In some embodiments, the GLP-2 analog peptide composition does not contain bacterial endotoxins at concentrations greater than 5.0 EU / mg.

[0169] In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including aerobic microorganisms, at a concentration of more than 1, 10, 50, 100, 150, or 200 CFU / 0.1 g. In some embodiments, the GLP-2 analog peptide composition does not contain aerobic microorganisms at a concentration of more than 100 CFU / 0.1 g.

[0170] In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including yeasts and molds, at a combined concentration of greater than 0.1, 1, 5, 10, 15, or 20 CFU / 0.1 g. In some embodiments, the GLP-2 analog peptide composition does not contain impurities, including yeasts and molds, at a combined concentration of greater than 10 CFU / 0.1 g.

[0171] In some embodiments, the GLP-2 analog peptide composition is free of impurities including acetate, trifluoroacetic acid, acetonitrile, bacterial endotoxins, aerobic microorganisms, yeast, mold, or any combination thereof.

[0172] After synthesis, the apraglutide active pharmaceutical ingredient ("API") is lyophilized and then formulated into a drug product.

[0173] compound The GLP-2 agonists of the present disclosure can be used alone as medicaments or in pharmaceutical compositions comprising one or more GLP-2 agonists as active ingredients and a pharma- ceutical acceptable adjuvant, excipient, diluent, or carrier. The pharmaceutical compositions may also contain other active ingredients.

[0174] Suitable pharma- ceutically acceptable carriers include carriers that are typically used with peptide-based drugs.For general guidance on drug formulation, see, for example, "Remington: The Science and Practice of Pharmacy" 22nd ed., Pharmaceutical Press, Philadelphia, PA, 2012.Non-limiting examples of suitable excipients include glycine, L-histidine, mannitol, and any combination thereof.

[0175] The preferred formulation uses glycine as a buffering agent. L-histidine is used in this formulation as a physical stabilizer. L-histidine also functions as a buffer to maintain the target pH. Mannitol is used in this formulation as a bulking agent in the lyophilization process step. Water for injection is the solvent for this formulation and is removed during the lyophilization step. Sodium hydroxide is used for pH adjustment. The pH can be measured and adjusted to 8.30±0.10 with sodium hydroxide (NaOH) solution.

[0176] The present disclosure provides a robust and reproducible lyophilization cycle for the manufacture of drug products. Two scaled-down development batches were filled using the same container closures intended to be used in the manufacture of clinical batches. The lyophilization cycle was optimized for primary drying (primary drying cycle) to define the process to be implemented in clinical manufacturing of the drug product. The optimized lyophilization cycle resulted in a drug product with expected quality attributes (i.e., assay, purity and impurity levels, reconstitution time and moisture content) and was deemed appropriate for clinical batches of the drug product. Stability testing performed on the second scaled-down development batch demonstrated that the drug product remained unchanged at the intended storage temperature (2-8°C) for up to three months.

[0177] In some aspects, the compositions of injectable apraglutide and reconstitution solutions shown in Table 3 are exemplary compositions that may be produced using the methods of the present disclosure.

[0178] [Table 5] a The amount of drug substance used is calculated based on the apraglutide content in the corresponding drug substance batch. b An ingredient used during the manufacture of a drug product that does not appear in the final product.

[0179] In some embodiments, the apraglutide composition comprises glycine, L-histidine, mannitol, water for injection (WFI), sterile water for injection (sWFI). , sodium hydroxide, sucrose, or any combination thereof.

[0180] In one embodiment, the present disclosure provides a pharmaceutical composition comprising the apraglutide of the present disclosure, glycine, L-histidine and mannitol dissolved in water, wherein the concentration of apraglutide is 25mg / mL, the concentration of glycine is 3.75mg / mL, the concentration of L-histidine is 7.75mg / mL water, and the concentration of mannitol is 115.0mg / mL. In some embodiments, the volume of water can be 0.5mL. In some embodiments, the above pharmaceutical composition can further comprise sodium hydroxide in an amount such that the pH of the solution is about pH 8.3.

[0181] In some embodiments, the osmolality of the pharmaceutical composition of the present disclosure is 290-780 mOsmol / kg. In some embodiments, the osmolality of the pharmaceutical composition of the present disclosure is about 780 mOsmol / kg. In some embodiments, the osmolality of the pharmaceutical composition of the present disclosure is about 780±160 mOsmol / kg.

[0182] The injectable GLP-2 analog peptide composition may be a lyophilized powder for an injectable solution produced aseptically. It may be provided in a colorless glass vial suitable for lyophilized sterile products, closed with a rubber stopper, and sealed with an aluminum cap. Before administration, the injectable GLP-2 analog peptide may be dissolved in 0.5 mL of sterile water for injection (sWFI). The reconstituted solution may be administered subcutaneously.

[0183] Administration In some embodiments, the GLP-2 agonist, such as apraglutide, is administered parenterally, for example, by injection. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Liquid carriers for injectable solutions include Examples of aqueous solutions include, but are not limited to, water, saline, aqueous dextrose, and glycols.

[0184] In some embodiments, apraglutide is delivered via a two-chamber syringe or a dual cartridge injector, one example of which is described in PCT / EP2012 / 000787, the contents of which are incorporated herein by reference.

[0185] dosage The amount that constitutes an effective dose may depend on various factors, such as the disease and clinical condition of the patient (e.g., body weight) and the route of administration. Doses may be administered, for example, twice daily, once daily, twice weekly, weekly, biweekly, once or twice monthly, etc. Doses generally range from about 1 mg to about 10 mg per week for a period of about 1 week to about 100 weeks. In some embodiments, the weekly dose is about 1 mg to 10 mg. In some embodiments, the subject is medicated for about 1 week to about 100 weeks, about 1 week to about 80 weeks, about 1 week to about 60 weeks, about 1 week to about 48 weeks, about 2 weeks to about 24 weeks, about 2 weeks to about 20 weeks, or about 2 weeks to about 16 weeks. In some embodiments, the subject is administered a dose about once per week. In some embodiments, the subject is administered a dose about once every two weeks or about twice per month. About once per two weeks or about twice per month.

[0186] Apraglutide increases citrulline levels in a dose-dependent manner. Citrulline is a marker of small intestinal enterocyte mass. Dosing of apraglutide at 1 mg, 5 mg and 10 mg induces a long-term increase in citrulline concentrations in patients.

[0187] In some embodiments, apraglutide is administered parenterally (e.g., subcutaneously, intravenously, intramuscularly, or orally). In some embodiments, apraglutide is administered intravenously. In some embodiments, apraglutide is administered subcutaneously.

[0188] As body weight decreased, exposure (AUC and C max ) increases nonlinearly, it is advisable to dose patients weighing less than 50 kg with 2.5 mg to prevent high exposure. Patients weighing 50 kg or more may receive doses of 5 mg or more.

[0189] effect In some embodiments, a subject administered an effective dose of a GLP-2 agonist requires less nutritional support compared to a control. For example, in some embodiments, a subject administered an effective dose of a GLP-2 agonist requires at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% less nutritional support compared to a control. In one embodiment, a subject administered an effective dose of a GLP-2 agonist requires 100% less nutritional support compared to a control (i.e., enteral autonomy).

[0190] In some embodiments, subjects administered an effective dose of a GLP-2 agonist require fewer days of complete parenteral support compared to a control. For example, in some embodiments, administration of an effective dose of a GLP-2 agonist may reduce the length of time complete parenteral support is required by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% compared to a control. In one embodiment, administration of an effective dose of a GLP-2 agonist reduces the length of time complete parenteral support is required by 100% compared to a control (i.e., enteral autonomy). In some embodiments, subjects administered an effective dose of a GLP-2 agonist require fewer days of complete parenteral support compared to a control (i.e., enteral autonomy). Requires total parenteral support for less than 80 days per year, less than 75 days per year, less than 70 days per year, less than 65 days per year, less than 60 days per year, less than 55 days per year, less than 50 days per year, less than 45 days per year, less than 40 days per year, less than 35 days per year, less than 30 days per year, less than 25 days per year, less than 20 days per year, less than 15 days per year, less than 10 days per year, or less than 5 days per year.

[0191] In some embodiments, a subject administered an effective dose of a GLP-2 agonist does not require total parenteral nutrition following treatment.

[0192] In some embodiments, patients receiving apraglutide: a. Has intact colonic continuity ("CIC"), no stoma (small bowel less than 200 cm from the duodenojejunal curvature based on available medical / surgical records) and last bowel resection at least 12 months prior to screening, OR b. Include male and female subjects with short bowel syndrome-associated intestinal failure ("SBS-IF") receiving parenteral support following surgical resection of the small intestine, either by jejunostomy or ileostomy (<200 cm from the duodenal-jejunal flexure, based on available medical / surgical records), with the most recent bowel resection at least 6 months prior to screening.

[0193] In some embodiments, administration of an effective dose of a GLP-2 agonist results in a reduction in hospital stay compared to a control. For example, in some embodiments, administration of an effective dose of a GLP-2 agonist can reduce hospital stay by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% compared to a control. In some embodiments, subjects administered an effective dose of a GLP-2 agonist are hospitalized for less than 50 days, less than 45 days, less than 40 days, less than 35 days, less than 30 days, less than 25 days, less than 20 days, less than 15 days, less than 10 days, or less than 5 days.

[0194] In some embodiments, administration of an effective dose of a GLP-2 agonist to a population of subjects reduces mortality compared to a population of subjects receiving standard care treatments (e.g., surgical treatments, nutritional support, and wound care) that do not include administration of an effective dose of a GLP-2 agonist. For example, in some embodiments, the mortality rate is reduced to less than 15%, less than 12%, less than 10%, less than 8%, less than 5%, less than 3%, less than 2%, or less than 1%. SC injections are typically administered in the abdominal region or thigh. The injection site should be rotated so that the injection is administered at least 5 cm away from where the last injection was administered.

[0195] It is believed that apraglutide can demonstrate clinical efficacy (reduction in PS volume) in SBS-IF subjects with either CIC or stoma after 24 weeks of weekly dosing.Thus, in one embodiment, a single 2.5mg dose (for subjects with body weight less than 50kg at the most recent study visit) or 5mg dose (for subjects with body weight 50kg or more at the most recent study visit) of apraglutide or corresponding placebo is administered by subcutaneous ("SC") injection once a week for a treatment period of 24 weeks (stoma) or 48 weeks (CIC).

[0196] Apraglutide demonstrated superior effects on energy absorption at less frequent dosing (i.e., once weekly) compared to other GLP-2 analogues. At a dose of 5 mg once weekly, apraglutide caused a 140.1% (+15.8) change from baseline increase in urinary flow, a baseline change in wet weight absorption of 760.4 g / day (+236.1), and a baseline change in energy absorption of 1,074 kJ / day (+377).

[0197] Apraglutide promoted an increase in absolute urinary output by an adjusted mean of 711 mL / day (95% CI 132 to 1,289; P = .021) compared with placebo, which corresponds to a 48% daily increase (95% CI 12 to 84; P = .014), as shown in Table 9. Apraglutide promoted an increase in absolute urinary output by 714 mL / day (95% CI 132 to 1,289; P = .021) compared with placebo, which corresponds to a 48% daily increase (95% CI 12 to 84; P = .014), as shown in Table 10. Treatment with 10 mg of apraglutide promoted an increase in absolute urinary output by an adjusted mean of 795 mL (95% CI, 195 to 1,394; P = .014) compared with placebo. The corresponding change in relative urine production was 34% (95% CI, -4 to 71; P = .072).

[0198] Apraglutide increased urinary sodium excretion by an adjusted mean of 56 mmol / day (95% CI -10 to 123; P = .087) compared with placebo, as shown in Table 9. Apraglutide at 5 mg increased urinary sodium excretion by an adjusted mean of 66 mmol / day (95% CI -69 to 201; P = .171) compared with placebo, as shown in Table 10. In the 10 mg dose group, absolute urinary sodium excretion increased by an adjusted mean of 88 mmol / day (95% CI 20 to 156; P = .017) compared with placebo, as shown in Table 9.

[0199] Apraglutide increased intestinal energy absorption by 1,095 kJ / day (95% CI 196 to 1,994; P = .024), as shown in Table 19.

[0200] In contrast, teduglutide dosed once daily caused a 139.3% change from baseline increase in urinary flow, a baseline change in wet weight absorption of 743 g / day (+119.25), and a baseline change in energy absorption of 792 kJ / day (+570), as reported in Jeppesen, Gut, 54, pp. 1224-1231 (2005).

[0201] Similarly, in contrast, once-daily administered grepaglutide was As reported in Gastroenterol Hepatol, 4, pp. 354-363 (2019), this resulted in a change from baseline increase in urinary output of 111% (0.1 mg dose), 140% (1.0 mg dose), or 132% (10 mg dose), a baseline change in wet weight absorption of -211 g / day (0.1 mg dose), 650 g / day (1.0 mg dose), or 786 g / day (10 mg dose), and a baseline change in energy absorption of -377 kJ / day (0.1 mg dose), 435 kJ / day (1.0 mg dose), or 588 kJ / day (10 mg dose).

[0202] Any use of growth factors such as natural GLP-2, GLP-1 or GLP-2, GLP-1 analogues, glutamine or growth hormones other than the investigational IMP must be discontinued for 12 months (CIC subjects) and 6 months (stoma subjects) prior to administration of apraglutide.

[0203] In some embodiments, administration of an effective dose of a GLP-2 agonist to a population of subjects reduces mortality as compared to a population of subjects receiving standard care treatments (e.g., surgical treatments, nutritional support, and wound care) that do not include administration of an effective dose of a GLP-2 agonist. For example, in some embodiments, morbidity is reduced by less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%.

[0204] In some embodiments, apraglutide is used for postoperative intestinal autonomy restoration or for the treatment of enterocutaneous fistula. In some embodiments, apraglutide is used for the treatment of anastomotic leak, functional intestinal failure, intestinal dysfunction, necrotizing enterocolitis, graft-versus-host disease, Crohn's disease, or celiac disease. Used for treatment.

[0205] The present disclosure provides methods of treating short bowel syndrome in a subject, comprising administering at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure.

[0206] The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in treating short bowel syndrome in a subject, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0207] The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for the manufacture of a medicament for treating short bowel syndrome in a subject, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0208] The present disclosure provides a method for increasing wet weight intestinal absorption in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing wet weight intestinal absorption in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing wet weight intestinal absorption in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0209] In some aspects, the increase in wet weight intestinal absorption can be at least about 100 g / day, or at least about 200 g / day, or at least about 300 g / day, or at least about 400 g / day, or at least about 500 g / day, or at least about 600 g / day, or at least about 700 g / day, or at least about 800 g / day, or at least about 900 g / day, or at least about 1000 g / day.

[0210] The present disclosure provides a method for reducing fecal output in a subject with short bowel syndrome, comprising administering at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure to the subject. The present disclosure provides a method for reducing stoma output in a subject with short bowel syndrome, comprising administering at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure to the subject. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for reducing fecal output in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for reducing fecal output in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0211] In some aspects, the reduction in fecal output can be at least about 100 g / day, or at least about 200 g / day, or at least about 300 g / day, or at least about 400 g / day, or at least about 500 g / day, or at least about 600 g / day, or at least about 700 g / day, or at least about 800 g / day, or at least about 900 g / day, or at least about 1000 g / day.

[0212] The present disclosure provides a method for increasing urine production in a subject having short bowel syndrome, comprising: The present disclosure provides a method comprising administering at least one therapeutically effective amount of the GLP-2 analog peptide composition of the present disclosure to a subject.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing urine production in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing urine production in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0213] In some aspects, the increase in urine production can be at least about 100 g / day, or at least about 200 g / day, or at least about 300 g / day, or at least about 400 g / day, or at least about 500 g / day, or at least about 600 g / day, or at least about 700 g / day, or at least about 800 g / day, or at least about 900 g / day, or at least about 1000 g / day.

[0214] The present disclosure provides a method for increasing sodium and / or potassium absorption in a subject having short bowel syndrome, the method comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing sodium and / or potassium absorption in a subject having short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing sodium and / or potassium absorption in a subject having short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0215] In some aspects, the increase in sodium and / or potassium absorption can be at least about 5 mmol / day, or at least about 10 mmol / day, or at least about 15 mmol / day, or at least about 20 mmol / day, or at least about 25 mmol / day, or at least about 30 mmol / day, or at least about 35 mmol / day, or at least about 40 mmol / day, or at least about 45 mmol / day, or at least about 50 mmol / day.

[0216] The present disclosure provides a method for increasing urinary sodium and / or potassium excretion in a subject having short bowel syndrome, the method comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing urinary sodium and / or potassium excretion in a subject having short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing urinary sodium and / or potassium excretion in a subject having short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0217] In some aspects, the increase in urinary sodium and / or potassium excretion can be at least about 5 mmol / day, or at least about 10 mmol / day, or at least about 15 mmol / day, or at least about 20 mmol / day, or at least about 25 mmol / day, or at least about 30 mmol / day, or at least about 35 mmol / day, or at least about 40 mmol / day, or at least about 45 mmol / day, or at least about 50 mmol / day.

[0218] The present disclosure provides a method for increasing intestinal absorption of energy in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing intestinal absorption of energy in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for intestinal absorption of energy in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0219] In some aspects, the increase in intestinal absorption of energy can be at least about 500 kJ / day, or at least about 600 kJ / day, or at least about 700 kJ / day, or at least about 800 kJ / day, or at least about 900 kJ / day, or at least about 1000 kJ / day, or at least about 1100 kJ / day, or at least about 1200 kJ / day.

[0220] The present disclosure provides a method for reducing the energy content of fecal output in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for reducing the energy content of fecal output in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for reducing the energy content of fecal output in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0221] In some aspects, the reduction in the energy content of fecal excretion can be at least about 500 kJ / day, or at least about 600 kJ / day, or at least about 700 kJ / day, or at least about 800 kJ / day, or at least about 900 kJ / day, or at least about 1000 kJ / day, or at least about 1100 kJ / day, or at least about 1200 kJ / day.

[0222] The present disclosure provides a method for increasing carbohydrate and / or lipid absorption in a subject having short bowel syndrome, the method comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing carbohydrate and / or lipid absorption in a subject having short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing carbohydrate and / or lipid absorption in a subject having short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0223] In some aspects, the increase in carbohydrate and / or lipid absorption can be at least about 100 kj / day, or at least about 200 kj / day, or at least about 300 kj / day, or at least about 400 kj / day, or at least about 500 kj / day, or at least about 600 kj / day, or at least about 700 kj / day, or at least about 800 kj / day, or at least about 900 kj / day, or at least about 1000 kj / day, or at least about 1100 kj / day, or at least about 1200 kj / day.

[0224] The present disclosure provides a method for increasing protein absorption in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing protein absorption in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing protein absorption in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0225] In some aspects, the increase in protein absorption can be at least about 100 kJ / day, or at least about 200 kJ / day, or at least about 300 kJ / day, or at least about 400 kJ / day, or at least about 500 kJ / day, or at least about 600 kJ / day, or at least about 700 kJ / day, or at least about 800 kJ / day, or at least about 900 kJ / day, or at least about 1000 kJ / day, or at least about 1100 kJ / day, or at least about 1200 kJ / day.

[0226] The present disclosure provides a method for increasing weight in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing weight in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing weight in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0227] In some aspects, the weight gain can be at least about 0.5 kg, or at least about 1.0 kg, or at least about 1.5 kg, or at least about 2.0 kg, or at least about 2.5 kg, or at least about 3.0 kg, or at least about 3.5 kg, or at least about 4.0 kg, or at least about 4.5 kg, or at least about 5.0 kg.

[0228] The present disclosure provides a method for increasing lean body mass in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing lean body mass in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing lean body mass in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0229] In some aspects, the increase in lean body mass may be at least about 0.5 kg, or at least about 1.0 kg, or at least about 1.5 kg, or at least about 2.0 kg, or at least about 2.5 kg, or at least about 3.0 kg, or at least about 3.5 kg, or at least about 4.0 kg, or at least about 4.5 kg, or at least about 5.0 kg.

[0230] The present disclosure provides a method for reducing fat mass in a subject having short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a method for reducing body fat mass in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for reducing fat mass in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject at least one therapeutically effective amount.

[0231] In some aspects, the loss of fat mass can be at least about 0.5 kg, or at least about 1.0 kg, or at least about 1.5 kg, or at least about 2.0 kg, or at least about 2.5 kg, or at least about 3.0 kg, or at least about 3.5 kg, or at least about 4.0 kg, or at least about 4.5 kg, or at least about 5.0 kg.

[0232] The present disclosure provides a method for increasing the concentration of L-citrulline in plasma in a subject with short bowel syndrome, comprising administering at least one therapeutically effective amount of the GLP-2 analog peptide composition of the present disclosure to the subject.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing the concentration of L-citrulline in plasma in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing the concentration of L-citrulline in plasma in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0233] In some aspects, the increase in plasma L-citrulline concentration can be at least about 5 μmol / L, or at least about 10 μmol / L, or at least about 15 μmol / L, or at least about 20 μmol / L.

[0234] The present disclosure provides a method for increasing urine output in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for increasing urine output in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for increasing urine output in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0235] In some aspects, the increase in urine output can be at least about 400 mL / day, or at least about 500 mL / day, or at least about 600 mL / day, or at least about 700 mL / day, or at least about 800 mL / day, or at least about 900 mL / day, or at least about 1000 mL / day.

[0236] The present disclosure provides a method for reducing oral fluid intake in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for reducing oral fluid intake in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for reducing oral fluid intake in a subject with short bowel syndrome. The present disclosure provides a method for administering a GLP-2 analog peptide composition to a subject in at least one therapeutically effective amount.

[0237] In some aspects, the reduction in oral fluid intake can be at least about 400 mL / day, or at least about 500 mL / day, or at least about 600 mL / day, or at least about 700 mL / day, or at least about 800 mL / day, or at least about 900 mL / day, or at least about 1000 mL / day.

[0238] The present disclosure provides a method for reducing parenteral support in a subject with short bowel syndrome, comprising administering to the subject at least one therapeutically effective amount of a GLP-2 analog peptide composition of the present disclosure. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for reducing parenteral support in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount. The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for reducing parenteral support in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0239] In some aspects, the reduction in parenteral support can be at least about 400 mL / day, or at least about 500 mL / day, or at least about 600 mL / day, or at least about 700 mL / day, or at least about 800 mL / day, or at least about 900 mL / day, or at least about 1000 mL / day.

[0240] The present disclosure provides a method for reducing plasma aldosterone concentration in a subject with short bowel syndrome, comprising administering at least one therapeutically effective amount of the GLP-2 analog peptide composition of the present disclosure to the subject.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in a method for reducing plasma aldosterone concentration in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.The present disclosure provides a GLP-2 analog peptide composition of the present disclosure for use in the manufacture of a medicament for reducing plasma aldosterone concentration in a subject with short bowel syndrome, wherein the GLP-2 analog peptide composition of the present disclosure is for administration to the subject in at least one therapeutically effective amount.

[0241] In some aspects, the decrease in plasma aldosterone concentration can be at least about 500 pmol / L, or at least about 750 pmol / L, or at least about 1000 pmol / L, or at least about 1250 pmol / L, or at least about 1500 pmol / L, or at least about 1750 pmol / L, or at least about 2000 pmol / L, or at least about 2250 pmol / L, or at least about 2500 pmol / L, or at least about 2750 pmol / L, or at least about 3000 pmol / L.

[0242] In some embodiments of the above method, the increase can be at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% increase compared to the control level. In some embodiments, the control level is the amount before administration of the GLP-2 analog peptide composition of the present disclosure.

[0243] In some embodiments of the above method, the decrease can be at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% decrease compared to the control level. In some embodiments, the control level is the amount before administration of the GLP-2 analog peptide composition of the present disclosure.

[0244] In some embodiments, the increase or decrease recited by the above methods is after at least about 4 weeks of treatment with the GLP-2 analog peptide composition of the present disclosure.

[0245] In some aspects, the short bowel syndrome can be short bowel syndrome intestinal dysfunction (SBS-II). In some aspects, the short bowel syndrome can be short bowel syndrome intestinal failure.

[0246] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0247] Exemplary embodiments Embodiment 1. A composition comprising a sodium salt of apraglutide, wherein the sodium salt of apraglutide has a purity of 95% or greater, and the apraglutide has the structure:

[0248] [ka]

[0249] Embodiment 2. The composition of embodiment 1, wherein the sodium salt of apraglutide has a purity of 97% or greater.

[0250] Embodiment 3. The composition contains 3% or less Des-Gly. 4 3. The composition of embodiment 1 or 2, comprising an apraglutide impurity.

[0251] Embodiment 4. Aspartimide in the composition 3 Apraglutide, Asp 33 -OH Aplug Lutide and Des-Ser 7 The composition of any one of embodiments 1 to 3, wherein the total of apraglutide impurities is 2% or less.

[0252] Embodiment 5. The composition comprises 2% or less of [Trp 25 5. The composition of any one of embodiments 1 to 4, comprising an apraglutide impurity of (1,2-(2,4,6-trimethoxyphenyl)).

[0253] Embodiment 6. The composition contains no more than 1.5% β-Asp 3 6. The composition of any one of embodiments 1 to 5, comprising an apraglutide impurity.

[0254] Embodiment 7. The composition contains 1% or less β-Asp 3 7. The composition of any one of embodiments 1 to 6, comprising an apraglutide impurity.

[0255] Embodiment 8. The composition of any one of embodiments 1-7, wherein the composition contains 1% or less of a D-His apraglutide impurity.

[0256] Embodiment 9. The composition comprises: Asp below 1% 33 -OH apraglutide impurity, Des-Ser below 1% 7 Apraglutide impurities, Less than 1% D-aspartimide 3 Apraglutide impurities, Less than 1% [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity, Des-Gly in the composition 4 Apraglutide and Aspartimide 3 The composition of any one of embodiments 1 to 8, wherein the total of apraglutide impurities is 1% or less.

[0257] Embodiment 10. The composition of any one of embodiments 1-9, wherein the sodium salt of apraglutide is provided as a lyophilized powder.

[0258] Embodiment 11. A pharmaceutical composition comprising the composition according to any one of embodiments 1 to 10.

[0259] Embodiment 12. The pharmaceutical composition of embodiment 11, further comprising at least one of glycine, L-histidine, and mannitol.

[0260] Embodiment 13. The pharmaceutical composition comprises: Approximately 12.5 mg of apraglutide (sodium salt), Approximately 1.88 mg of glycine, Approximately 3.88 mg of L-histidine, and about 57.5 mg of mannitol.

[0261] Embodiment 14. The pharmaceutical composition of embodiment 12, wherein the pharmaceutical composition is provided as a lyophilized powder.

[0262] Embodiment 15. A dual-chamber powder syringe comprising a composition according to any one of embodiments 1 to 10 or a pharmaceutical composition according to any one of embodiments 11 to 14.

[0263] Embodiment 16. A method for making a GLP-2 analog peptide, comprising: a) carrying out solid phase peptide synthesis (SPPS) to synthesize a GLP-2 analog peptide on Fmoc-Rink-amide-methylbenzhydrylamine (MBHA) resin; b) Treating the resin with a solution containing trifluoroacetic acid (TFA), water, and anisole. cleaving the synthesized GLP-2 analog peptide from the resin and deprotecting the side chains of the synthesized GLP-2 analog peptide; c) purifying the synthesized GLP-2 analog peptide from step (b) by performing a first preparative reversed-phase high performance liquid chromatography (RP-HPLC) purification using a TFA-based mobile phase, thereby producing a solution containing the GLP-2 analog peptide having a purity of 90% or more; d) purifying the product of step (c) by performing a second RP-HPLC purification using a NaHCO3-based mobile phase, thereby producing a solution containing a GLP-2 analog peptide having a purity of 97% or more.

[0264] Embodiment 17. The method of embodiment 16, further comprising: e) further purifying the product from step (d) by performing a third RP-HPLC purification using a NaOAc-based mobile phase, thereby producing a solution comprising the sodium salt of the GLP-2 analog peptide having a purity of 97% or greater.

[0265] Embodiment 18. f) adjusting the pH of the solution containing the sodium salt of the GLP-2 analog peptide to about pH 7.9 using 0.1% AcOH in water; g) passing the product of step (f) through a filter having a pore size of 0.2 μm; 18. The method of embodiment 17, further comprising: h) lyophilizing the product of step (g), thereby producing a lyophilized sodium salt of the GLP-2 analog peptide having a purity of 97% or greater.

[0266] Embodiment 19.

[0267] The method of embodiment 16, further comprising: (i) decarboxylating the synthesized GLP-2 analog peptide by solubilizing the peptide in a solution comprising water and acetonitrile in an ammonia buffer.

[0268] Embodiment 20. The method of embodiment 19, wherein the pH of the solution comprising water and acetonitrile in ammonia buffer is adjusted to about pH 8.0.

[0269] Embodiment 21. Step (a) comprises: i) preparing an MBHA resin on which SPPS is performed; ii) performing an initial Fmoc deprotection reaction followed by a coupling reaction to add a first Fmoc-protected amino acid to the resin, thereby forming a protected peptide on the resin; iii) performing an Fmoc deprotection reaction followed by a coupling reaction to add at least one Fmoc-protected amino acid to the protected peptide; iv) repeating step iii until the GLP-2 analog peptide has been synthesized on the resin to produce a resin-linked Fmoc-protected and side-chain-protected GLP-2 analog peptide; v) performing an Fmoc deprotection reaction to produce a resin-linked side-chain protected GLP-2 analog peptide; The method according to any one of embodiments 16 to 20, comprising: vi) drying the resin-linked side-chain protected GLP-2 analog peptide.

[0270] Embodiment 22. Step (a)(i) comprises: (a1) The resin is dissolved in dimethylformamide (DMF) and N,N-diisopropyl alcohol under a N2 atmosphere. Washing with a solution containing propylethylamine (DIEA) at 5 mL per gram of resin; (b1) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in DMF, coupling the Rink amide linker to the resin in a solution containing hexafluorophosphate benzotriazole tetramethyluronium (HBTU), DIEA, and hydroxybenzotriazole (HOBt); (c1) washing the product formed in step (b1) with DMF; (d1) carrying out a reduction reaction by contacting the resin with a solution containing acetic anhydride (AcO) and DIEA in DMF; (e1) washing the product formed in step (d1) with DMF.

[0271] Embodiment 23. Carrying out an Fmoc deprotection reaction followed by a coupling reaction comprises: (a2) treating the resin with a solution of piperidine in DMF; (b2) washing the resin with DMF; (c2) washing the resin with a solution containing DMF and oxymer; (d2) contacting the resin with at least one Fmoc-protected amino acid and a first amount of a solution comprising diisopropylcarbodiimide (DIC) and ethyl cyanohydroxyiminoacetate (Oxima); (e2) contacting the resin with a second amount of a solution comprising DIC and Oxyma; 23. The method of any one of embodiments 21-22, comprising: (f2) washing the product formed in step (e2) with DMF.

[0272] Embodiment 24. The method of embodiment 23, wherein the resin is contacted with a solution comprising a second amount of DIC and Oxyma about 30 minutes after contacting the resin with the solution comprising the first amount of DIC and Oxyma.

[0273] Embodiment 25. Carrying out an Fmoc deprotection reaction followed by a coupling reaction comprises: (a2) treating the resin with a solution of piperidine and oxymer in DMF; (b2) washing the resin with DMF; (c2) washing the resin with a solution containing DMF and oxymer; (d2) contacting the resin with at least one Fmoc-protected amino acid and a first amount of a solution comprising diisopropylcarbodiimide (DIC) and ethyl cyanohydroxyiminoacetate (Oxima); (e2) contacting the resin with a second amount of a solution comprising DIC and Oxyma; 23. The method of any one of embodiments 21-22, comprising: (f2) washing the product formed in step (e2) with DMF.

[0274] Embodiment 26. The method of embodiment 25, wherein the resin is contacted with a first amount of a solution comprising piperidine and oxyma in DMF for 15 minutes, and then the resin is contacted with a second amount of a solution comprising piperidine and oxyma in DMF for 30 minutes.

[0275] Embodiment 27. At least one Fmoc-protected amino acid is Fmoc-Gln(Trt)-Thr(Ψ Me,Me 27. The method of any one of embodiments 23-26, wherein pro)-OH.

[0276] Embodiment 28. At least one Fmoc-protected amino acid is Fmoc-Gly(Tmb 27. The method of any one of embodiments 23-26, wherein R is —OH.

[0277] Embodiment 29. The method of any one of embodiments 23 to 26, wherein at least one protected amino acid is Boc-His(Trt)-Gly-OH.

[0278] Embodiment 30. The method of any one of embodiments 23 to 29, wherein the method further comprises performing a coupling test between steps (e2) and (f2), wherein the coupling test is a Kaiser test.

[0279] Embodiment 31. The method of any one of embodiments 16 to 30, wherein the GLP-2 analog peptide is apraglutide.

[0280] Embodiment 32. A composition comprising a GLP-2 analog peptide produced using the method according to any one of embodiments 16 to 31.

[0281] Embodiment 33. A method of treating short bowel syndrome associated intestinal failure (SBS-IF) or short bowel syndrome associated intestinal dysfunction (SBS-II) in a subject, comprising administering apraglutide or a pharma- ceutical acceptable salt thereof to the subject, Apraglutide or a pharma- ceutically acceptable salt thereof is administered at a dose of about 2.5 mg / week if the subject has a body weight of less than 50 kg; or The method, wherein apraglutide or a pharma- ceutically acceptable salt thereof is administered at a dose of about 5 mg / week when the subject has a body weight of 50 kg or more.

[0282] Embodiment 34. The method of embodiment 33, wherein the apraglutide or a pharma- ceutically acceptable salt thereof is administered by subcutaneous injection.

[0283] Embodiment 35. The method of embodiment 33 or 34, wherein the subject has colonic continuity and the apraglutide or a pharma- ceutically acceptable salt thereof is administered for about 48 weeks.

[0284] Embodiment 36. The method of embodiment 35, wherein the subject has greater than 50% colonic continuity.

[0285] Embodiment 37. The method of embodiment 33 or 34, wherein the subject has at least one stoma and the apraglutide is administered for about 24 weeks.

[0286] Embodiment 38. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of a meal-ingested wet weight in the subject compared to untreated or placebo-treated subjects.

[0287] Embodiment 39. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof reduces fecal output in the subject compared to untreated or placebo-treated subjects.

[0288] Embodiment 40. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof increases absolute urinary output in the subject compared to untreated or placebo-treated subjects.

[0289] Embodiment 41. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of sodium and potassium in the subject compared to untreated or placebo-treated subjects.

[0290] Embodiment 42. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof increases urinary excretion of sodium and potassium in the subject compared to untreated or placebo-treated subjects.

[0291] Embodiment 43. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of energy in the subject compared to untreated or placebo-treated subjects.

[0292] Embodiment 44. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof reduces the energy content of fecal excretion in the subject compared to untreated or placebo-treated subjects.

[0293] Embodiment 45. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of carbohydrates, proteins, and lipids in the subject compared to untreated or placebo-treated subjects.

[0294] Embodiment 46. The method of embodiment 33, wherein administration of apraglutide or a pharma- ceutically acceptable salt thereof increases citrulline concentrations in the subject compared to untreated or placebo-treated subjects.

[0295] Embodiment 47. The sodium salt of apraglutide having a purity of 95% or greater, wherein the apraglutide has the following structure:

[0296] [ka]

[0297] Embodiment 48. The sodium salt of apraglutide of embodiment 47, wherein the sodium salt of apraglutide has a purity of 97% or more.

[0298] Embodiment 49. The sodium salt of apraglutide contains 3% or less of Des-Gly. 4 The sodium salt of apraglutide according to any one of embodiments 1 to 48, comprising an apraglutide impurity.

[0299] Embodiment 50. Aspartimide in the sodium salt of apraglutide 3 Apraglutide, Asp 33 -OH Apraglutide and Des-Ser 7 The sodium salt of apraglutide according to any one of embodiments 1 to 49, wherein the total of apraglutide impurities is 2% or less.

[0300] Embodiment 51. The composition of sodium salt of apraglutide contains 2% or less of [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity.

[0301] Embodiment 52. The sodium salt of apraglutide contains 1.5% or less β-Asp 3 The sodium salt of apraglutide according to any one of embodiments 1 to 51, comprising an apraglutide impurity.

[0302] Embodiment 53. The sodium salt of apraglutide contains 1% or less of β-Asp 3 The sodium salt of apraglutide according to any one of embodiments 1 to 52, comprising an apraglutide impurity.

[0303] Embodiment 54. The sodium salt of apraglutide of any one of embodiments 1 to 53, wherein the sodium salt of apraglutide contains 1% or less of D-His apraglutide impurity.

[0304] Embodiment 55. The sodium salt of apraglutide is Asp below 1% 33 -OH apraglutide impurity, Des-Ser below 1% 7 Apraglutide impurities, Less than 1% D-aspartimide 3 Apraglutide impurities, Less than 1% [Trp 25 ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity, Des-Gly in the sodium salt of apraglutide 4 Apraglutide and Aspartimide 3 The sodium salt of apraglutide according to any one of embodiments 1 to 54, wherein the total of apraglutide impurities is 1% or less.

[0305] Embodiment 56. The sodium salt of apraglutide according to any one of embodiments 1 to 55, wherein the sodium salt of apraglutide is provided as a lyophilized powder.

[0306] Embodiment 57. A pharmaceutical composition comprising the sodium salt of apraglutide according to any one of embodiments 1 to 56.

[0307] Embodiment 58. The pharmaceutical composition of embodiment 57, further comprising at least one of glycine, L-histidine, and mannitol.

[0308] Embodiment 59. The pharmaceutical composition comprises: Approximately 12.5 mg of apraglutide (sodium salt), Approximately 1.88 mg of glycine, Approximately 3.88 mg of L-histidine, and about 57.5 mg of mannitol.

[0309] Embodiment 60. The pharmaceutical composition according to any one of embodiments 57 to 59, wherein the pharmaceutical composition is provided as a lyophilized powder.

[0310] Embodiment 61. A two-chamber powder syringe comprising a composition according to any one of embodiments 47 to 56 or a pharmaceutical composition according to any one of embodiments 57 to 60.

[0311] Working Example The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be merely exemplary of the invention and are not intended to limit the scope of what is regarded as the invention.

[0312] Example 1: Apraglutide manufacturing process (I) Solid Phase Peptide Synthesis (Step 1) The following is an exemplary method of the present disclosure for producing apraglutide at improved purity levels compared to previously described synthetic routes (e.g., U.S. Patent No. 8,580,918). SPPS is the sequential synthesis of peptide chains immobilized on a solid support by repeated cycles comprising the following steps: 1. Removal of the N-terminal Fmoc protecting group from peptide resin 2. DMF Washing 3. Coupling of Fmoc-AA-OH 4. Coupling Test 5. DMF Wash

[0313] This cycle is repeated until the peptide sequence is completed.

[0314] The α-amino group of the amino acid is protected with the base-sensitive 9-fluorenylmethyloxycarbonyl (Fmoc) group. The side chain functional groups are protected with acid-labile groups. All amino acid derivatives used in this process are commercially available.

[0315] SPPS is a stepwise synthesis of peptide chains immobilized on a solid support. In the synthesis, a peptide sequence can be assembled using MBHA resin. After swelling the resin and washing with DMF, then DMF / DIEA under nitrogen atmosphere, the Fmoc-Rink-amide linker can be coupled using HBTU / DIPEA / HOBt in DMF. After coupling, the resin can be washed with DMF and then acetylated using Ac2O / DIPEA. A Kaiser test can be performed to confirm the completion of coupling.

[0316] After washing the resin with DMF, each Fmoc-protected amino acid is coupled to the resin-bound peptide according to the following cycle: 1. The Fmoc-protecting group is removed with piperidine in DMF and the resin is washed extensively with DMF. 2. Couplings are carried out in DMF with varying amino acid equivalents using DIC / Oxyma for activation. 3. The coupling of amino acids is monitored by using a ninhydrin assay performed during each synthesis cycle.

[0317] At the end of the assembly, after the last amino acid is coupled and deprotected, the resin is washed with DMF and isopropanol and dried under vacuum.

[0318] Cleavage of the peptide from the resin and deprotection (Step 2) The protected peptide can be simultaneously cleaved from the resin and deprotected by treatment with a mixture of TFA / water / anisole. MTBE is then added to the peptide / TFA slurry to precipitate the crude peptide in the presence of the cleaved resin. The tide is filtered, washed with MTBE, and dried under vacuum to constant weight.

[0319] Decarboxylation Reaction (Step 3a) The crude peptide is solubilized in a mixture of HO / ACN (80:20 ratio) in ammonia buffer. The solution is adjusted to a target pH ≥ 7 using 25% NH4OH in HO. The decarboxylation reaction is maintained at room temperature for 24 hours. The crude peptide is washed with a solution of HO / ACN (80:20 ratio) in ammonia buffer at approximately pH 10 and stored at room temperature.

[0320] Purification by preparative RP-HPLC (step 3b) The crude peptide is dissolved in a mixture of water / acetonitrile / NH4OH. The solution is diluted with acetic acid and then filtered.

[0321] Primary purification is performed by preparative RP-HPLC using NaHCO3 / H2O / CH3CN as eluent. Elution from the column is monitored by UV and the obtained fractions are analyzed by RP-HPLC. Fractions that meet the monitoring criteria are mixed in a combined pool. Fractions that do not meet the monitoring criteria can be recycled by repeating the purification step. The purity of the pool is controlled by analytical RP-HPLC.

[0322] Conversion of the sodium salt by preparative RP-HPLC (Step 4) This step can be performed to exchange the peptide counterion from TFA anion to sodium cation via pH change and further purify the peptide. The combined pool from step 3 is diluted with water and re-purified by preparative RP-HPLC using NaOAc eluent.

[0323] The purified peptide solution is then subjected to evaporation under vacuum to reduce the amount of acetonitrile in the solution, and then the purified peptide solution is adjusted to a target pH of 7.9 using 0.1% AcOH.

[0324] The pure pool can be concentrated and lyophilized. The purity of the pool is analyzed by RP-HPLC.

[0325] Freeze-drying and packaging (Step 5) Prior to lyophilization, the purified peptide in solution is filtered through a 0.2 μm membrane. Lyophilization is performed at low pressure. The resulting lyophilized final peptide is packed under argon. The lyophilized apraglutide is controlled according to the apraglutide standard.

[0326] Reprocessing Lyophilized apraglutide that does not meet the standards established in the apraglutide specification can be subjected to repurification.

[0327] Repurification can be carried out after reconstitution of the peptide by repeating the purification and counterion exchange steps as described above.

[0328] After repurification, the material is lyophilized according to the procedure described above.

[0329] Freeze-dried apraglutide that does not meet the criteria established in the apraglutide specification may be subjected to re-lyophilization.

[0330] Re-lyophilization may be performed after reconstitution of the peptide by repeating the lyophilization step, as described above.

[0331] impurities In some aspects of the methods of the present disclosure, the apraglutide is substantially free of organic impurities, including, but not limited to, residues of reagents and materials (including by-products) used in the manufacturing process.

[0332] In some aspects of the methods of the present disclosure, the apraglutide is substantially free of non-peptide impurities.

[0333] In some aspects of the methods of the present disclosure, the apraglutide is substantially free of residual solvent.

[0334] Des-Gly 4 Apraglutide, Aspartimide 3 Apraglutide, Asp 33 -OH Apraglutide, Des-Ser 7Apraglutide and [Trp 25 Peptide impurities, including but not limited to, apraglutide, may be observed in apraglutide compositions.

[0335] Apraglutide for injection may be a lyophilized powder stored under long-term storage conditions (5° C.±3° C.). The stability results provided herein (Table 5) demonstrate that no degradation was observed under long-term storage conditions.

[0336] As required for parenteral apraglutide compositions, testing for bacterial endotoxins may be preferred to ensure the microbial quality of the apraglutide composition after manufacture, packaging, storage, and distribution. <85> This is carried out in accordance with the following:

[0337] Chemical properties of the product The compounds of the present disclosure are 33 amino acid synthetic peptide analogs of glucagon-like peptide-2 (GLP-2) that act as selective full agonists of the GLP-2 receptor with potency and selectivity comparable to that of native GLP-2.

[0338] The peptide of the present disclosure is a linear peptide. The peptide sequence contains one D stereoisomeric amino acid (D-phenylalanine), one unnatural amino acid (norleucine) and two achiral amino acids (glycine). All other amino acids are in the L-configuration. The peptide of the present disclosure is synthesized as a single enantiomer with all stereocenters of defined chirality. It can be isolated in its sodium salt form with some residual water as a natural component.

[0339] The disclosed peptide appears as a white to off-white homogeneous powder. It can be isolated by reverse phase chromatographic purification followed by lyophilization. No crystalline or polymorphic forms are known.

[0340] The solubility of the disclosed peptides in purified water is greater than 100 mg / mL.

[0341] The pH of a 100 mg / mL aqueous solution of the disclosed peptide is 7.6-8.4. The calculated isoelectric point is 4.3a.

[0342] The optimum solubility and chemical stability of apraglutide was observed at pH 8.0-8.5.

[0343] In-process management In some embodiments, the purity of the purified peptide pool as measured by RP-HPLC is greater than 95%, 97% or 99%. In some embodiments, the purity of the purified peptide pool as measured by RP-HPLC is greater than 95%.

[0344] In some embodiments, the in-process controls presented in Table 4 can be used in the methods of the present disclosure. In some embodiments, the in-process controls presented in Table 4 can have the purity presented in the "Acceptance Criteria" column of Table 4.

[0345] [Table 6]

[0346] The manufacturing process described in this example is referred to herein as Process A. Table 2a shows the purity of the apraglutide product produced using Process A, as well as the levels of major contaminants. As shown in Tables 2a and 2b, Process A can obtain apraglutide with a purity of 95% or more and low levels of contaminants. Furthermore, Process A shows product yields ranging from 15% to 22%.

[0347] Example 2: Measurement of in vitro GLP-2 agonist activity To determine the activity of the GLP-2 agonist of the present disclosure on the hGLP-2 receptor, a transcriptional reporter gene assay is used. Two constructs, an hGLP-2 receptor expression DNA construct and a reporter DNA construct containing an intracellular cAMP-responsive promoter element that controls the expression of firefly luciferase, are transiently transfected into human embryonic kidney cell line (HEK-293). (See, for example, HimmLer et al., J.Recept.Res., (1993), 13, 79-74 for details of this assay.) Cells are exposed to serial dilutions of compound at half-logarithmic dilutions per dose for 5 hours. After compound exposure, cells are lysed, luciferase activity is measured, and compound potency and EC50 values ​​are determined by nonlinear regression analysis. hGLP-2, a natural 33 amino acid peptide ligand, is used as an internal control for each experiment.

[0348] Example 3: Formulations To produce a formulation suitable for subcutaneous injection, apraglutide is aseptically manufactured and lyophilized with the pharmaceutical excipients glycine, L-histidine, and mannitol into a powder that is reconstituted with sterile water or buffer for injection.

[0349] To develop the lyophilized formulation, a screening study was conducted to evaluate the optimal bulking agents and excipients to achieve the desired physical stability of apraglutide in the liquid phase. Initial testing of the bulking agents sucrose and mannitol demonstrated that both were suitable for the lyophilization process as well as in terms of reconstitution time, moisture content, and visual inspection of the lyophilized product. The effect of concentrations and combinations of the stabilizers with the two bulking agents, sucrose and mannitol, was investigated. Glycine and Tris were added at 20 mM (1.50 mg / mL for glycine and 2.4 mg / mL for Tris) and 40 mM (3.0 mg / mL for glycine and 40 mM for Tris). The formulations were tested as buffers at concentrations of 4.8 mg / mL for Tris and pH 8.2-8.5. All formulations were tested with either mannitol or sucrose as bulking agents in the presence or absence of L-histidine at a concentration of 20 mM (3.10 mg / mL). A total of 11 formulations were prepared using a factorial design. The formulations were placed on an orbiting table and subjected to physical stress at an accelerated temperature of 40 °C for 3 days. The physical stability of the formulations was evaluated by size exclusion chromatography, optical density and viscosity. The chemical stability of apraglutide was evaluated by reversed-phase liquid chromatography and size exclusion chromatography. The chemical and physical stability of the formulations containing glycine in combination with histidine was superior to that of the formulations containing Tris buffer. Both mannitol and sucrose were suitable as bulking agents. However, mannitol was chosen due to its better processability during lyophilization.

[0350] Since the effect of glycine concentration could not be concluded from the screening study, a laboratory scale stability study was set up with two formulations containing glycine at concentrations of 20 Mm (1.50 mg / mL) and 40 Mm (3.0 mg / mL), respectively. Both formulations contained L-histidine, mannitol, apraglutide, and were pH adjusted to 8.3. The formulations were lyophilized and followed for 3 months at -20°C, 5°C, 25°C, and 40°C. In conclusion, at -20°C, 5°C, and 25°C, both formulations were chemically and physically stable throughout the 3-month study period. At 40°C, both formulations showed signs of reduced physical stability at 3 months. The formulation containing the lower concentration of 20 mM glycine had a favorable higher crystallinity of mannitol as assessed by X-ray powder diffraction. Therefore, 20 mM glycine was selected for the formulation.

[0351] Example 4: Citrulline as a marker of apraglutide effect Using plasma citrulline as a pharmacodynamic ("PD") marker , explained the effect of apraglutide. Similar to the pharmacokinetic ("PK") model development, PK / PD models were developed for healthy individuals from studies GLY-101-2015 and TA799-002 only. For the development of the PD model, the PD observations were fitted together with the PK observations using the final PK model with its structure, residual-, covariate-, and random effect-models. The starting model was a turnover model in which plasma citrulline was constantly synthesized and degraded with a synthesis rate of ksyn and a degradation rate of kdeg. Plasma apraglutide stimulated citrulline synthesis via a sigmoidal Emax relationship with baseline citrulline levels R0, half-maximal effect at concentration EC50, maximum effect Emax, and Hill coefficient gamma. Citrulline turnover was assumed to be at steady state such that citrulline synthesis was determined only by citrulline baseline and degradation. The population PK / PD of apraglutide in plasma was established using data from two studies in healthy volunteers and two studies with SBS patients testing doses of 2.5 to 50 mg SC. Concentrations of apraglutide are due to two-compartment and dose-nonlinear kinetics. Apraglutide clearance was estimated to be 16.8 L / day, with a median volume of distribution of 31.5 L for a 70 kg individual receiving 5 mg SC. The half-life was estimated to be 1.3 days. Volume of distribution and clearance were calculated using the coefficient β V1 / F,BW = 1.94, and β Cl / F,BW = 1.8 and was weight dependent. These coefficients showed strong weight dependence. The zero-order absorption duration was β Tk0,Dose It was dose-dependent with a coefficient of =0.249.

[0352] The effect of apraglutide on plasma citrulline was found to be sigmoidal, with citrulline saturation occurring at a dose of 5 mg. This phenomenon is consistent with the fact that its synthesis is increased by apraglutide. max A stimulated citrulline turnover model was used to account for this. Citrulline baseline was found to be 5.12 μg / mL, while the maximum effect was 0.626, with a half-maximal effective apraglutide concentration of 13.7 ng / mL.

[0353] A population PK / PD model was used to predict plasma apraglutide exposure and plasma citrulline trough concentration-time profiles with and without inter-individual variability for individuals weighing 40-120 kg receiving weekly doses of 2.5, 5, or 10 mg SC. Predictions without inter-individual variability indicated that the effect of apraglutide on citrulline began to saturate for 40 kg individuals receiving 5 mg apraglutide SC weekly, i.e., as body weight decreased, the same apraglutide dose resulted in a decreasing effect on citrulline. This saturation effect was found to be even more pronounced for individuals receiving 10 mg apraglutide SC weekly. Predictions with inter-individual variability showed large variability in plasma citrulline concentration-time profiles and plasma citrulline trough levels. Apraglutide exposure for individuals weighing 40-120 kg and receiving 2.5, 5, or 10 mg apraglutide SC dosing was compared to post-hoc exposure estimates in SBS patients. Bracketing between doses of 2.5 and 5 mg at 50 kg body weight was simulated. This bracketing variant resulted in apraglutide exposure within the range of the posterior AUC estimates in SBS patients receiving apraglutide 5 mg SC weekly. As a clinical biomarker, urinary excretion was correlated with posterior estimates of plasma apraglutide exposure and plasma citrulline trough levels in SBS patients. Apraglutide exposure and citrulline trough levels were found to be only weakly correlated with urinary excretion.

[0354] Plasma citrulline, a biomarker of intestinal cell mass, is increased following administration of GLP-2 analogues in human clinical trials.

[0355] A pharmacokinetic / pharmacodynamic (PK / PD) study of apraglutide, a novel long-acting GLP-2 analogue being developed for short bowel syndrome, and citrulline. The PK (prothrombin time) and PD (prothrombin time) relationships were evaluated in a randomized, double-blind, parallel-group, placebo-controlled, multiple-dose study. Twenty-three healthy adult volunteers received six weekly subcutaneous doses of apraglutide (1, 5, or 10 mg) or placebo and were followed for an additional six weeks after the last dose. Blood sampling was controlled for dietary, lifestyle, and diurnal effects. L-citrulline was quantified using a validated LC-MS method. PK data were subjected to noncompartmental analysis and PD data were analyzed by ANCOVA.

[0356] PK parameters showed a half-life of 72 hours. Increases in citrulline were observed 2 days after the first apraglutide dose, with maximal effects achieved after 4 or 7 days in most subjects. Mean citrulline levels increased from baseline in all apraglutide arms and remained elevated throughout the 6-week treatment period. Increases in citrulline were significantly greater with apraglutide 5 mg and 10 mg than with apraglutide 1 mg versus placebo (Table 5). There were no statistically significant differences between the 5 mg and 10 mg apraglutide dose levels. Plasma citrulline levels remained elevated for 10 to 17 days after the last apraglutide dose. Apraglutide was safe and well tolerated, with no serious AEs.

[0357] [Table 7]

[0358] Apraglutide was safe and well tolerated, and demonstrated longer PD responses than plasma exposure. These PK and PD data confirm the feasibility of once-weekly subcutaneous dosing of apraglutide.

[0359] Example 5: Phase 2 human clinical trial results of administration of the apraglutide composition of the present disclosure The following non-limiting examples describe results from a Phase 2 clinical trial in which subjects with SBS-IF were treated with an apraglutide formulation of the present disclosure. The Phase 2 trial investigated the safety and efficacy of 5 and 10 mg apraglutide in patients with SBS-IF.

[0360] Without wishing to be bound by theory, the beneficial effect of GLP-2 analogues in SBS patients may include dynamic changes in body fluid homeostasis, which can be considered when evaluating treatment results.Improved intestinal absorption reduces fecal excretion.Since a large amount of water and sodium absorbed in the intestine is excreted by the kidney through systemic circulation, increased absorption in SBS patients may lead to increased urine production and sodium excretion.Therefore, the hydration status of SBS patients can generally be monitored by measuring urine volume and urinary sodium.

[0361] Eight adult patients with SBS-IF were treated with the apraglutide compositions of the present disclosure according to Table 6. Briefly, patients with SBS-IF were administered 5 mg of the apraglutide of the present disclosure or a placebo once weekly for four weeks. The patients were then administered 10 mg of the apraglutide of the present disclosure once weekly for four weeks. Additionally, there was a 6-10 week washout period between treatments.

[0362] [Table 8]

[0363] result A total of 12 patients were screened as part of the study, of which 8 were randomized for treatment. All 8 patients continued the open-label regimen with 10 mg apraglutide (Part B) in the additional treatment period. One patient discontinued the study after the first drug dose in the Part B treatment period due to fatigue from study procedures and lack of response. The patient provided data for immediate, not post-treatment, measurements. Eight patients comprised the safety and full analysis sets. Patient demographics and baseline characteristics are summarized in Table 7. Data in Table 7 are mean (SD) or N (%), and parenteral support (PS) was the PS scheduled at study entry based on weekly averages.

[0364] Three patients had been previously treated (>6 months prior) with GLP-2 analogues in clinical trials. Individual patient plots showed that all effects returned to baseline after the washout period. Thus, no carryover effects were observed.

[0365] [Table 9]

[0366] safety results Common associated adverse events are reported in Table 8 (data are N or N (%)). Adverse events occurring in more than two patients included polyuria (n=7), stoma complications (n=6), gastrointestinal stoma complications (n=5), decreased gastrointestinal stoma drainage (n=6), abnormal gastrointestinal stoma drainage (n=5), decreased dry mouth (n=5), edema (n=4), weight gain (n=3), and anorexia (n=3). Three patients experienced injection site reactions. All injection site reactions occurred with active treatment. One patient had an injection site reaction in the 5 mg dose group and three patients had injection site reactions in the 10 mg dose group. A total of eight cases of serious adverse events were reported by five patients. None of these were considered related to the study drug. Two serious adverse events involved mechanical complications related to the tunneled central venous catheter used by the patients for PS administration, both of which required hospitalization for catheter exchange. Six serious adverse events were catheter-related bloodstream infections (CRBSI). One patient had four recurrent events of CRBSI. Serious adverse events were distributed equally between the placebo and treatment periods. Most patients had at least one treatment-related adverse event, all of which were mild to moderate, with no clear differences between active dose levels.

[0367] [Table 10]

[0368] There were no study discontinuations or attrition due to related adverse events. One patient omitted the third dose of apraglutide during the placebo period due to peripheral edema. No safety concerns arose from vital signs, blood samples, ECGs, dipstick urinalysis, or weights. There were no deaths.

[0369] Three patients developed anti-apraglutide antibodies during the study. Two patients were positive for anti-apraglutide antibodies at the end of the 5 mg treatment period and one patient was positive at the end of the 10 mg treatment period. The one patient who had positive antibodies at the end of the 5 mg treatment period also had positive antibodies at the end of the 10 mg treatment period. For the two patients who developed positive antibodies during the 5 mg treatment period, they were negative after a 6- to 10-week washout period. For the two patients with positive antibodies at the end of the 10 mg treatment, both were still positive after a 4- to 6-week washout period, but the titers were lower than at the end of the 10 mg treatment period. No effect of anti-apraglutide antibodies was detected on the PK profile, pharmacodynamic response to apraglutide, or the number or duration of adverse events.

[0370] Urine output and urinary sodium excretion: Change from baseline to end of treatment (days 27-29) The individual changes from baseline to the end of each treatment in absolute urinary output, including the mean change for each treatment, are plotted in Figure 5. The mean changes were different from the adjusted means calculated in the statistical analysis comparing treatment to placebo. For the statistical analysis of Parts A+B, as shown in Table 9, apraglutide 5 mg promoted an increase in absolute urinary output by an adjusted mean of 711 mL / day (95% CI 132 to 1,289; P = .021) compared to placebo, which corresponds to a daily increase of 48% (95% CI 12 to 84; P = .014). For the statistical analysis of Part A, as shown in Table 10, apraglutide 5 mg promoted an increase in absolute urinary output by an adjusted mean of 714 mL / day (95% CI 490 to 939; P = .002), which corresponds to a daily increase of 49% (95% CI 4 to 94; P = .041). Treatment with 10 mg apraglutide promoted an increase in absolute urine output by an adjusted mean of 795 mL (95% CI 195 to 1,394; P = .014) compared with placebo. The corresponding change in relative urine production was 34% (95% CI -4 to 71; P = .072). As shown in Table 9, no differences were seen between the 5 mg and 10 mg dose groups. In Table 9, data are presented as adjusted means (95% CI), calculations are based on change from baseline to or near the end of treatment for each dose group, and N indicates the number of patients in the complete analysis set. Results from the statistical analysis of Parts A+B are presented graphically in Figure 6.

[0371] [Table 11]

[0372] The individual changes from baseline to the end of each treatment in absolute urinary sodium excretion are plotted in Figure 7. For the statistical analysis of Parts A+B, apraglutide at 5 mg increased urinary sodium excretion by an adjusted mean of 56 mmol / day (95% CI -10 to 123; P = .087) compared to placebo, as shown in Table 9. For the statistical analysis of Part A, apraglutide at 5 mg increased urinary sodium excretion by an adjusted mean of 66 mmol / day (95% CI -69 to 201; P = .171) compared to placebo, as shown in Table 10. In Table 10, data are presented as adjusted means (95% CI), calculations are based on the change from baseline to the end of treatment for each dose group, and N indicates the number of patients in the complete analysis set. In the 10 mg dose group, absolute urinary sodium excretion increased by an adjusted mean of 88 mmol / day (95% CI 20 to 156; P = .017) compared with placebo, as shown in Table 9. Relative urinary sodium excretion did not change after apraglutide treatment, as shown in Tables 9 and 10. No differences were found between the 5 mg and 10 mg dose groups, as further shown in Table 9.

[0373] [Table 12]

[0374] Urine output and urinary sodium excretion: Change from baseline to immediately after the first treatment injection (days 1-3) Individual changes in absolute urinary output and urinary sodium excretion from baseline to immediately after the first treatment injection are plotted in Figures 8 and 9, respectively. As shown in Table 11, no immediate changes were observed in the 5 mg or 10 mg dose groups compared to placebo, and no significant changes were observed in the active dose groups. There were no differences between the groups. In Table 11, data are presented as adjusted means (95% CI), calculations are based on the change from baseline to immediately after the first treatment injection for each dose group, and N indicates the number of patients in the complete analysis set.

[0375] [Table 13]

[0376] Oral fluid intake: Change from baseline to near the end of treatment (days 20-22) On days 20 to 22, treatment with 10 mg apraglutide reduced absolute and relative oral fluid intake by adjusted means of 363 mL / day (95% CI, -641 to -86; P = .015) and -15% (95% CI, -25 to -5; P = .006), respectively. In the analysis of Part A+B, a mean reduction of 244 mL / day (95% CI, -512 to 23; P = .070), as shown in Table 9, was seen for the 5 mg dose group, and a mean reduction of 242 mL / day (95% CI, -560 to 76; P = .103), as shown in Table 10. Furthermore, no differences were found between the active dose groups, as shown in Table 9.

[0377] PS volume: change from baseline to near the end of treatment (days 20-22) On days 20-22, 10 mg apraglutide reduced relative daily PS volume by -28% (-51 to -4; P = .025) compared with placebo. A similar trend was seen for the absolute change in daily PS volume, which was reduced by 469 mL / day (-941 to 4; P = .052), as shown in Table 9. No change in PS volume was observed for the 5 mg dose group, as shown in Tables 9 and 10, and no differences were seen between the 5 mg and 10 mg dose groups, as shown in Table 9.

[0378] Fluid composite effect: Change from baseline to near the end of treatment (days 20-22) A post-hoc analysis for parts A+B was performed to calculate the combined fluid effect, defined as the sum of the increase in urine production (days 27–29), the decrease in PS volume, and the decrease in spontaneous oral fluid intake (days 20–22). Spontaneous oral fluid intake and the decrease in PS volume remained unchanged between urine collections and were therefore assessed near the end of the treatment period. Administration of 5 mg and 10 mg apraglutide resulted in a combined fluid effect of 1,036 mL / day (95% CI 262 to 1,810; P = .014) and 1,630 mL / day (95% CI 262 to 1,810; P = .014), respectively, compared with placebo. The effect of the two doses was 0.827 to 2,433; P = .001). There was no difference between the two doses, with an estimated difference of 594 (95% CI -207 to 1,396; P = .129). Individual plots of the combined fluid effects, along with urinary output at the end of treatment, are shown in Figures 10A-C.

[0379] Hydration state parameters In a post-hoc analysis of Part A+B apraglutide, 5 mg and 10 mg reduced plasma aldosterone by 2,894 pmol / L (95% CI, -6,247 to 458; P = .083) and 3,045 pmol / L (95% CI, -6,460 to 370; P = .075), respectively. Apraglutide did not alter other hydration parameters, including creatinine, urinary, hematocrit, albumin, protein, and CO2 total, as shown in Table 12. In Table 12, data are presented as adjusted means (95% CI) and calculations were based on baseline. Based on the change from to the end of treatment for each dose group, N indicates the number of patients in the complete analysis set.

[0380] [Table 14]

[0381] Plasma L-citrulline In statistical analysis of Parts A+B, apraglutide at 5 mg increased absolute plasma L-citrulline by an adjusted mean of 17.6 μmol / L (95% CI 2.0 to 33.2; P = .031), corresponding to a relative increase of 65% (95% CI 15 to 115; P = .015). In statistical analysis of Part A, apraglutide at 5 mg increased absolute plasma L-citrulline by an adjusted mean of 17.7 μmol / L (95% CI -6.3 to 41.7; P = .100), corresponding to a relative increase of 66% (95% CI -13 to 145; P = .077). The difference in change from baseline for the 10 mg dose versus placebo was smaller, as shown in Table 13. In Table 13, data are shown as adjusted mean (95%CI), calculation is based on the change from baseline to the end of treatment of each dose group, N indicates the number of patients in the complete analysis set. Individual changes from baseline in absolute plasma L-citrulline concentration are plotted in Figure 11. Mean changes from baseline and individual patient plasma L-citrulline changes are plotted in Figure 27. Starting 2 days after the first dose, a dose-dependent increase in plasma citrulline levels was observed with apraglutide. As shown in Figure 28, citrulline levels increased significantly after 3 weekly doses of apraglutide compared to placebo.

[0382] [Table 15]

[0383] Weight and body composition In the Part A+B analysis, 5 mg of apraglutide increased absolute and relative fat mass, respectively. 5 mg apraglutide significantly increased fat mass and body weight by 1.77 kg (95% CI 0.29 to 3.24; P = .024) and 10% (95% 1 to 18; P = .035). Corresponding to the increase in fat mass, apraglutide 5 mg increased body weight by 1.9 kg (95% CI 0.1 to 3.7; P = .043). No changes in lean body mass or bone mineral mass were observed for either dose group compared with placebo. In statistical analysis of Part A, trends toward increased fat mass and body weight were observed for apraglutide 5 mg, as shown in Table 14. In Table 14, data are presented as adjusted means (95% CI), calculations are based on change from baseline to end of treatment for each dose group, and N indicates the number of patients in the complete analysis set.

[0384] [Table 16]

[0385] Pharmacokinetics The plasma concentration of apraglutide increased after dosing, with the maximum mean concentration measured 72 hours after dosing. The mean plasma concentration of apraglutide before the second dose (C trough ) were 4.5 ng / mL and 8.9 ng / mL during the 5 mg and 10 mg treatment periods, respectively.

[0386] Summary of Example 5 The results described in Example 5 demonstrate that administration of the apraglutide composition of the present disclosure to subjects was safe, well tolerated, and induced beneficial changes in intestinal fluid absorption, including, but not limited to, increased urinary volume.

[0387] Treatment-related adverse events were mild to moderate and consistent with the physiological effects of GLP-2. There was no dose-dependence of adverse events. Injection site reactions were minor and reflective of the weekly dosing regimen. There were no treatment-related events of abdominal pain or abdominal distension.

[0388] Treatment with apraglutide doses of 5 and 10 mg once weekly increased urine output when compared with placebo. Apraglutide at 10 mg significantly increased urinary sodium excretion and reduced oral fluid intake as well as PS at the end of treatment (assessed near the end of treatment). Similar trends were seen with the 5 mg dose for urinary sodium excretion and oral fluid intake.

[0389] Overall, these increases in urine volume reflect increased intestinal fluid and sodium absorption. Without wishing to be bound by theory, this is an important effect for SBS patients who are at high risk for dehydration, sodium depletion, and renal dysfunction. Fluid and electrolyte abnormalities may also be a cause of morbidity and hospitalization.

[0390] Due to the relatively short treatment period of 4 weeks, if clinical signs of fluid retention were present, A reduction in PS volume was performed. It is assumed that during long-term apraglutide treatment, increased urine output may allow for further reduction in PS volume, as evidenced by a larger fluid combination effect. Treatment with 5 mg apraglutide increased relative urine output in 6 of 8 patients compared with placebo, exceeding the 10% increase that would have induced a reduction in PS volume.

[0391] Plasma concentrations of L-citrulline increased during both active treatment periods. Without wishing to be bound by theory, this indicates an increase in enterocyte mass, consistent with an enterotrophic effect of apraglutide on the intestinal epithelium. A reduced effect of 10 mg was observed in two patients who had a clinically significant increase in urinary output (assessed on days 27-29) during the 5 mg treatment period. However, these patients had a concomitant decrease in PS volume and oral fluid intake (assessed on days 20-22) during the 10 mg treatment period, resulting in a positive fluid composite effect, defined as the sum of the beneficial effects including increased urine production, decreased need for PS volume, and decreased oral fluid intake.

[0392] As understood by those skilled in the art, patients with SBS may suffer from secondary hyperaldosteronism due to excessive fecal sodium loss and chronic sodium depletion.In the study described in Example 5, it was found that administration of apraglutide tends to reduce aldosterone levels in both dose groups compared with placebo.Without wishing to be bound by theory, this indicates that apraglutide alleviates secondary hyperaldosteronism due to improved fluid and sodium absorption.

[0393] Treatment with 5 mg of apraglutide was associated with increases in fat mass and body weight. This is the first time that an increase in fat mass has been observed in subjects following treatment with GLP-2 or a GLP-2 analog. The increase in body weight correlates well with the corresponding changes in fat mass and / or lean mass.

[0394] The statistical analysis described in Example 5 was performed in two versions, where Part A compared 5 mg with placebo, while Part A+B included placebo, 5 mg, and 10 mg, estimating contrasts between the three treatments. The results of the Part A and Part A+B analyses were generally similar.

[0395] In summary, the results described in Example 5 demonstrate that apraglutide dosing at 5 and 10 mg once a week for 4 weeks was well tolerated and safe. The results also demonstrate the clinical efficacy of GLP-2 analogs with weekly dosing. Weekly treatment with apraglutide at both doses tested (5 and 10 mg) resulted in increased urine output and improved other markers of intestinal rehabilitation in patients with SBS, but no difference in response was observed. It is envisioned that the apraglutide composition of the present disclosure may contribute to patient care and compliance by allowing for weekly administration as opposed to daily, which in turn may improve quality of life. Reduced injection frequency may increase patient acceptance and reduce the risk of injection site reactions. These results indicate that apraglutide treatment leads to increased intestinal absorption by promoting intestinal adaptation. Without wishing to be bound by theory, these results indicate that in vivo apraglutide treatment can improve intestinal wet weight absorption in patients with SBS-IF and therefore can be implemented in a therapeutic setting.

[0396] method Study design and participants The study described in Example 5 was a double-blind, crossover, randomized, placebo-controlled, phase 2 study followed by an additional treatment period with an open-label regimen. Eight adult patients (ages ≥18 and ≤80 years) with SBS-IF were enrolled in the study. Patient eligibility was screened. Patients were assessed during cleaning visits. The main inclusion criteria were: patients with SBS secondary to surgical resection of the small intestine with jejunostomy or ileostomy, at least 6 months since last bowel resection, a documented fecal output of at least 1500 g / day within the past 18 months, and PS infusions ≥3 times per week for ≥12 months according to the patient's medical records. Patients were excluded if they had clinical signs of activity, a history of cancer within 5 years, or inadequate liver, kidney, or heart function. Patients were also excluded if they had received native GLP-2 or GLP-2 analogs within the past 3 months.

[0397] As shown in Figure 4, the study was organized into two parts, namely Parts A and B. Part A was a double-blind, crossover, randomized, placebo-controlled study. In Part A, patients were treated with 5 mg apraglutide or placebo once weekly for 4 weeks. After a washout period of 6–10 weeks, the alternative treatment was administered. Part A was followed by a second washout period of 6–10 weeks before patients entered Part B. Part B was an open-label dosing regimen of 10 mg apraglutide administered once weekly for 4 weeks. An additional open-label treatment period (Part B) was included to investigate the safety and tolerability of the 10 mg dosing. Procedures during each treatment period were consistent. The study drug was provided as a lyophilized powder for reconstitution and administered as a subcutaneous injection in the abdominal area.

[0398] procedure Safety assessments were performed during each treatment period and included injection site reactions, vital signs, blood samples, electrocardiogram (ECG), urine dipstick tests, body weight, and liver enzymes. Observations on liver enzymes were included that were performed at baseline, during each injection of study drug (pre-dose and successive time points), 4 days after the first treatment injection, at the end of each treatment period, and 4-6 weeks after the last dose at the end of the study, as shown in Table 15. In addition, liver enzymes were measured before each drug administration.

[0399] [Table 17]

[0400] Efficacy assessments were performed for each of the 29-day treatment periods. Patients received a paper diary to record PS volume administration, urine collections, and oral fluid intake at specific time points. Patients were assessed at baseline (days -2 to 1), immediately after the first treatment injection (days 1 to 3), and at the end of the treatment period. A 48-hour urine collection was performed at home on days 27-29 (5 days after the fourth dose of study drug). Urine was collected by the patient in a urine container with volume markings, and the 48-hour urine volume was reported in the diary. After completing the 48-hour collection, the patient transferred approximately 100 mL of urine from the container to a sample, which was delivered at the next patient visit. The amount of sodium excreted per 48 hours was then calculated as the concentration of sodium in the sample multiplied by the total volume of urine collected. During each urine collection, the weekly PS volume and content and the daily oral fluid intake were kept constant. As they were kept constant, an increase in urine volume was a sign of increased fluid absorption. The patient was informed to create a 24-hour drinking menu based on their habitual oral fluid intake at the baseline visit (day -3). The patient adhered to their drinking menu during each urine collection, and the accompanying 48-hour oral fluid intake was reported in the diary during each urine collection.

[0401] On days 20–22, patients were asked to record their spontaneous oral fluid intake over a 48-h period. This measurement was included to investigate whether increased intestinal absorption was associated with a decrease in spontaneous oral fluid intake.

[0402] Reductions in PS volume were allowed between days 4 and 24, if deemed clinically necessary by the investigator. Due to the relatively short treatment period of 4 weeks, PS volume was only reduced if the patient had clinical signs of fluid retention (such as edema and excessive unintentional weight gain). PS volume reduction was assessed between days 20 and 22. Patients returned to their baseline PS volume and content during the end of treatment urine collection (days 27–29). Concomitant medications, including proton pump inhibitors, loperamide, and opiates, were kept unchanged and stable throughout the study. If necessary, adjustments could be made in the drinking menu and prescribed PS volume and content during the treatment period based on discussion between the patient and the investigator.

[0403] Body weight was measured using a horizontal platform scale.Body composition was measured by dual energy X-ray absorptiometry (Norland XR-36 DXA densitometer, Ford Atkinson, WI, USA) at baseline and end of treatment.

[0404] Blood samples for analysis of plasma concentrations of apraglutide and fasting plasma L-citrulline (a marker of intestinal cell mass) were collected at baseline, during the first and second treatment injections, 4 days after the first treatment injection, and at the end of treatment. Blood samples for fasting plasma L-citrulline were also collected 4-6 weeks after the last dose at the end of the study. Blood samples for anti-apraglutide antibodies were collected at baseline, end of treatment, and 4-6 weeks after the last dose at the end of the study.

[0405] Analysis of urinary sodium concentration Urinary sodium was measured on a COBAS 8000 modular analyzer series using an ion-specific electrode system (Roche Diagnostics, Indianapolis, IN) with a lower limit of quantification of 10 mmol / liter. If urinary sodium was below the lower limit of quantification, samples were analyzed by flame photometry.

[0406] Analysis of Plasma Apraglutide Concentration and L-Citrulline Apraglutide and L-citrulline were quantified using a validated LC-MS based method. For Apraglutide, the analytical method used solid phase extraction purification of intact Apraglutide and its internal standard. Compounds were identified and quantified using reversed phase HPLC with MS / MS detection using an AB Sciex API 5000 quadrupole mass spectrometer. For samples below the quantification limit of 5.00 ng / mL, compounds were identified and quantified using reversed phase UHPLC with MS / MS detection using an AB Sciex API 5500 quadrupole mass spectrometer. For L-citrulline, the analytical method used protein precipitation extraction of L-citrulline and its internal standard. AB Sciex API 5000 quadrupole mass spectrometer. Compounds were identified and quantified using Hilic HPLC with MS / MS detection using a spectrometer.

[0407] Analysis of anti-apraglutide antibodies A fully validated ELISA method was used for the detection of anti-apraglutide antibodies in serum, following a three-stage assay approach. In the first stage, anti-apraglutide antibodies present in samples and controls were bound to apraglutide immobilized on a microtiter plate. The bound anti-apraglutide antibodies were then detected using Protein A / G and Protein L. Samples with a signal above the validated screening cut-point were considered potentially positive and were then tested in the second stage, the confirmatory assay. For this, the samples were pretreated with an excess of apraglutide before being tested in the above assay. Inhibition of the signal above the validated confirmatory cut-point confirmed the presence of the antibodies. The samples were then reported as anti-apraglutide positive. The titers were then determined by serial dilutions of all confirmed positive samples.

[0408] statistical analysis A crossover design was applied, where each patient served as his own control to eliminate intersubject variability and reduce the effects of confounding covariates.

[0409] Safety was evaluated in all patients who received at least one dose of study drug (active or placebo). Efficacy was assessed according to a modified intention-to-treat principle, including all randomized patients with at least one valid post-baseline efficacy measurement (full analysis set).

[0410] Two different statistical analyses were performed: a version for Part A only and a full version for Part A+B. The Part A version was based on a 2 × 2 crossover design, whereas the Part A+B version did not assume a period effect to estimate the contrasts between the three different treatments (placebo, 5 mg, and 10 mg). Both the Part A and Part A+B versions of the statistical analysis were included in the results. The analysis for Part A was randomized and blinded. The analysis for Part A+B was included to perform a comparison between placebo, 5 mg, and 10 mg. Analysis of variance was used to evaluate the effect of apraglutide. Period-specific baseline measurements of outcome variables, oral fluid intake, and PS volume were included in the analysis. All statistical tests were performed using two-sided tests at a significance level of 5%. Estimates were presented with approximately 95% confidence intervals and p-values. SAS version 9.4 was used for the analysis.

[0411] Example 6: Phase 2 metabolic balance study The following non-limiting examples describe results from a Phase 2 clinical trial in which subjects with short bowel syndrome intestinal failure and short bowel syndrome intestinal failure were treated with the apraglutide formulation of the present disclosure. The Phase 2 trial investigated the safety and efficacy of the apraglutide formulation of the present disclosure.

[0412] Treatment with glucagon-like peptide-2 (GLP-2) analogues promotes intestinal adaptation in patients with short bowel syndrome (SBS). Apraglutide is a novel long-acting GLP-2 analogue designed to allow once-weekly dosing. This Phase 2 study investigated the safety and efficacy of apraglutide 5 mg once-weekly in patients with SBS intestinal failure (SBS-IF) and SBS intestinal dysfunction (SBS-II). In this open-label study, eight adult patients with SBS-IF (n=4) or SBS-II (n=4) and fecal wet weight ≥ 1500 g / day were treated with apraglutide 5 mg subcutaneously once-weekly for 4 weeks. Safety was the primary endpoint. As secondary endpoints, a 72-hour metabolic balance study was used to examine intestinal absorption of wet weight, energy (measured by bomb calorimetry) and electrolytes, as well as changes from baseline in fecal wet weight and urine production. General Potential treatment-related adverse events were consistent with the physiological effects of GLP-2 and included decreased stoma output (n=6), stoma complications (n=6), nausea (n=5), flatulence (n=4), polyuria (n=3), and abdominal pain (n=3). Once-weekly apraglutide significantly increased energy, wet weight, and electrolyte absorption, decreased fecal wet weight, and increased urine production (Table 16).

[0413] [Table 18]

[0414] result Nine patients were screened. One patient did not meet the inclusion criteria of fecal output ≥ 1,500 g / day and urine output < 2,000 mL / day (assessed during baseline metabolic balance testing). Eight patients were medicated in the study. As shown in Figure 13, all eight patients completed the study and constituted the safety and complete analysis set. Patient demographics and baseline characteristics are summarized in Table 17. Data in Table 17 are mean (SD) or N (%), and parenteral support (PS) was the PS scheduled at the start of the study based on weekly averages.

[0415] [Table 19]

[0416] safety results All patients experienced at least one treatment-related adverse event. Adverse events were mild to moderate. Common adverse events are summarized in Table 18 (data are N or N (%)). Adverse events occurring in more than two patients included decreased gastrointestinal stoma output (n=6), stoma complications (n=6), gastrointestinal stoma complications (n=5), nausea (n=5), abnormal gastrointestinal stoma output (n=4), flatulence (n=4), polyuria (n=3), and abdominal pain (n=3). One patient experienced a transient injection site reaction (localized erythema and pruritus) after one injection, which was unrelated to the presence of anti-apraglutide antibodies. Three patients had previously received native GLP-2 or another GLP-2 analog in a clinical trial (minimum 16 months prior to study enrollment). A total of three serious adverse events (SAEs) were reported. ) occurred in two patients. One SAE, an event of abdominal pain requiring hospitalization, was assessed as related to study drug. The abdominal pain was treated conservatively and the patient was discharged within 24 hours. A temporary interruption and rechallenge at a reduced dose allowed the patient to complete the study. The remaining two SAEs were not considered related to apraglutide. They included one event of acute kidney injury due to dehydration in a patient with SBS-II and one event of CRBSI in a patient with SBS-IF. Two additional patients required dose reduction to complete the study. One patient with SBS-II had signs of fluid retention after the first drug dose and therefore the second and third doses were given at a reduced dose. The fourth / last dose was given at the full dose (5 mg) without further complications. One patient experienced constipation after the first drug dose and as a result the second dose was given at a reduced dose. The full dose was reintroduced for the third and fourth / last doses without further complications.

[0417] [Table 20]

[0418] No safety concerns arose from vital signs, blood samples, ECG, urine dipstick urinalysis, or weight. One patient with SBS-IF developed anti-apraglutide antibodies during the study. The antibodies were negative after a 4-6 week washout. The patient had previously been treated with a GLP-2 analogue in a clinical trial setting in 2016. No effect of anti-apraglutide antibodies on the pharmacokinetic profile, pharmacodynamic response to apraglutide, or number or duration of adverse events was detected. No patients discontinued the study due to adverse events, and no deaths occurred.

[0419] Efficacy Endpoints wet weight Individual changes from baseline in dietary intake, fecal excretion, urine, and wet weight absorption after treatment with apraglutide are shown in Figure 14. Apraglutide did not change wet weight of dietary intake, as shown in Table 19. In Table 19, data are presented as adjusted means (95% CI ) and calculations are based on change from baseline to end of treatment. Apraglutide increased wet weight intestinal absorption by 741 g / day (95% CI, 194 to 1287; P = .015), as shown in Table 19. Apraglutide significantly reduced fecal output by 680 g / day (95% CI, -1200 to -159; P = .018) and increased urine production by 560 g / day (95% CI, 72 to 1048; P = .030), as shown in Table 19.

[0420] [Table 21]

[0421] electrolyte Apraglutide increased sodium and potassium absorption by 38 mmol / day (95% CI 3 to 74; P = .039) and 18 mmol / day (95% CI 4 to 32; P = .020), respectively, as shown in Table 19. Urinary sodium and potassium excretion increased by 27 mmol / day (5 to 49; P = .024) and 13 mmol / day (95% CI 6 to 20; P = .003), as shown in Table 19. There were no changes in magnesium and calcium absorption or urinary excretion, as shown in Table 19. The electrolyte content of dietary intake and fecal excretion were unchanged, as shown in Table 19. Individual changes from baseline in electrolyte absorption are shown in FIG. 15.

[0422] Energy and macronutrients FIG. 16 shows the individual changes from baseline in the energy content of dietary intake, fecal excretion, and absorption of energy. Apraglutide did not alter total dietary energy intake or any individual macronutrients, as shown in Table 19. Compared to baseline, apraglutide increased intestinal absorption of energy by 1,095 kj / day (95% CI 196 to 1,994; P=.024), as shown in Table 19. Suggesting an improvement in energy absorption, the energy content of fecal excretion was reduced by 941 kj / day (95% CI -2,438 to 556 P=.181), as shown in Table 19. Carbohydrate and lipid absorption were significantly increased by 518 kj / day (95% CI 112 to 924; P=.019) and 376 kj / day (95% CI 61 to 691; P=.026), respectively, as shown in Table 19. Protein absorption increased by 104 kj / day (95% CI -205 to 412; P = .453), as shown in Table 19. Individual changes in macronutrient absorption are plotted in Figure 17.

[0423] Weight and body composition Body weight increased by 1.8 kg (95% CI, 0.4 to 3.1; P = .016) after 4 weeks of apraglutide treatment. Lean body mass increased significantly by 1.7 kg (95% CI, 0.8 to 2.6; P = .003), and fat mass decreased by 1.1 kg (95% CI, -2.1 to -0.0; P = .044). There was no significant change in bone mineral content, which changed by -20 g (95% CI, -58 to 19; P = .268).

[0424] Citrulline Compared with baseline, apraglutide increased absolute and relative plasma concentrations of L-citrulline by 15.2 μmol / L (95% CI, 3.3 to 27.1; P = .019) and 66% (95% CI, 3 to 128; P = .043), respectively.

[0425] Pharmacokinetics Plasma concentrations of apraglutide increased rapidly after dosing, reaching a maximum mean concentration of 118.0 ng / mL after a mean of 29.5 hours. The mean elimination half-life was 27.0 hours, and the mean clearance was 0.9 L / h.

[0426] Summary of Example 6 The results described in Example 6 demonstrate that 5 mg of apraglutide administered once weekly was safe and well tolerated in patients with SBS-II and SBS-IF after 4 weeks of treatment and showed a positive effect on intestinal absorption, which eliminates or reduces the need for PS in a clinical setting.

[0427] Adverse events were consistent with the known physiological effects of GLP-2. Frequently reported associated adverse events were of gastrointestinal origin, but they were generally mild and transient.

[0428] Apraglutide significantly increased intestinal absorption of wet weight, energy and electrolytes (sodium and potassium). The improved absorption was accompanied by a significant decrease in fecal wet weight and an increase in urine production and urinary electrolyte excretion (sodium and potassium). The changes in urine production found in this study are considered clinically relevant as they may allow a decrease in PS or help restore intestinal autonomy in patients with SBS-IF. It is envisioned that these improvements may also ultimately reduce the risk of developing intermittent or chronic IF in patients with SBS-II, reducing the symptom burden of malabsorption.

[0429] The pharmacokinetic profile of apraglutide supported a once-weekly dosing regimen.

[0430] Apraglutide is the first GLP-2 analogue that improves energy absorption across the entire patient range in SBS populations, as measured by bomb calorimetry, which is considered the gold standard laboratory method for quantifying intestinal energy absorption.For individual macronutrients, Apraglutide increases carbohydrate and lipid absorption, but does not change protein absorption.As will be understood by those skilled in the art, in the later stages of clinical development, the withdrawal of PS with the reduction of energy requirement is considered as evidence of improved energy absorption.

[0431] The PK profile of apraglutide allowed for consistent exposure which may explain the improved effect on energy absorption.

[0432] Apraglutide significantly increased body weight and lean body mass and decreased fat mass, indicating a potential for improved hydration status, as will be appreciated by those skilled in the art.

[0433] Apraglutide did not change the wet weight or energy content of food intake. Oral fluid intake was fixed, but solid food intake was not restricted. GLP-2 did not significantly affect appetite or postprandial satiety in healthy humans. Reduction in food intake may be an undesirable side effect in SBS patients who rely on binge eating to compensate for intestinal losses. Long-term GLP-2 treatment allowed patients to reduce food intake while maintaining a similar level of absorption. It is therefore envisioned that administration of apraglutide may alleviate the need for severe binge eating, which is a predominant symptom in patients with particularly severe SBS-II.

[0434] Apraglutide significantly increased plasma L-citrulline, a marker of enterocyte mass, supporting its expected adaptogenic effects. GLP-2 could also inhibit gastrointestinal motility and reduce gastric acid secretion, thereby increasing the exposure of enterocytes to luminal contents. Mesenteric blood flow could also be stimulated by GLP-2, which could increase nutrient absorption. GLP-2 could also upregulate transport proteins, as shown in animal studies.

[0435] The results described in Example 6 show that apraglutide treatment at 5 mg administered weekly for 4 weeks is safe and well tolerated in patients with SBS-IF and SBS-II. These results demonstrate for the first time that a once-weekly GLP-2 analogue significantly improves wet weight, energy, and electrolyte absorption, and increases urine production. Apraglutide only requires weekly injections, which may reduce the burden of injections and contribute to patient care and compliance. The reduction in injection frequency may also increase patient acceptance and reduce the risk of injection site reactions.

[0436] Treatment with apraglutide once a week increased intestinal absorption of fluid, electrolytes and energy.Without wishing to be bound by theory, these results indicate that treatment of patients with the apraglutide composition of the present disclosure can improve intestinal wet weight absorption and energy in patients with SBS-IF, and therefore can be implemented in a therapeutic setting.

[0437] method Study design and participants A total of nine adult patients (aged ≥18 and ≤80 years) with SBS were screened, eight of whom were enrolled in the study. Four patients had SBS-II and four had SBS-IF. Both subgroups of patients were included to investigate the safety and efficacy of apraglutide across the spectrum of disease. Patient eligibility was assessed during the screening visit. The main inclusion criteria were SBS secondary to surgical resection of the small intestine with or without the colon, at least 6 months since last surgical bowel resection, and severe malabsorption defined as fecal wet weight output ≥1,500 g / day and urine volume output <2000 mL / day. Fecal output and urine volume output were confirmed during baseline examination. Patients were excluded if they had clinical signs of activity, a history of cancer within 5 years, or inadequate liver, kidney, or heart function. Patients were also excluded if they were pregnant or lactating, had a positive HIV test, a positive hepatitis B or C test, had been hospitalized within 1 month prior to the screening visit, or had received native GLP-2 or a GLP-2 analog within the past 3 months.

[0438] procedure The patient was treated with 5 mg of apraglutide once a week for 4 weeks. Apraglutide was provided as a lyophilized powder to be reconstituted in sterile water prior to injection, and treatment was administered as a subcutaneous injection in the abdominal area. A 72-hour metabolic balance study was performed at baseline and at the end of the treatment period (starting 1 day after the 4th and final apraglutide injection). Each 72-hour metabolic balance study was performed during the 5-day hospital stay. On the day of admission, the patient was instructed to create a 24-hour drinking menu based on habitual oral fluid intake. The drinking menu had to be followed during each balance study. On the second day of admission, the balance study was started immediately after the patient urinated to empty the stoma bag or had a bowel movement. The patient was instructed to collect an exact replica (fluids and solids were separated) of fecal output, urine, and food intake in respective buckets that were replaced after 24 hours. Patients had free access to food, but daily PS (volume and content) and oral fluid intake were kept constant during the baseline and post-treatment metabolic balance study periods. Baseline and post-treatment measurements were ensured to be comparable for measuring treatment effects. Daily PS and compliance with prescribed drinking menu were recorded during hospitalization. Concomitant medications, including proton pump inhibitors, loperamide, and opiates, remained unchanged throughout the study.

[0439] The contents of the buckets were weighed, processed into dry matter, and analyzed as previously described. Energy was analyzed by bomb calorimetry, nitrogen by Kjeldahl method, lipids by modified Van de Kamer titration technique, carbohydrates by Englist method, sodium and potassium by flame photometry, and calcium and magnesium by atomic absorption spectrometry. Twenty-four-hour averages were calculated based on the three 24-hour periods. Absolute changes were calculated as the difference between baseline and post-treatment values. Relative changes were calculated as the absolute change divided by the baseline value and multiplied by 100.

[0440] Body weight was measured using a horizontal platform scale.Body composition was measured by dual energy X-ray absorptiometry (Norland XR-36 DXA densitometer, Norland, Ford Atkinson, WI, USA) at baseline and after treatment.

[0441] The first, second and fourth apraglutide injections were administered in-house. Safety assessments included observations of injection site reactions, vital signs, blood samples, electrocardiograms (ECGs), urinalysis and body weight. They were performed at baseline, during each injection of study drug, 4 days after the first injection, at the end of treatment and 4-6 weeks after the last dose at the end of the study, as shown in Table 20. Liver enzymes were measured before each drug administration.

[0442] [Table 22]

[0443] Plasma concentrations of apraglutide and fasting L-citrulline (a marker of intestinal cell mass) Blood samples for analysis of midpoint concentrations were collected at baseline, during the first and second injections (pre-dose and consecutive time points), 4 days after the first injection, and at the end of treatment. Blood samples for apraglutide pharmacokinetics were also collected after the fourth and final injection over a 96-hour period. Blood samples for fasting plasma L-citrulline were also collected 4-6 weeks after the last dose at the end of the study. Blood samples for anti-apraglutide antibodies were collected at baseline, end of treatment, and at 4-6 week follow-up visits. All eight patients tested negative for anti-apraglutide antibodies at baseline.

[0444] result The primary endpoint of this study was safety.Secondary endpoints were absolute and relative changes from baseline in food intake, wet weight fecal excretion and absorption, energy, macronutrients and electrolytes, urine production, urinary electrolyte excretion, body weight, body composition, bone mineral content, plasma L-citrulline, and pharmacokinetic profile.Except for the change from baseline in plasma L-citrulline, only absolute changes are presented within the scope of this disclosure.

[0445] Analysis of Plasma Concentrations of Apraglutide and L-Citrulline Apraglutide and L-citrulline were quantified using a validated LC-MS based method. For Apraglutide, the analytical method used solid phase extraction purification of intact Apraglutide and its internal standard. Compounds were identified and quantified using reversed phase HPLC with MS / MS detection using an AB Sciex API 5000 quadrupole mass spectrometer. For samples below the quantification limit of 5.00 ng / mL, compounds were identified and quantified using reversed phase UHPLC with MS / MS detection using an AB Sciex API 5500 quadrupole mass spectrometer. For L-citrulline, the analytical method used protein precipitation extraction of L-citrulline and its internal standard. Compounds were identified and quantified using Hilic HPLC with MS / MS detection using an AB Sciex API 5000 quadrupole mass spectrometer.

[0446] Analysis of anti-apraglutide antibodies A fully validated ELISA method was used for the detection of anti-apraglutide antibodies in serum, following a three-stage assay approach. In the first stage, anti-apraglutide antibodies present in samples and controls were bound to apraglutide immobilized on a microtiter plate. The bound anti-apraglutide antibodies were then detected using Protein A / G and Protein L. Samples with a signal above the validated screening cut-point were considered potentially positive and were then tested in the second stage, the confirmatory assay. For this, the samples were pretreated with an excess of apraglutide before being tested in the above assay. Inhibition of the signal above the validated confirmatory cut-point confirmed the presence of the antibodies. The samples were then reported as anti-apraglutide positive. The titers were then determined by serial dilutions of all confirmed positive samples.

[0447] statistical analysis Statistical tests of adjusted mean changes from baseline to end of treatment were analyzed using paired t-tests. All statistical tests were performed using two-sided tests at the 5% significance level. Estimates were presented with approximately 95% confidence intervals and p-values. SAS version 9.4 was used for the analyses.

[0448] Example 7: Pharmacokinetic / Pharmacodynamic Evaluation Demographics and other baseline characteristics Eleven women and 13 men were included in the study. Demographic and baseline data are summarized in Table 21. No relevant medical history was reported. The most frequently reported previous concomitant medications were for birth control. After the first dose, the most frequently reported concomitant medications were Paracetamol. No relevant differences in concomitant medications were noted between treatment groups.

[0449] [Table 23] BMI=body mass index, SD=standard deviation.

[0450] Measuring Treatment Compliance Treatment was administered to subjects subcutaneously (SC) by a member of clinical staff and there was full treatment compliance.

[0451] Pharmacodynamic and efficacy results Plasma citrulline concentrations versus time after the first dose of apraglutide per dose level are shown in Figure 18. The corresponding least squares (LS) means plots of the change from baseline are provided in Figure 19. Summary pharmacokinetic parameters for citrulline are provided in Table 22.

[0452] The endogenous marker citrulline was chosen to assess the pharmacodynamic effects of apraglutide. Citrulline was detected in all baseline and post-dose samples. Three baseline citrulline samples were obtained during two baseline visits between screening and the first dosing day, and before dosing on the first dosing day. Thirteen of the 24 subjects had a difference of <0.5 μg / mL for the three baseline samples, with the smallest difference being 0.061 μg / mL observed for the subject dosed with 10 mg. Another five and four subjects had differences between 0.5-1 μg / mL and 1-2 μg / mL, respectively, across baseline assessments. The remaining two subjects had differences of >2 μg / mL, with the largest difference being 2.762 μg / mL observed for the subject dosed with 1 mg. At baseline, mean citrulline ranges were 5.3825-5.6207 μg / mL for placebo, 5.3900-6.2028 μg / mL for 1 mg apraglutide, 4.8983-5.3023 μg / mL for 5 mg apraglutide, and 5.0730-5.4690 μg / mL for 10 mg apraglutide. In the placebo group, mean citrulline concentrations remained relatively stable over the study, ranging from 4.9735-5.8253 μg / mL.

[0453] After the first weekly SC dose of apraglutide, citrulline increased in a dose-dependent manner, with peak citrulline levels measured after 4 or 7 days, except for one subject in the 1 mg group who reached maximum citrulline concentrations after day 2. The highest mean citrulline values ​​were 6.3920 μg / mL (after 4 days), 7.1933 μg / mL (after 4 days), and 7.8487 μg / mL (after 7 days) for the 1, 5, and 10 mg doses, respectively, compared with 5.8325 μg / mL (after 1 day) for placebo.

[0454] Concentration at the end of each dosing interval, R pre-dose was evaluated for each medication and generally demonstrated that citrulline levels were similar across the entire treatment period (Table 22).

[0455] After the sixth and final weekly SC dose of apraglutide, all dose levels showed Increases were also observed at higher doses. max The Rs were 7.1702, 8.1577, and 8.7254 μg / mL, reached 2.0, 3.9, and 3.9 days after the last dose of 1, 5, and 10 mg, respectively (Table 22). The corresponding placebo level was 6.3707 μg / mL, 16.9 days after the last dose. These Rs max Values ​​are the average R evaluated across the entire study. max The mean R max occurred at approximately the same time for all apraglutide groups, ranging from 35.9 to 38.9 days after the first dose of 1 and 5 mg, respectively, whereas placebo peaked at 50.4 days after the first dose.

[0456] The mean citrulline increase observed after the last dose appeared to decline more rapidly at the lowest dose compared to the 10 mg dose. Citrulline was assessed through day 77. The lowest mean citrulline levels after the last dose were reached after 17 days for both 1 and 10 mg. For the 5 mg dose, the lowest mean citrulline levels were reached after an additional week. Rt 1 / 2 were evaluated for two subjects, both of whom received the 10 mg dose, and were 6.5 and 11.0 days (Table 22).

[0457] [Table 24] NA = not applicable, evaluated throughout the entire study period; ND = no data available; R max = maximum response, R pre-dose = response immediately before dosing, Rtmax = time to reach maximum response, Rt1 / 2 = response half-life. The number of subjects (N) was 6 unless otherwise indicated. * One missing subject (N=5) and ** There were four missing subjects (N=2).

[0458] R expressed as coefficient of variation (%CV) pre-dose and R max The variability over time (both after the last dose and throughout the study) ranged from 16.0 to 31.9% for placebo (Table 22). This was consistent with the %CV range of 18.1 to 29.2% observed for the 1 and 5 mg doses. The %CV for the 10 mg dose was slightly higher, ranging from 32.4 to 44.0%.

[0459] Statistical steady-state analysis per dose level was performed using the Helmert approach. Briefly, the first contrast tested was the mean R pre-dose is the mean R pooled across all remaining time points (weeks 2–6) pre-doseFor the second contrast, the week 2 mean was compared to the pooled mean across weeks 3–6, and so on until the contrast was no longer statistically significant. pre-dose Average R at 6 weeks for 10mg pre-dose (0.8720, 95% CI: 0.1590; 1.5850 and p=0.0172) was statistically significant. Visual inspection of the data suggests that steady state concentrations were reached after the first dose at all dose levels investigated.

[0460] Citrulline data was analyzed using a mixed model analysis of variance. A summary of the analysis results is shown in Table 23. Statistical analysis clearly showed a significant overall treatment effect of apraglutide on citrulline (p=0.0007). Although the difference between placebo and 1 mg was not statistically significant, an increase was observed in all dose groups compared to placebo. The increase compared to placebo became more pronounced and significant with increasing dose, i.e., an increase of 1.2574 μg / mL (p=0.0025) for the 5 mg dose and 1.6343 μg / mL (p=0.0002) for the 10 mg dose (Table 23). Furthermore, when comparing dose levels with each other, the 5 and 10 mg doses induced significantly higher citrulline levels compared to the 1 mg dose (0.9429 μg / mL higher [p=0.0186] and 1.3198 μg / mL higher [p=0.0018], respectively). The difference between the 5 mg and 10 mg doses was not statistically significant, although the effect after 10 mg appeared to be slightly greater than after 5 mg.

[0461] [Table 25] Overall treatment p-values ​​(all dose groups compared to placebo) and subsequent treatment group comparisons are shown. Estimates of differences with 95% confidence intervals and p-values ​​are shown for each comparison.

[0462] In addition to citrulline, Bristol stool scale and body weight were evaluated as pharmacodynamic endpoints. In contrast to citrulline and body weight, Bristol stool scale is a discrete variable. Subjects assessed stool in the past 24 hours using the Bristol stool scale, which ranges from constipation (type 1) to diarrhea (type 7). No stool in the past 24 hours was recorded as 0. Two subjects (allocated to either 1 mg or 5 mg) reported no stool before the first dose, so the screening Bristol stool scale score was also taken into account when determining the baseline Bristol stool scale score.

[0463] A significant overall treatment effect of apraglutide on the Bristol Stool Scale was observed, with a p-value of 0.0189 (Table 23). Specific placebo vs. dose level contrasts showed significant reductions in the 1 and 5 mg groups compared to placebo (-0.9, p=0.0092 and -1.0, p=0.0046). The reduction in the 10 mg group compared to placebo was not statistically significant. This pattern was likely driven by a small number of subjects who reported occasional absence of stools. Following the first dose, Bristol Stool Scale assessments were scheduled at 11 visits and at study discharge. Of these visits, no stools were reported in the past 24 hours 6 times (2 subjects), 13 times (4 subjects), and 1 time (1 subject) in the 1 mg, 5 mg, and 10 mg groups, respectively. In the placebo group, all subjects reported no stools in the past 24 hours at each visit. Nine missed stools were reported during the six weeks of dosing and 11 missed stools in the six weeks thereafter, indicating no association with dosing.

[0464] Given the potential impact of scoring no stool as 0 when a subject may not have had a normal bowel movement every 24 hours (supported by the observation that 2 of 24 subjects reported 0 before the first dose), the analysis was repeated as a sensitivity analysis setting no stool to missing. In this adjusted analysis, no significant treatment effects were found, either overall or for any of the specific treatment contrasts (Table 23).

[0465] Body weight appeared to be fairly constant over the entire study period, with no statistically significant treatment effects of apraglutide on overall body weight or for any of the specific treatment contrasts (Table 23). Considering individual body weight values, placebo subject 1007 showed an increase in weight from 92.90 kg at screening to 99.30 kg at the study discharge visit.

[0466] Citrulline, BSS and weight data were analyzed with a mixed model analysis of variance with fixed factors treatment, time and treatment by time, random factor subject and mean contingency value as a covariate. Mean contingency values ​​were calculated from all post-screening and pre-dosing contingency values, except for BSS, where the screening value was also used to calculate the mean baseline.

[0467] MCP-MOD analysis The MCP-MOD method was applied. This statistical method consisted of two steps: a multiple comparison step and a modeling step. The first step of the procedure (MCP) is used to test for a significant dose-response by evaluating pre-specified candidate models. Once the dose-response is established, the second step (MOD) is used to estimate the dose-response curve and estimate the target dose of interest. The following five candidate models were identified: -Linear model with maximum effect for the 10mg dose -Log-linear model with maximum effect for the 10mg dose -5mg ED 50 The maximum effect (E max ) Dose to induce the model -1mg ED 50 and a sigmoid E with a Hill coefficient (HILL) of 2 max Model -5mg ED 50 and a sigmoid E with a HILL of 5 max Model

[0468] Results from the MCP step showed that all five candidate dose-response models produced significant contrasts with p-values ​​of <0.0001 (Table 24). The estimates of the differences were slightly different, resulting in a set of models with better fit and a set with slightly worse fit. The candidate dose-response model producing the greatest estimate of the difference was 1) ED 50 E = 5 mg max model, 2) log-linear model, and 3) ED of 1 mg. 50 and a sigmoid E with HILL of 2 max The estimates of these differences were very similar, so all three models were included in the MOD part. The models were: 1) E max , 2) LogLin and 3) Sigmoid E max It is called a model.

[0469] [Table 26] CI = confidence interval, ED50 = dose inducing 50% of the maximum effect, Emax = maximum effect, H = Hill coefficient, MCP-MOD = multiple comparison procedure-modeling. Doses consisted of 0 (placebo), 1, 5 and 10 mg apraglutide. Citrulline response data included mean baseline values ​​and data from days 35 to 42 (=week 6). For each candidate dose-response model, analysis results are characterized by test statistics, p-values ​​for contrasts, and estimates of differences with 95% CI.

[0470] A subset of citrulline data was created using the mean reserve value and data from days 35-42 = week 6. Coefficients were run in a mixed model analysis of variance with fixed factors treatment, time and treatment by time, random factor subject, and mean reserve value as a covariate. The mean reserve value was calculated from all post-screening and pre-dosing reserve values.

[0471] The following contrasts are calculated in the model: -Linear (coefficients -0.508 -0.381 0.127 0.762) -LinLog(coefficient -0.654 -0.283 0.306 0.631) -Emax(coefficient -0.632 -0.316 0.316 0.632) -Sigmoid Emax Hill=2 ED50=1 (coefficients -0.759-0.140 0.432 0.467) -Sigmoid Emax Hill=5 ED50=5 (coefficients -0.456-0.455 0.164 0.747)

[0472] The MOD step showed that the predicted citrulline changes from placebo for the 1, 5, and 10 mg doses were fairly similar for all three models (Table 25). At the 1 mg dose, the predicted placebo- and baseline-corrected changes in citrulline across the three models ranged from 0.7125 to 0.8712 μg / mL, with E max The results were significant for the log-linear (p=0.0238) and log-linear (p=0.0184) models. max For the model, the p-value was 0.0645. With increasing dose to 5 mg, the predicted change from placebo increased to a range of 2.1652 to 2.3327 μg / mL. For the 10 mg dose, the predicted change from placebo was slightly higher, ranging from 2.6946 to 2.8604 μg / mL. The predicted response to placebo for both the 5 and 10 mg doses was statistically significant for all models with a p-value of <0.0001.

[0473] The apraglutide dose levels required to achieve baseline and placebo-corrected citrulline increases of 1, 2, and 3 μg / mL were predicted (Table 25). max Model E max The predicted apraglutide dose for a 1 μg / mL increase in citrulline was <3 μg / mL, so a change of 2.5 μg / mL was selected as the maximum response for this model. max , LogLin and Sigmoid E maxFor the model, the predicted apraglutide doses were 1.2333 mg, 1.2249 mg, and 1.3606 mg, respectively, to induce a 2 μg / mL increase in citrulline. max , LogLin and Sigmoid E max For the model, the predicted apraxon doses for the change from placebo of 2.5 or 3 μg / mL were 3.8471 mg, 4.2011 mg, and 3.4042 mg, respectively. Finally, the predicted apraxon doses for the change from placebo of 2.5 or 3 μg / mL were E max , LogLin and Sigmoid E max Using the model, they were 13.1053 mg, 11.4325 mg, and 6.4619 mg, respectively. It should be noted that there is considerable uncertainty in the predicted apraglutide doses, especially to achieve an efficacy of 2.5 or 3 μg / mL.

[0474] [Table 27] CI=confidence interval, E max = maximal effect. ND = not determined. * Sigmoid E max Model E max was <3 μg / mL, so the maximum response was set at 2.5 μg / mL. Predictions were performed based on apraglutide doses of 0 (placebo), 1, 5, and 10 mg, yielding citrulline response data consisting of mean baseline values ​​and data from days 35 to 42 (=week 6). Estimated change / dose with 95% CI and p-values ​​are shown for each prediction.

[0475] Adjustment for covariates For all pharmacodynamic endpoints (citrulline, Bristol stool scale, and body weight), contingency values ​​were used as covariates. Mean contingency values ​​were calculated from all post-screening and pre-dose contingency values, except for Bristol stool scale, where the screening value was also used to calculate the mean baseline. The mean contingency values ​​for citrulline were also calculated using the sigmoid E max Except for the model, MCP-MOD was used as a covariate in the model. This was addressed by subtracting the preliminary value multiplied by the slope of the linear model of the MCP portion from the value at week 6.

[0476] Relationship between drug dose, drug concentration, and response Plasma apraglutide concentrations versus time after the first dose of apraglutide by dose level are shown in Figures 20A-B. Summary pharmacokinetic parameters of apraglutide are provided in Table 26.

[0477] The mean apparent total clearance (CL / F) appeared to be constant across the three dose levels, ranging from 16.480 (5 mg) to 20.747 L / d (10 mg) (Table 26). z The mean apparent volume of distribution between the 5 and 10 mg dose groups (V / F) appeared to be dose-dependent with values ​​of 55.426 and 105.021 L for the 5 and 10 mg dose groups, respectively. z The data points available for determining / F are insufficient (an insufficient number of samples were above the lower limit of quantification during the sampling period after the last dose);

[0478] After the first weekly SC dose of apraglutide, plasma apraglutide concentrations increased without any apparent lag time in all subjects (except for subject 1002 at 1 mg, who did not have detectable concentrations until the next dose). max was achieved by 4, 4, and 1 subjects receiving apraglutide at 1 mg, 5 mg, and 10 mg, respectively, on day 1 (Figure 20). The remaining subjects achieved C max The 1 mg subjects were below the limit of quantification (LOQ) on the 4th or 7th day, but the 5 mg and 10 mg subjects were below the limit of quantification (LOQ) on the 4th or 7th day. Subjects in the 10 mg group remained above the LOQ until the next dose. Exposure after the first dose was dose-dependent, with mean C of 13.918 ± 11.2, 94.088 ± 50.5, and 136.855 ± 55.0 ng / mL for the 1, 5, and 10 mg dose groups, respectively. max , and 35.214, 300.269 and 476.937d, respectively. * AUC in ng / mL tauThe half-life (h) measured after dose #6 in the 5 mg dose group was measured to be 72.7 ± 23.0, and the half-life measured after dose #6 in the 10 mg dose group was measured to be 76.3 ± 27.6.

[0479] The next 5 doses of C trough In the 1 mg dose, five subjects (all dosed with 1 mg) had one or more samples that were undetectable for apraglutide. All other subjects had measurable apraglutide concentrations. Mean C across the six doses trough ranged from 0.000 (week 1) to 0.873 ng / mL (week 4) for 1 mg apraglutide, 7.605 (week 5) to 10.163 ng / mL (week 4) for 5 mg apraglutide, and 13.710 (week 6) to 18.460 ng / mL (week 2) for 10 mg apraglutide (Table 26).

[0480] After the sixth and final weekly SC dose of apraglutide, all subjects (subject 3004 was excluded due to discontinuation) showed an increase in apraglutide concentrations with no apparent lag time. max Except for 2 and 3 subjects at 5 and 10 mg, respectively, who reached C max Pharmacokinetic parameters were assessed up to 2 weeks after the final dose. All subjects at 1 mg and one subject at 5 mg had non-quantifiable concentrations in the last two or three samples. The remaining subjects (including all subjects at 10 mg) still had quantifiable concentrations up to 4.46 ng / mL in the last pharmacokinetic sample.

[0481] [Table 28-1]

[0482] [Table 28-2] AUC last= area under the plasma concentration-time curve from time 0 to the last measurable concentration, AUC tau = area under the plasma concentration-time curve until the end of the treatment period of the corresponding medication, CL / F = apparent total clearance, C max = maximum concentration, C トラフ = plasma concentration immediately before the next dose, lambda z = terminal elimination rate constant, PK = pharmacokinetics, t1 / 2 = terminal elimination half-life, t max = time to reach maximum plasma concentration, V z / F = apparent volume of distribution during the terminal elimination phase. * 1 missing subject (N=5), ** 2 missing subjects (N=4) and *** There were 3 missing subjects (N=3, PK parameters not calculated). Values ​​below the limit of quantification (<1 ng / mL) were set to 0. The number of subjects is 6 unless otherwise indicated.

[0483] The concentration profiles observed after the sixth dose appeared comparable to the first dose, although exposures were slightly higher (Figure 20). Exposures were 0.02424, 0.02496, and 0.01824 ng / mL / μg mean C for the 1, 5, and 10 mg dose groups, respectively. max / dose and 0.05944, 0.07153 and 0.05824d * AUC in ng / mL / μg tau Dose proportionality with dose was demonstrated (Table 26).

[0484] In three of six subjects dosed with 1 mg, insufficient data points were available to determine the terminal elimination rate constant (an insufficient number of samples were above the lower limit of quantification during the sampling period after the last dose). Mean half-lives were subsequently determined only for the 5 and 10 mg dose groups and were comparable between the two groups (3.04 and 3.18 days, respectively).

[0485] C max and AUC tauThe variability in was most pronounced after the first dose of 1 mg with %CVs of 145.5 and 145.3%, respectively (Table 26). The variability was lower after the last dose of 1 mg and both the first and last doses at the 5 and 10 mg dose levels. max The %CV ranged from 39.9 to 64.6%, and the AUC tau The corresponding range of %CV was 23.1-52.4%.

[0486] Dose-normalized pharmacokinetic parameter C max and AUC tau Visual inspection of the values ​​suggested that apraglutide followed linear kinetics over the dose range of 1 to 10 mg, which is consistent with the associated constant CL / F.

[0487] Similar to citrulline, a statistical steady-state analysis was performed for apraglutide by dose level using the Helmert approach. This steady-state analysis included mean C trough (-3.616, 95%CI:-5.893;-1.399 and p=0.0023) vs. mean C trough None of the contrasts revealed statistical significance, except for . Visual inspection of the data suggests that steady state was reached after the first dose at all dose levels investigated.

[0488] Graphical analysis was performed to evaluate the apraglutide concentration-citrulline effect relationship. Some individual citrulline parameters (R max and R pre-dose ) was used as the pharmacokinetic parameters of apraglutide (C max , C trough and AUC tau ) were plotted against the mean mean of the ... max About R max The AUC tau is R maxThe regression line showed that the AUC tau Each 100d * For each increase in ng / mL, the corresponding maximum citrulline response increased by 0.39 μg / mL. The R-squared value was R max 26% of the variability in AUC tau As shown in Figures 23, 24, and 25, correlation plots of all apraglutide and citrulline concentrations assessed by dose level at week 6 revealed counterclockwise hysteresis as there was a time delay between the measured apraglutide concentrations and the citrulline effect.

[0489] Pharmacokinetics The mean CL / F was consistent across the three dose levels, ranging from 16.480 (5 mg) to 20.747 L / day (10 mg). The mean Vz / F was determined only for the 5 and 10 mg dose groups and was dose-dependent with values ​​of 55.426 and 105.021 L for the 5 and 10 mg dose groups, respectively. The mean t1 / 2 could only be determined for the 5 and 10 mg apraglutide dose groups and was comparable for these groups (3.04 and 3.18 days, respectively).

[0490] Exposure following the first weekly SC dose of apraglutide was assessed in the 1, 5, and 10 mg dose groups. The mean C of 13.918, 94.088 and 136.855 ng / mL, respectively max , and 35.214, 300.269 and 476.937d, respectively. * AUC in ng / mL tau The concentration profiles observed after the sixth dose were comparable to the first dose, although exposures were numerically higher. Exposures were 0.02424, 0.02496, and 0.01824 ng / mL / μg mean C for the 1, 5, and 10 mg dose groups, respectively. max / dose and 0.05944, 0.07153 and 0.05824d * AUC in ng / mL / μg tauAt doses 1 and 6, t max was approximately 1 day for the 1 mg and 5 mg apraglutide doses and 2 days for the 10 mg dose.

[0491] Dose-normalized pharmacokinetic parameter C max and AUC tau Visual inspection of the values ​​suggests that apraglutide follows linear kinetics over the dose range of 1 to 10 mg. Visual inspection of the data suggests that steady state was reached after the first dose at all dose levels investigated.

[0492] Pharmacodynamics After the first and final dose of apraglutide, citrulline concentration increased in a dose-dependent manner.The average citrulline increase observed after the final dose appeared to decline more rapidly with 1mg and 5mg doses compared to 10mg dose.Rt1 / 2 could only be assessed for two subjects who both received 10mg, and was found to be 6.5 and 11.0 days.

[0493] At dose 6, the mean R max R was 7.1702, 8.1577, and 8.7254 μg / mL after the final doses of 1, 5, and 10 mg, respectively, and reached on days 2.0, 3.9, and 3.9. The corresponding placebo value was 6.3707 μg / mL, 16.9 days after the last dose. These R max The value is the average R max Visual inspection of the data suggests that steady state was reached after the first dose at all dose levels investigated.

[0494] A significant treatment effect of apraglutide was observed for citrulline (p=0.0007). Apraglutide induced an increase in citrulline. This response was statistically significant with an increase of 1.2574 μg / mL (95% CI: 0.5037; 2.0112; p=0.0025) for apraglutide 5 mg and 1.6343 μg / mL (95% CI: 0.8809; 2.3878; p=0.0002) for apraglutide 10 mg compared to placebo, whereas the increase was not statistically significant with apraglutide 1 mg compared to placebo (0.3146 μg / mL, 95% CI: -0.4371; 1.0663; p=0.3910). Contrasts between dose groups showed that the 5 and 10 mg doses induced significantly higher citrulline levels compared to the 1 mg dose (0.9429 μg / mL, 95% CI: 0.1768; 1.7089; p=0.0186 and 1.3198 μg / mL, 95% CI: 0.5586; 2.0810; p=0.0018, respectively). Importantly, the difference between 5 mg and 10 mg apraglutide was not statistically significant, although the effect after 10 mg appeared to be slightly higher than after 5 mg.

[0495] A significant treatment effect of apraglutide on the Bristol Stool Scale was observed with a p-value of 0.0189 (all dose groups compared to placebo). This effect was not dose-dependent, as only significant reductions compared to placebo were observed in the 1 and 5 mg dose groups (-0.9, 95% CI: -1.6; -0.3; p=0.0092 and -1.0, 95% CI -1.7; -0.4; p=0.0046, respectively).

[0496] Exploratory graphical analysis showed that R max C against max The slope of the regression line for revealed that the AUC tau is R max There was a positive correlation with the p-value of 0.0377. According to the regression line, the AUC tau Each 100d *For each increase in ng / mL, the corresponding maximum citrulline response increased by 0.39 μg / mL. The R-squared value was R max 26% of the variability in AUC tau Correlation plots of all apraglutide and citrulline concentrations assessed at 6 weeks revealed a counterclockwise hysteresis.

[0497] The MCP-MOD procedure uses three candidate pre-specified models: 1) E max , 2) LogLin and 3) Sigmoid E max The models provided similar results with the best fit to the dose-response in the MCP portion. All three models were applied to the MOD portion and predicted similar citrulline changes from placebo for the 1, 5, and 10 mg doses of apraglutide. At the 1 mg dose, predicted placebo- and baseline-corrected changes in citrulline across the three models ranged from 0.7125 to 0.8712 μg / mL, with E max The results were significant for the LogLin (p=0.0184) and LogLin (p=0.0238) models. max For the model, the p-value was 0.0645. For the 5 mg dose, the response ranged from 2.1652 to 2.3327 μg / mL. For the 10 mg dose, a slightly stronger response was obtained, ranging from 2.6946 to 2.8604 μg / mL. The predicted response versus placebo for both the 5 and 10 mg doses was statistically significant for all models with a p-value of <0.0001.

[0498] The apraglutide dose levels required to achieve baseline and placebo-corrected citrulline increases of 1, 2, and 3 μg / mL were predicted. max Model E max The predicted apraglutide dose for a 1 μg / mL increase in citrulline was 1.0 μg / mL, 1.5 μg / mL, and 1.6 μg / mL, respectively. max , LogLin and Sigmoid E maxFor the model, the predicted apraglutide doses to induce a 2 μg / mL increase in citrulline were 1.2333 mg (95% CI: -0.04246; 2.5091), 1.2249 mg (95% CI: -0.2169; 2.6667), and 1.3606 mg (95% CI: 0.2680; 2.4532), respectively. max , LogLin and Sigmoid E max For the model, predicted apraxurtide doses for change from placebo of 2.5 or 3 μg / mL were 3.8471 mg (95% CI: 0.3534; 7.3409), 4.2011 mg (95% CI: 0.6406; 7.7617), and 3.4042 mg (95% CI: -0.1290; 6.9374), respectively. max , LogLin and Sigmoid E max Using the model, the results were 13.1053 mg (95% CI: -6.3068; 32.5173), 11.4325 mg (95% CI: 1.5808; 21.2843) and 6.4619 mg (95% CI: -0.5521; 13.4759).

[0499] PD / PK model The clinical pharmacokinetic observations were best matched by a model with correlations between V1 / F and Cl / F and dose covariates for duration of absorption and body weight covariates for V1 / F and Cl / F. The pharmacodynamic observations were then added to this model to generate the PK / PD model. Plasma citrulline was described by a turnover model and a maximum PD effect model.

[0500] The population PK / PD model showed that a 70 kg individual receiving a subcutaneous injection of apraglutide 5 mg would achieve a volume of distribution of 31.3 L and a peak plasma concentration (C max Simulated plasma concentration profiles of apraglutide and citrulline from weekly subcutaneous injections of apraglutide 2.5, 5, or 10 mg are shown in FIG.

[0501] PK / PD models did not show any accumulation of apraglutide over time. Citrulline accumulation was evident during the first 3 weeks of treatment, but steady-state concentrations were not reached thereafter. The simulated apraglutide concentration-time profiles by body weight showed lower area-under-the curve (AUC) and C at steady state with increasing body weight. max showed.

[0502] Example 8: Apraglutide manufacturing process-(II) Below is an exemplary method of the present disclosure for producing apraglutide at improved purity levels compared to previously described synthetic routes (e.g., U.S. Pat. No. 8,580,918).

[0503] Solid Phase Peptide Synthesis (Step 1) SPPS is the sequential synthesis of peptide chains immobilized on a solid support by repeated cycles involving the following steps: 1. Removal of the N-terminal Fmoc protecting group from peptide resin 1a. In some embodiments, the residue Asp 3 The Fmoc deprotection reaction involves treating the resin with a solution of 10% piperidine and 2% oxymer in DMF. The deprotection reaction is carried out in two cycles, the first cycle for 15 minutes and the second cycle for 30 minutes. 2. DMF Washing 3. Coupling of Fmoc-AA-OH 4. Coupling Test 5. DMF Wash

[0504] This cycle is repeated until the peptide sequence is completed.

[0505] The α-amino group of the amino acid is protected with the base-sensitive 9-fluorenylmethyloxycarbonyl (Fmoc) group. The side chain functional groups are protected with acid-labile groups. All amino acid derivatives used in this process are commercially available.

[0506] SPPS is a stepwise synthesis of peptide chains immobilized on a solid support. In the synthesis, a peptide sequence can be assembled using MBHA resin. After swelling the resin and washing with DMF, then DMF / DIEA under nitrogen atmosphere, the Fmoc-Rink-amide linker can be coupled using HBTU / DIPEA / HOBt in DMF. After coupling, the resin can be washed with DMF and then acetylated using Ac2O / DIPEA. A Kaiser test can be performed to confirm the completion of coupling.

[0507] After washing the resin with DMF, each Fmoc-protected amino acid is coupled to the resin-bound peptide according to the following cycle: 1. The Fmoc-protecting group is removed with piperidine in DMF and the resin is washed extensively with DMF. 2. Couplings are carried out in DMF with varying amino acid equivalents using DIC / Oxyma for activation. In some embodiments, the dipeptide Boc-His(Trt)-Gly-OH is coupled to the resin instead of the sequential assembly of Gly and His amino acids. The dipeptide is preactivated with Boc-His(Trt)-Gly-OH / Oxima / DIC (2.5 mmol / 2.5 mmol / 2.5 mmol) in 7 mL of DMF at 20° C.±2° C. for 1 hour before being added to the coupling reaction. A Kaiser test can be performed to confirm the completion of the coupling. 3. The coupling of amino acids is monitored by using a ninhydrin assay performed during each synthesis cycle.

[0508] At the end of the assembly, after the last amino acid is coupled and deprotected, the resin is washed with DMF and isopropanol and dried under vacuum.

[0509] Cleavage of the peptide from the resin and deprotection (Step 2) The protected peptide can be simultaneously cleaved from the resin and deprotected by treatment with a mixture of TFA / water / anisole. MTBE is then added to the peptide / TFA slurry to precipitate the crude peptide in the presence of the cleaved resin. The resulting crude peptide is filtered, washed with MTBE, and dried under vacuum to constant weight.

[0510] Decarboxylation Reaction (Step 3a) The crude peptide is solubilized in a mixture of H2O / ACN (70:30 ratio) in ammonia buffer. The solution is adjusted to a target pH of 8.0±0.1 using 25% acetic acid or 25% NH4OH in H2O. The decarboxylation reaction is maintained at 50°C for 65 minutes. The crude peptide is washed with a solution of H2O / ACN (70:30 ratio) in ammonia buffer at pH 8.0±0.1 and stored at 5°C±3°C.

[0511] Purification by preparative RP-HPLC (step 3b) The crude peptide is dissolved in a mixture of water / acetonitrile / NH4OH. The solution is diluted with acetic acid and then filtered.

[0512] Primary purification is performed by preparative RP-HPLC using NaHCO3 / H2O / CH3CN as eluent. Elution from the column is monitored by UV and the obtained fractions are analyzed by RP-HPLC. Fractions that meet the monitoring criteria are mixed in a combined pool. Fractions that do not meet the monitoring criteria can be recycled by repeating the purification step. The purity of the pool is controlled by analytical RP-HPLC.

[0513] Conversion of the sodium salt by preparative RP-HPLC (Step 4) This step can be performed to exchange the peptide counterion from TFA anion to sodium cation via pH change and further purify the peptide. The combined pool from step 3 is diluted with water and re-purified by preparative RP-HPLC using NaOAc eluent.

[0514] The purified peptide solution is then subjected to evaporation under vacuum to reduce the amount of acetonitrile in the solution, and then the purified peptide solution is adjusted to a target pH of 7.9 using 0.1% AcOH.

[0515] The pure pool can be concentrated and lyophilized. The purity of the pool is analyzed by RP-HPLC.

[0516] Freeze-drying and packaging (Step 5) Prior to lyophilization, the purified peptide in solution is filtered through a 0.2 μm membrane. Lyophilization is performed at low pressure. The resulting lyophilized final peptide is packed under argon. The lyophilized apraglutide is controlled according to the apraglutide standard.

[0517] Reprocessing Lyophilized apraglutide that does not meet the standards established in the apraglutide specification can be subjected to repurification.

[0518] Repurification can be carried out after reconstitution of the peptide by repeating the purification and counterion exchange steps as described above.

[0519] After repurification, the material is lyophilized according to the procedure described above.

[0520] Freeze-dried apraglutide that does not meet the criteria established in the apraglutide specification may be subjected to re-lyophilization.

[0521] Re-lyophilization may be performed after reconstitution of the peptide by repeating the lyophilization step, as described above.

[0522] impurities The process for producing apraglutide described in Example 8 was carried out to obtain β-Asp in apraglutide. 3 The level of peptide isomer impurity was reduced to less than 1.5%. In some embodiments, the apraglutide manufacturing process described in Example 8 reduces the β-Asp in apraglutide. 3The level of peptide isomeric impurities was reduced to less than 1%.

[0523] β-Asp 3 The formation of the impurity is due to high pH and the sequence Asp 3 -Gly 4 This is supported by the presence of. Asp-Gly sequences are particularly susceptible to aspartimide formation due to ring closure (attack of the nitrogen from the α-carboxyamide bond on the β-carboxy side chain). Aspartimides are susceptible to base-catalyzed epimerization and can undergo ring-opening reactions, which can result in the formation of multiple by-products. Attack by water can generate β-aspartyl peptides.

[0524] β-Asp during the decarboxylation / extraction step (step 3a) upon cleavage of the peptide from the resin 3 The formation of impurities can reach levels >1% and is accelerated by prolonged exposure to extremes of pH (especially strong base solutions of ammonia in HO / ACN 80:20, pH approx. 10 at room temperature). The apraglutide manufacturing process described in Example 8 involved lowering the pH from 10 to 8, shortening the decarboxylation reaction time from 24 hours to 65 minutes, changing the reaction temperature from room temperature to 50° C., and lowering the storage temperature from room temperature to 5° C. These changes resulted in the formation of β-Asp at levels below 1.5%. 3 In some embodiments, these changes resulted in impurities at levels of less than 1% β-Asp. 3 Impurities were produced.

[0525] The process for producing apraglutide described in Example 8 contains the residue Asp 3 The Fmoc deprotection reaction of was modified to use a solution of 10% piperidine and 2% oxymer in DMF, and two cycles of deprotection (first cycle for 15 min, second cycle for 30 min). These milder basic conditions allowed the deprotection of Asp 3 allowing for the complete deprotection of aspartimide and then β-Asp 3 By-product formation was reduced.

[0526] The formation of D-His impurity may result from racemization of amino acids during the coupling reaction using DIC / Oxima. The process of sequential assembly of individual Gly and His amino acids showed variability in the level of D-His impurity between 0.3% and 1.2%, sometimes accompanied by incomplete coupling. Incorporation of Fmoc-His(Trt)-OH using the coupling reagent DIC / Oxima may favor racemization of His to the D form.

[0527] The apraglutide manufacturing process as described in Example 8 used the dipeptide Boc-His(Trt)-Gly-OH as starting material for this reaction, instead of the sequential assembly of Gly and His amino acids. This allows the racemization level in His to be reduced to less than 1% (0.3% in one working batch) and incorporates the final D-His level into the dipeptide (Boc-D-His(Trt)-Gly-OH) specification. This also allows a reduction in the number of deprotection steps, and therefore, as mentioned above, β-Asp 3 The contributing effect of the deprotection conditions (high pH) on impurity formation is minimized.

[0528] Example 9: Phase 3 human clinical trial of administration of the apraglutide composition of the present disclosure The following non-limiting examples describe a Phase 3 clinical trial in which subjects with SBS-IF are treated with the disclosed apraglutide formulation. This Phase 2 clinical trial investigates the effectiveness of weekly subcutaneous apraglutide in reducing parenteral support dependency in patients with SBS-IF.

[0529] 144 adult patients with SBS-IF are treated with the apraglutide composition of the present disclosure or a placebo. Patients with SBS-IF are administered 2.5 mg of the apraglutide of the present disclosure if the subject weighs less than 50 kg, or 5 mg of the apraglutide of the present disclosure if the subject weighs 50 kg or more, or a placebo, once a week for 48 weeks.

[0530] The study is a multicenter, double-blind, randomized, placebo-controlled, Phase 3 study. Participant randomization includes anatomy-specific randomization of stoma and colonic continuity.

[0531] The primary endpoint efficacy assessment will be performed at week 24. The primary endpoint will assess the relative change from baseline in actual weekly PS. Secondary endpoint efficacy assessments will be performed at weeks 24 and 48. Anatomy-specific secondary endpoints as well as secondary endpoints common to all patients will be assessed.

[0532] Secondary endpoints evaluated include, but are not limited to, the following: 1.Subjects who achieve at least 1 day / week reduction in PS at Week 24 / 48. 2. Relative change from baseline in actual weekly PS at weeks 12, 24 and 48. 3.SBS-IF patients achieved intestinal autonomy at 24 / 48 weeks. 4. At least 20% reduction in PS volume from baseline at weeks 20 / 24. 5. Calorie reduction in parenteral nutrition (PN) at 24 weeks. 6. Patient Global Impression of Severity (PGIS) ) change from baseline 7. Change from baseline in Pittsburgh Sleep Quality Inventory (PSQI) 8. Patient Global Impression of Change (PGIC) A change from Sline. 9. Absorption rate constant (ka) of apraglutide based on population PK data analysis 10. Apparent clearance (CL / F) of apraglutide based on population PK data analysis 11. Apparent volume of distribution (Vz / F) of apraglutide based on population PK data analysis

[0533] Example 10: Phase 1 human clinical trial of administration of the apraglutide composition of the present disclosure The following non-limiting examples describe a Phase 1 clinical trial in which subjects with normal and impaired renal function were treated with the disclosed apraglutide formulation. This Phase 1 clinical trial investigates the pharmacokinetics and safety of a single subcutaneous dose of 5 mg apraglutide in subjects with varying degrees of renal function. Renal function is a marker of renal function in accordance with the Chronic Kidney Disease Epidemiology (CKD) guidelines. Estimated glomerular filtration rate (KD-EPI) was calculated according to the creatinine equation. It is calculated using the eGFR (epithelial filtration rate).

[0534] The study is a two-stage, open-label, multicenter, non-randomized, Phase 1 study. In the first part of the study, eight subjects with severe renal impairment (Cohort 1) and six subjects with normal renal function (Cohort 2) will be administered a single 5 mg dose of the disclosed apraglutide. In the second part, eight subjects with moderate renal impairment (Cohort 3) and eight subjects with mild renal impairment (Cohort 4) will be administered a single 5 mg dose of the disclosed apraglutide. AUC inf or AUC last Subjects will be enrolled if the geometric mean ratio (GMR) is >=2.

[0535] Patient eligibility will be assessed during the screening visit. Main inclusion criteria are: 1. All participants A. 18 years old or older and under 75 years old b. Subjects who are willing and able to comply with the test procedures c. Subjects who are able to understand the consent form and are willing to sign it d. Body mass index (BMI) ≥ 17.5 to ≤ 40 kg / m2; and total weight ≥ 50 kg (110 lb). e. Women of childbearing potential (WOCBP) who are using a highly effective method of contraception during the study and for one month after the end of trial (EOT) visit. Infertile or subfertile or postmenopausal women. f. Male subjects with a female partner of childbearing potential: highly effective contraception and no sperm donation during the study and for one month following the (EOT) visit. 2. Healthy participants a. No clinically relevant abnormalities (medical history, vital signs, ECG, safety tests) b. eGFR ≥ 90 mL / min / 1.73 m2 measured by CKD-EPI at two screening visits c. Demographically comparable to the group of subjects with renal impairment 3.Participants with renal dysfunction a. Severe renal dysfunction: eGFR < 30 mL / min / 1.73 m2 but not requiring hemodialysis b. Moderate renal dysfunction: eGFR ≥ 30 mL / min / 1.73 m2 and < 60 mL / min / 1.73 m2 c. Mild renal dysfunction: eGFR ≥ 60 and < 90mL / min / 1.73m2

[0536] The primary endpoint efficacy assessment will be performed 240 hours after a single dose. The primary endpoints are maximum plasma (Cmax), area under the concentration-time curve from 0 to infinity (AUC inf ) and the area under the concentration-time curve from 0 to the last measurable concentration from 240 hours (AUC last ) will be evaluated. Secondary endpoint efficacy assessments will be performed at 240 hours, 7 days, 14 days, and 28 days. The primary and secondary endpoints will be evaluated across all subjects.

[0537] Secondary endpoints evaluated include, but are not limited to, the following: 1. Area under the concentration-time curve (AUC) from time 0 to the last measurable concentration on day 7 0~7日Samples were collected over 168 hours after a single dose. 2. Time to maximum concentration (T max Samples were collected over 240 hours after a single dose. 3. Terminal elimination rate constant. Samples were collected over 240 hours after a single dose. 4. Terminal elimination half-life. Samples were collected over 240 hours after a single dose. 5. Apparent clearance (CL / F) after extravascular administration. Samples were collected over 240 hours after a single dose. 6. Apparent volume of distribution (Vz / F) after extravascular administration. Samples were collected over 240 hours after a single dose. 7. Number of participants with adverse events or adverse events of special interest at Days 14-28 8. Clinically significant changes from baseline in vital signs on days 14-28 9. Clinically significant changes from baseline in triplicate 12-lead ECGs recorded on days 14-28 10. Number of participants who experienced clinically significant changes from baseline in clinical laboratory assessments on days 14-28

[0538] As will be understood by those skilled in the art, renal dysfunction may adversely affect some pathways of hepatic / intestinal drug metabolism and is also associated with other changes, such as changes in absorption, plasma protein binding, transport, and tissue distribution, so patients with renal dysfunction usually require adjustment of drug dosing. Even though pharmacokinetic data shows that renal excretion is not the main excretion pathway of apraglutide, other GLP-2 analogues (i.e., teduglutide) require dose reduction in patients with moderate and severe renal dysfunction and end-stage renal disease. In the study described in Example 10, it was found that dosing apraglutide 5 mg to patients with severe CKD is safe without risk of overdosing in this population. Without wishing to be bound by theory, this indicates that apraglutide has the potential to be administered to patients with renal dysfunction without adaptation.

[0539] Pharmacokinetics Plasma concentrations of apraglutide following a single subcutaneous dose of 5 mg apraglutide were monitored over 240 hours in both healthy subjects (cohort 2) and subjects with severe CKD (cohort 1) and are shown in FIG.

[0540] A summary of the pharmacokinetic parameters of apraglutide is shown in Table 27. When analyzing the individual apraglutide plasma concentration curves, two outliers were identified (one in each cohort). The outlier in cohort 1 had the highest body weight (BW) and body mass index (BMI) in this group, while the outlier in cohort 2 had the lowest BW and BMI in this group. Geometric mean ratios were calculated with and without the outlier subjects.

[0541] [Table 29] Cmax=maximal plasma, AUC inf = area under the concentration-time curve from 0 to infinity.

[0542] For the entire population, C max The geometric mean ratio for was 0.572, with a 90% CI of 0.37 to 0.885, and the AUC inf For the , it was 0.614, with a 90% CI of 0.388 to 0.972. On the other hand, when the data from outliers were removed, C max The geometric mean ratio of was 0.774, with a 90% CI of 0.556 to 1.077, and the AUC inf The geometric mean ratio of was 0.849, with a 90% CI of 0.657 to 1.098. As will be understood by those skilled in the art, patients with severe renal dysfunction have higher body weights and body mass indexes than healthy subjects. Without wishing to be bound by theory, this may indicate that subjects in cohort 1 have lower exposure to apraglutide due to lower plasma concentrations.

[0543] Example 11: Apraglutide production process-(III) 2A and 2B are schematic diagrams of an exemplary method of the present disclosure for producing apraglutide at improved purity levels compared to previously described synthetic routes (e.g., U.S. Patent No. 8,580,918). The manufacturing process described in FIG. 2A and FIG. 2B is referred to herein as Process B, and includes a primary purification by RP-HPLC (C18) chromatography in a TFA-based mobile phase (HO / acetonitrile) (adjusting the pH of the fractions using sodium bicarbonate (NaHCO3) to a purity of ≧90%), followed by a secondary purification by RP-HPLC (C18) in a NaHCO3 mobile phase (HO / acetonitrile) (to a purity of ≧97%), followed by desalting / buffer exchange by RP-HPLC (C18) in a sodium acetate (NaOAc) / HO / acetonitrile mobile phase. Table 2a shows the purity of the apraglutide product produced using Process B, as well as the levels of major contaminants. Table 2a shows the purity of the apraglutide product produced using Process B, as well as the overall product yield of this process. As shown in Tables 2a and 2b, Process B can obtain apraglutide with a purity of 97% or more and low levels of contaminants. Furthermore, Process B shows a product yield of about 20%.

[0544] impurities The process for producing apraglutide described in FIG. 2A and FIG. 2B was further modified to obtain β-Asp in apraglutide. 3 The level of peptide isomer impurities can be reduced to less than 1.5%. In some embodiments, the process for producing apraglutide described in Example 8 can reduce the β-Asp in apraglutide. 3 The level of peptide isomeric impurities was reduced to less than 1%.

[0545] β-Asp 3 The formation of the impurity is due to high pH and the sequence Asp 3 -Gly 4This is supported by the presence of. Asp-Gly sequences are particularly susceptible to aspartimide formation due to ring closure (attack of the nitrogen from the α-carboxyamide bond on the β-carboxy side chain). Aspartimides are susceptible to base-catalyzed epimerization and can undergo ring-opening reactions, which can result in the formation of multiple by-products. Attack by water can generate β-aspartyl peptides.

[0546] β-Asp during the decarboxylation / extraction step upon peptide cleavage from the resin (step 3(1), Figure 2B) 3 The formation of impurities can reach levels >1% and is accelerated by prolonged exposure to extreme pH (especially a strong base solution of ammonia in HO / ACN 80:20, pH approx. 10 at room temperature). The aplaglutide manufacturing process described in Figures 2A and 2B can be modified to lower the pH from 10 to 8, shorten the decarboxylation reaction time from 24 hours to 65 minutes, change the reaction temperature from room temperature to 50°C, and reduce the storage temperature from room temperature to 5°C. These changes result in the formation of β-Asp at levels below 1.5%. 3 In some embodiments, these changes may result in impurities such as β-Asp at levels less than 1%. 3 Impurities were produced.

[0547] The process for producing apraglutide described in FIG. 2A and FIG. 2B also includes the residue Asp 3 The Fmoc deprotection reaction of can be modified to involve two cycles of deprotection (first cycle for 15 min, second cycle for 30 min) using a solution of 10% piperidine and 2% oxymer in DMF. These milder basic conditions afford the deprotection of Asp 3 allowing for the complete deprotection of aspartimide and then β-Asp 3 Reduces by-product formation.

[0548] The formation of D-His impurity may result from racemization of amino acids during the coupling reaction using DIC / Oxima. The process of sequential assembly of individual Gly and His amino acids showed variability in the level of D-His impurity between 0.3% and 1.2%, sometimes accompanied by incomplete coupling. Incorporation of Fmoc-His(Trt)-OH using the coupling reagent DIC / Oxima may favor racemization of His to the D form.

[0549] The aplaglutide manufacturing process as described in Figures 2A and 2B can be modified to use the dipeptide Boc-His(Trt)-Gly-OH as the starting material for this reaction, instead of the sequential assembly of Gly and His amino acids. This allows the racemization level in His to be reduced to less than 1% (0.3% in one working batch) and incorporates the final D-His level into the dipeptide (Boc-D-His(Trt)-Gly-OH) specification. This also allows a reduction in the number of deprotection steps, thus allowing the β-Asp, as described above, to be reduced. 3 The contributing effect of the deprotection conditions (high pH) on impurity formation is minimized.

[0550] The process for producing apraglutide as described in Figures 2A and 2B and modified as described above is referred to herein as "Process C." As shown in Table 2b, Process C can produce apraglutide having a purity of 97% or more and a product yield of about 22%.

[0551] Equivalent The foregoing description is provided for illustrative purposes only and is not intended to limit the disclosure to the precise form disclosed. Details of one or more embodiments and / or aspects of the present disclosure are set forth in the accompanying description above. Any one of the embodiments and / or aspects described herein can be combined with any other embodiment and / or aspect described herein, and any number of embodiments and / or aspects can be combined. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. Other features, objects, and advantages of the present disclosure will become apparent from the description and claims. In this specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference.

Claims

1. A sodium salt of apraglutide having a purity of 95% or more, wherein the sodium salt of apraglutide has the following structure: 【Chemistry 1】

2. The sodium salt of apraglutide according to claim 1, wherein the sodium salt of apraglutide has a purity of 97% or more.

3. The sodium salt of apraglutide contains 3% or less of Des-Gly. ４ The sodium salt of apraglutide according to claim 1 or 2, containing apraglutide impurities.

4. Aspartimide in the sodium salt of apraglutide ３ Apraglutide impurity, Asp ３３ -OH apraglutide impurity and Des-Ser ７ The sodium salt of apraglutide according to any one of claims 1 to 3, wherein the total amount of apraglutide impurities is 2% or less.

5. The sodium salt of apraglutide contains 2% or less of [Trp ２５ 5. The sodium salt of apraglutide according to claim 1, comprising an apraglutide impurity, wherein the apraglutide is 2-(2,4,6-trimethoxyphenyl).

6. The sodium salt of apraglutide contains 1.5% or less of β-Asp ３ The sodium salt of apraglutide according to any one of claims 1 to 5, containing apraglutide impurities.

7. The sodium salt of apraglutide contains 1% or less of β-Asp ３ The sodium salt of apraglutide according to any one of claims 1 to 6, containing apraglutide impurities.

8. The sodium salt of apraglutide according to any one of claims 1 to 7, wherein the sodium salt of apraglutide contains 1% or less of D-His apraglutide impurity.

9. The sodium salt of apraglutide is Less than 1% Asp ３３ -OH apraglutide impurity; Des-Ser of 1% or less ７ Apraglutide impurities, Less than 1% D-aspartimide ３ Apraglutide impurities, Less than 1% [Trp ２５ ,2-(2,4,6-trimethoxyphenyl)]apraglutide impurity, Des-Gly in the sodium salt of apraglutide ４ Apraglutide impurities and aspartimide ３ The sodium salt of apraglutide according to any one of claims 1 to 8, wherein the total amount of apraglutide impurities is 1% or less.

10. The sodium salt of apraglutide according to any one of claims 1 to 9, wherein the sodium salt of apraglutide is provided as a lyophilized powder.

11. A pharmaceutical composition comprising the sodium salt of apraglutide according to any one of claims 1 to 10.

12. 12. The pharmaceutical composition of claim 11, further comprising at least one of glycine, L-histidine, and mannitol.

13. The pharmaceutical composition comprises: Approximately 12.5 mg of apraglutide (sodium salt); Approximately 1.88 mg of glycine; Approximately 3.88 mg of L-histidine; and about 57.5 mg of mannitol.

14. 14. The pharmaceutical composition of claim 12 or 13, wherein the pharmaceutical composition is provided as a lyophilized powder.

15. A dual-chamber powder syringe comprising the sodium salt of apraglutide according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 14.

16. 1. A method for making a GLP-2 analog peptide, comprising: a) carrying out solid phase peptide synthesis (SPPS) to synthesize said GLP-2 analog peptide on Fmoc-Rink-amide-methylbenzhydrylamine (MBHA) resin; b) cleaving the synthesized GLP-2 analog peptide from the resin and deprotecting the side chains of the synthesized GLP-2 analog peptide by treating the resin with a solution containing trifluoroacetic acid (TFA), water, and anisole; c) purifying the synthesized GLP-2 analog peptide from step (b) by performing a first preparative reversed-phase high performance liquid chromatography (RP-HPLC) purification using a TFA-based mobile phase, thereby producing a solution containing the GLP-2 analog peptide having a purity of 90% or more; d) NaHCO ３ and purifying the product of step (c) by performing a second RP-HPLC purification using the system mobile phase, thereby producing a solution containing said GLP-2 analog peptide having a purity of 97% or greater.

17. 17. The method of claim 16, further comprising: e) further purifying the product from step (d) by performing a third RP-HPLC purification using a NaOAc-based mobile phase, thereby producing a solution comprising the sodium salt of said GLP-2 analog peptide having a purity of 97% or greater.

18. f) dissolving the sodium salt of the GLP-2 analog peptide using 0.1% AcOH in water; adjusting the pH of the solution containing the ammonium salt to about pH 7.9; g) passing the product of step (f) through a filter having a pore size of 0.2 μm; 18. The method of claim 17, further comprising: h) lyophilizing the product of step (g), thereby producing a lyophilized sodium salt of said GLP-2 analog peptide having a purity of 97% or greater.

19. 17. The method of claim 16, further comprising: c) (i) decarboxylating the synthesized GLP-2 analog peptide by solubilizing the peptide in a solution comprising water and acetonitrile in an ammonia buffer.

20. 20. The method of claim 19, wherein the pH of the solution of water and acetonitrile in ammonia buffer is adjusted to about pH 8.

0.

21. Step (a) i) preparing an MBHA resin on which the SPPS is carried out; ii) performing an initial Fmoc deprotection reaction followed by a coupling reaction to add a first Fmoc-protected amino acid to the resin, thereby forming a protected peptide on the resin; iii) performing an Fmoc deprotection reaction followed by a coupling reaction to add at least one Fmoc-protected amino acid to the protected peptide; iv) repeating step iii) until the GLP-2 analog peptide has been synthesized on the resin to produce a resin-linked Fmoc-protected and side-chain protected GLP-2 analog peptide; v) performing an Fmoc deprotection reaction to produce the resin-linked side-chain protected GLP-2 analog peptide; vi) drying the resin-linked side-chain protected GLP-2 analog peptide.

22. Step (a)(i) (a1) The resin is ２ Washing with a solution containing dimethylformamide (DMF) and N,N-diisopropylethylamine (DIEA) at 5 mL per gram of resin under atmospheric conditions; (b1) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in DMF, coupling a Rink amide linker to the resin in a solution containing hexafluorophosphate benzotriazole tetramethyluronium (HBTU), DIEA, and hydroxybenzotriazole (HOBt); (c1) washing the product formed in step (b1) with DMF; (d1) The resin is dissolved in DMF and acetic anhydride (Ac ２ carrying out a reduction reaction by contacting the mixture with a solution containing DIEA; (e1) washing the product formed in step (d1) with DMF.

23. Carrying out an Fmoc deprotection reaction followed by a coupling reaction; (a2) treating the resin with a solution of piperidine in DMF; (b2) washing the resin with DMF; (c2) washing the resin with a solution containing DMF and oxymer; (d2) contacting the resin with at least one Fmoc-protected amino acid and a first amount of a solution comprising diisopropylcarbodiimide (DIC) and ethyl cyanohydroxyiminoacetate (Oxima); (e2) contacting the resin with a second amount of a solution comprising DIC and Oxyma; 23. The method of claim 21 or 22, comprising: (f2) washing the product formed in step (e2) with DMF.

24. 24. The method of claim 23, wherein the resin is contacted with a second amount of solution containing the DIC and Oxyma about 30 minutes after contacting the resin with the first amount of solution containing the DIC and Oxyma.

25. carrying out an Fmoc deprotection reaction, followed by a coupling reaction; (a2) treating the resin with a solution of piperidine and oxymer in DMF; (b2) washing the resin with DMF; (c2) washing the resin with a solution containing DMF and oxymer; (d2) contacting the resin with at least one Fmoc-protected amino acid and a first amount of a solution comprising diisopropylcarbodiimide (DIC) and ethyl cyanohydroxyiminoacetate (Oxima); (e2) contacting the resin with a second amount of a solution comprising DIC and Oxyma; 23. The method of claim 21 or 22, comprising: (f2) washing the product formed in step (e2) with DMF.

26. 26. The method of claim 25, wherein the resin is contacted with a first amount of a solution comprising piperidine and oxyma in DMF for 15 minutes, followed by contacting the resin with a second amount of a solution comprising piperidine and oxyma in DMF for 30 minutes.

27. The at least one Fmoc-protected amino acid is Fmoc-Gln(Trt)-Thr(Ψ Ｍｅ，Ｍｅ The method according to any one of claims 23 to 26, wherein the aryl group is -(pro)-OH.

28. The method according to any one of claims 23 to 26, wherein the at least one Fmoc-protected amino acid is Fmoc-Gly(Tmb)-OH.

29. 27. The method of any one of claims 23 to 26, wherein the at least one protected amino acid is Boc-His(Trt)-Gly-OH.

30. 30. The method of any one of claims 23 to 29, wherein the method further comprises performing a coupling test between steps (e2) and (f2), the coupling test being a Kaiser test.

31. The method according to any one of claims 16 to 30, wherein the GLP-2 analogue peptide is apraglutide.

32. A composition comprising the GLP-2 analogue peptide produced using the method of any one of claims 16 to 31.

33. 1. A method of treating short bowel syndrome associated intestinal failure (SBS-IF) or short bowel syndrome associated intestinal dysfunction (SBS-II) in a subject, comprising administering apraglutide or a pharma- ceutical acceptable salt thereof to the subject; The apraglutide or a pharma- ceutically acceptable salt thereof is administered at a dose of about 2.5 mg / week when the subject has a body weight of less than 50 kg; or The method, wherein the apraglutide or a pharma- ceutically acceptable salt thereof is administered at a dose of about 5 mg / week when the subject has a body weight of 50 kg or more.

34. 34. The method of claim 33, wherein the apraglutide or a pharma- ceutically acceptable salt thereof is administered by subcutaneous injection.

35. 35. The method of claim 33 or 34, wherein the subject has colonic continuity and the apraglutide or a pharma- ceutically acceptable salt thereof is administered for about 48 weeks.

36. 36. The method of claim 35, wherein the subject has greater than 50% colonic continuity.

37. 35. The method of claim 33 or 34, wherein the subject has at least one stoma and the apraglutide is administered for about 24 weeks.

38. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of a meal-ingested wet weight in the subject compared to an untreated or placebo-treated subject.

39. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof reduces fecal output in the subject compared to untreated or placebo-treated subjects.

40. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof increases absolute urinary output in the subject compared to untreated or placebo-treated subjects.

41. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of sodium and potassium in the subject compared to untreated or placebo-treated subjects.

42. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof increases sodium and potassium urinary excretion in the subject compared to untreated or placebo-treated subjects.

43. 34. The method of claim 33, wherein said administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of energy in the subject compared to untreated or placebo-treated subjects.

44. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof reduces the energy content of fecal excretion in the subject compared to untreated or placebo-treated subjects.

45. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof increases intestinal absorption of carbohydrates, proteins, and lipids in the subject compared to untreated or placebo-treated subjects.

46. 34. The method of claim 33, wherein the administration of apraglutide or a pharma- ceutically acceptable salt thereof increases citrulline concentrations in the subject compared to untreated or placebo-treated subjects.

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