WO2025162422A1 - Glucagon-like peptide 2 derivative and use thereof - Google Patents

Abstract

Provided are a glucagon-like peptide 2 derivative and the use thereof. The glucagon-like peptide 2 derivative has an amino acid sequence of HGDGSFSDEMNTILDX₁₆LAARDFIX₂₄WLIQTKITD, wherein X₁₆ is selected from K or L, X₂₄ is selected from N or K, at least one of X₁₆ and X₂₄ is K, and amino acid K residues at positions 16 and/or 24 of the derivative are linked to a fatty acid side chain. The glucagon-like peptide 2 derivative can be used for promoting small intestine growth, and preventing and/or treating obesity and gastric and intestinal-related disorders.

Description

Glucagon-like peptide 2 derivatives and their applications Technical Field

The present disclosure belongs to the field of biomedicine and relates to glucagon-like peptide 2 (GLP-2) derivatives and applications thereof.

Background Art

Glucagon-like peptide 2 (GLP-2) is a polypeptide secreted by intestinal L cells in response to food. GLP-2 increases villus and crypt growth by binding to GLP-2 receptors located in enteroendocrine cells and enteric neurons. GLP-2 also increases intestinal and portal blood flow, inhibits gastric emptying and gastric acid secretion, and ultimately enhances intestinal absorption of nutrients.

Short bowel syndrome (SBS) is a rare, debilitating condition caused by the loss of a functional small intestine, affecting three million people worldwide. In adults, SBS often results from intestinal resection due to trauma, malignancy, or chronic enteritis. The short remnant intestine impairs the absorption of nutrients and fluids. SBS can lead to intestinal failure (IF), a condition in which intestinal function decreases to the minimum required for nutrient, water, and electrolyte absorption. This can lead to complications such as diarrhea, dehydration, and malnutrition, significantly impacting the quality of life and life expectancy of patients with SBS. To ensure adequate nutritional and energy support, intravenous nutrient infusion is a common treatment option, but this approach is inconvenient and unfriendly. Teduglutide (trade name: Gattex), developed by Takeda, is the only GLP-2 analogue approved for marketing in the EU in 2012 for the treatment of SBS in adults, with an expanded indication for SBS in children aged one year and older in 2019. It helps the intestine absorb more nutrients, reducing the frequency and volume of parenteral nutrition, making it the first disease-modifying therapy. However, the drug has a half-life of only approximately 1.3 hours ( https://www.accessdata.fda.gov/drugsatfda_docs/label/2012/203441Orig1s000lbl.pdf ) , requiring daily subcutaneous injection. To further reduce medication frequency, several long-acting GLP-2 receptor agonists are currently in development, including Apraglutide (VectiveBio) and Glepaglu tide (Zealand Pharma), both administered twice or once a week in Phase III clinical trials, and HM15912 (Hanmi), administered monthly in Phase II clinical trials.

GLP-2 also has anti-inflammatory properties, making it a potential target for the treatment of inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis. Furthermore, GLP-2 regulates intestinal motility and has the potential to be used to regulate irritable bowel syndrome (IBS). GLP-2 is also being studied for graft-versus-host disease (GvHD) and chemotherapy-related diarrhea.

Summary of the Invention

This disclosure provides a long-acting GLP-2 derivative that, compared to existing technologies, exhibits higher GLP-2R activation activity and a longer half-life, significantly reducing dosing frequency and improving patient compliance and experience. Furthermore, by incorporating NAC salt (N-[8-(2-hydroxybenzoyl)amino]caprylate) delivery technology, oral administration of the GLP-2 derivative is also possible, further enhancing patient compliance and convenience.

In a first aspect, the present disclosure provides a glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the glucagon-like peptide 2 derivative is:
HGDGSFSDEMNTILDX ₁₆ LAARDFIX ₂₄ WLIQTKITD,

wherein X ₁₆ is selected from K or L, X ₂₄ is selected from N or K, and at least one of X ₁₆ and X ₂₄ is K;

The amino acid K residue at position 16 and/or position 24 of the derivative is connected to a fatty acid side chain;

The connection is direct or indirect (eg, via a linker).

In some embodiments, in the above-mentioned glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof, X ₁₆ is K, X ₂₄ is N, and the amino acid sequence of the glucagon-like peptide 2 derivative is as shown in SEQ ID NO: 3.

In some embodiments, in the above-mentioned glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof, X ₁₆ is L, X ₂₄ is K, and the amino acid sequence of the glucagon-like peptide 2 derivative is as shown in SEQ ID NO: 4.

In some embodiments, in any of the above-mentioned glucagon-like peptide 2 derivatives or pharmaceutically acceptable salts thereof, the derivative is linked to the fatty acid side chain via the epsilon amino group of the amino acid K residue at position 16 and/or position 24.

In some embodiments, in any of the above-mentioned glucagon-like peptide 2 derivatives or pharmaceutically acceptable salts thereof, the fatty acid side chain is selected from

One or more of, wherein x is any integer from 4 to 38;

Preferably, the fatty acid side chain is selected from:

HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₅ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₇ CO-, HOOC(CH ₂ ) ₁₈ CO-, HOOC(CH ₂ ) ₁₉ CO-, HOOC(CH ₂ ) ₂₀ CO-, HOOC(CH ₂ ) ₂₁ CO- and HOOC(CH ₂ ) ₂₂ One or more of CO-;

More preferably, the fatty acid side chain is HOOC(CH ₂ ) ₁₆ CO-.

In some embodiments, in any of the above-mentioned glucagon-like peptide 2 derivatives or pharmaceutically acceptable salts thereof, the fatty acid side chain is connected to the amino acid K residue at position 16 and/or position 24 of the derivative via a linker.

In some embodiments, in the above-mentioned glucagon-like peptide 2 derivative or its pharmaceutically acceptable salt, the linker is selected from
One or more of, wherein m is 0, 1, 2 or 3; n is 1 or 2; p is any integer from 1 to 5;

Preferably, the connector is:

wherein m is 0, 1, 2 or 3, and n is 1; more preferably, wherein m is 1, and n is 1.

In some embodiments, the glucagon-like peptide 2 derivative is the polypeptide derivative M2 of the present disclosure, whose amino acid sequence is shown in SEQ ID NO: 3, and the epsilon amino group at the amino acid residue K at position 16 is connected to (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H(18-{[(23S)-23-carboxy-2,11,20-trioxo-10,19-diaza-4,7,13,16-tetraoxatricos-23-yl]amino}-18-oxooctadecanoic acid, i.e., 18-{[(23S)-23-carboxy-2,11,20-trioxyylidene-10,19-diaza-4,7,13,16-tetraoxatriacosan-23-yl]amino}-18-oxyylideneoctadecanoic acid); the structural formula of (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H is shown in formula (I).

In some embodiments, the glucagon-like peptide 2 derivative is the polypeptide derivative M3 of the present disclosure, whose amino acid sequence is shown in SEQ ID NO: 4, and the epsilon amino group at the 24th amino acid residue K is connected to (2-(2-(2-Aminoethoxy)ethoxy)acetyl) _2- (γGlu)-CO-( _CH2 ) ₁₆ -CO2H through an _amide bond; the structural formula of (2-(2-(2-Aminoethoxy)ethoxy)acetyl) _2- (γGlu)-CO-( _CH2 ) ₁₆ - _CO2H is also shown in formula (I).

In a second aspect, the present disclosure provides a method for preparing any of the above-mentioned glucagon-like peptide 2 derivatives or pharmaceutically acceptable salts thereof; the preparation method comprises the steps of preparing the glucagon-like peptide 2 derivative using a chemical method and/or a biological method;

Preferably, the chemical method includes liquid phase or solid phase polypeptide synthesis; the biological method includes molecular biology method and/or cell biology method.

In a third aspect, the present disclosure provides a pharmaceutical composition comprising any of the above-mentioned glucagon-like peptide 2 derivatives or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient;

The pharmaceutical compositions of the present disclosure may be administered by any suitable route known in the art, including, but not limited to, oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and/or cerebrospinal;

Preferably, the pharmaceutical composition is in the form of solid, liquid or semi-solid;

Preferably, the pharmaceutically acceptable excipients include one or more pharmaceutically acceptable lubricants, such as magnesium stearate;

Preferably, the pharmaceutical composition is an oral delivery composition; preferably, the oral delivery composition is in the form of solid, liquid, or semi-solid, more preferably solid, and further preferably an oral tablet;

Preferably, the oral delivery composition further comprises an oral absorption enhancer, which is an ingredient that can improve the oral absorption of the active ingredient of the drug, and is selected from one or more of the following: NAC salt, decanoate, Cu, Zn, Fe ions, reducing agents, tetrasodium ethylenediaminetetraacetic acid, sodium phosphate, tris(hydroxymethyl)aminomethane, lysine; the reducing agent is preferably ascorbic acid; preferably, the oral absorption enhancer is NAC salt (N-[8-(2-hydroxybenzoyl)amino]caprylate), and the NAC salt can be one or more of sodium salt (SNAC) and potassium salt (PNAC).

In some embodiments, the pharmaceutical composition is an oral tablet comprising any of the above-mentioned glucagon-like peptide 2 derivatives or a pharmaceutically acceptable salt thereof, NAC salt and magnesium stearate; in some embodiments, the oral tablet comprises any of the above-mentioned glucagon-like peptide 2 derivatives or a pharmaceutically acceptable salt thereof, PNAC and magnesium stearate.

In some embodiments, the pharmaceutical composition of the present disclosure further comprises one or more additional pharmaceutically active ingredients. In some embodiments, the pharmaceutically active ingredients may have beneficial effects on preventing and/or treating obesity and/or gastrointestinal and intestinal disorders, for example, they may be anti-inflammatory active ingredients;

Preferably, the stomach and intestine related disorders are ulcers, digestive disorders, malnutrition, malabsorption syndrome, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, abdominal splenosis, tropical splenosis, hypogammaglobulinemia splenosis, small intestinal damage, chemotherapy-induced diarrhea/mucositis, irritable bowel syndrome, graft-versus-host disease; preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

In some embodiments, pharmaceutical composition of the present disclosure can be used in combination with another one or more pharmaceutical compositions. In some embodiments, different pharmaceutical compositions can be applied to patients in need simultaneously, sequentially or respectively. In some embodiments, different pharmaceutical compositions are applied to patients in need in chronological order, for example, in 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, 2 months, 3 months or longer, simultaneously, sequentially or respectively apply each pharmaceutical composition once, twice, three times or more in one day.

In a fourth aspect, the present disclosure provides use of any of the above-mentioned glucagon-like peptide 2 derivatives or pharmaceutically acceptable salts thereof, or any of the above-mentioned pharmaceutical compositions in the preparation of drugs for promoting small intestinal growth, preventing and/or treating obesity, and preventing and/or treating gastric and intestinal related disorders;

Preferably, the stomach and intestine related disorders are ulcers, digestive disorders, malnutrition, malabsorption syndrome, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, abdominal splenomegaly, tropical splenomegaly, hypogammaglobulinemia splenomegaly, small intestinal damage, chemotherapy-induced diarrhea/mucositis, irritable bowel syndrome, and graft-versus-host disease; preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis;

Preferably, the drug is an oral drug;

Preferably, the oral medication is in the form of solid, liquid, or semisolid, more preferably solid, and even more preferably oral tablets;

Preferably, the promoting small intestine growth includes promoting the increase of small intestine weight and small intestine thickness.

In a fifth aspect, the present disclosure further provides any of the above-mentioned glucagon-like peptide 2 derivatives or pharmaceutically acceptable salts thereof or any of the above-mentioned pharmaceutical compositions for use in treatment.

In a sixth aspect, the present disclosure further provides a method for promoting small intestinal growth, preventing and/or treating obesity, and preventing and/or treating gastric and intestinal related disorders, comprising the step of administering a therapeutically effective amount of any of the above-mentioned glucagon-like peptide 2 derivatives or a pharmaceutically acceptable salt thereof, or any of the above-mentioned pharmaceutical compositions to a patient in need thereof;

Preferably, the stomach and intestine related disorders are ulcers, digestive disorders, malnutrition, malabsorption syndrome, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, abdominal splenomegaly, tropical splenomegaly, hypogammaglobulinemia splenomegaly, small intestinal damage, chemotherapy-induced diarrhea/mucositis, irritable bowel syndrome, and graft-versus-host disease; preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis;

Preferably, the promoting small intestine growth includes promoting the increase of small intestine weight and small intestine thickness.

The drugs of the present disclosure can be administered to the patient (e.g., mammal, such as human) by any suitable route known in the art, including but not limited to oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic and/or cerebrospinal.

In some embodiments, in any of the above methods, the administration cycle of the glucagon-like peptide 2 derivative or its pharmaceutically acceptable salt or the pharmaceutical composition is once or more every day, every week, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, for example, every day, every week, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111

In some embodiments, in any of the above methods, the total number of times the glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof or the pharmaceutical composition is administered can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

The present disclosure also provides a kit comprising any of the above-mentioned glucagon-like peptide 2 derivatives or pharmaceutically acceptable salts or pharmaceutical compositions thereof, and optionally instructions for use.

The present disclosure has the following beneficial effects:

1. Compared with the prior art, the polypeptide derivatives disclosed herein greatly enhance GLP-2R activation activity, providing highly effective pharmaceutical ingredients for the prevention and/or treatment of GLP-2R-related diseases.

2. The half-life of the polypeptide derivatives disclosed herein is significantly prolonged, thereby reducing the frequency of administration and improving patient compliance and experience.

3. At the same time, the polypeptide derivatives disclosed herein can be effectively compatible with oral delivery agents and thus can be applied to oral medications. For example, by combining the use of NAC salt (N-[8-(2-hydroxybenzoyl)amino]caprylate) delivery technology, oral absorption of the polypeptide derivatives is achieved, which can further increase patient compliance and convenience.

The glucagon-like peptide 2 derivatives disclosed herein can be used to promote small intestine growth, prevent and/or treat obesity and gastric and intestinal related disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the pharmacokinetic curve of Example 3 in rats.

FIG2 is the pharmacokinetic curve of beagle dogs in Example 4.

FIG3 is the experimental result of the small intestine growth promoting efficacy of Example 5.

FIG4 is the pharmacokinetic curve of oral administration to beagle dogs of Example 6.

DETAILED DESCRIPTION

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The present disclosure is further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure.

definition

Unless otherwise stated, the terms used herein have the following definitions.

Herein, the conventional one-letter and three-letter codes for the natural amino acids are used, as well as the commonly recognized three-letter codes for other amino acids, such as norleucine (Nle).All amino acid residues in the peptides of the present disclosure are in the L-configuration unless otherwise specified.

The terms "polypeptide" or "peptide" or "protein" are used interchangeably. A "polypeptide" or "peptide" or "protein" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring (e.g., synthetic) amino acids or amino acid analogs, regardless of post-translational modification (e.g., glycosylation or phosphorylation), wherein the amino acids in any chain are covalently linked by peptide bonds.

The term "fatty acid modified polypeptide" or "polypeptide derivative" refers to a polypeptide having a fatty acid side chain modification.

The terms “comprising,” “including,” or “containing” should be understood to imply the inclusion of specified components but not the exclusion of any other components.

The terms "patient," "subject," and "individual" are used interchangeably and include humans and non-human animals, including mammals such as monkeys, rats, mice, cows, pigs, goats, sheep, dogs, and cats.

When used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, "administering" and "treating" refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating a cell includes contacting an agent with a cell and contacting an agent with a fluid, wherein the fluid is contacted with the cell. "Administering" and "treating" also mean the in vitro and ex vivo treatment of a cell, for example, by an agent, diagnostic agent, binding composition, or by other cells.

As used herein, "preventing" or "treating" includes delaying the development of symptoms associated with a disease and/or lessening the severity of symptoms that will or are expected to develop due to the disease. The terms also encompass alleviating existing symptoms, preventing additional symptoms, and alleviating or preventing the underlying causes of the symptoms. Thus, the terms indicate that a beneficial result has been conferred on a vertebrate subject, such as a human, suffering from a disease.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a GLP-2 derivative or a pharmaceutically acceptable salt thereof that is effective in preventing or alleviating the disease or condition being treated when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. A therapeutically effective amount further refers to an amount of the GLP-2 derivative or a pharmaceutically acceptable salt thereof sufficient to result in a alleviation of symptoms, such as treatment, cure, prevention, or alleviation of the relevant medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of the symptoms of the condition. The effective amount for a particular subject may vary depending on a variety of factors, such as the disease being treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. The effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. When administered to an individual, the therapeutically effective amount refers to that individual ingredient. When administered in combination, the therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. A therapeutically effective amount will alleviate symptoms generally by at least 10%; usually by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.

GLP-2 derivatives and pharmaceutically acceptable salts thereof

The present invention discloses a glucagon-like peptide 2 derivative (GLP-2 derivative) with significantly improved GLP-2R activation activity, significantly prolonged half-life, and significantly increased plasma exposure compared to apraglutide, which is a fatty acid-modified polypeptide or polypeptide derivative.

Experiments have confirmed that the polypeptide derivatives M2 and M3 disclosed in the present invention have higher GLP-2R activation activity, longer half-life and higher plasma exposure than Apraglutide.

The amino acid sequence of the GLP-2 derivative M2 of the present disclosure is shown in SEQ ID NO: 3, and the epsilon amino group on the amino acid residue K at position 16 is bonded to (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H(18-{[(23S)-23-carboxy-2,11,20-trioxo-10,19-diaza-4,7,13,16-tetraoxatricos-23-yl]amino}-18-oxooctadecanoic acid, i.e., 18-{[(23S)-23-carboxy-2,11,20-trioxyylidene-10,19-diaza-4,7,13,16-tetraoxatriacosan-23-yl]amino}-18-oxyylideneoctadecanoic acid); the structural formula of (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H is shown in formula (I) herein.

The amino acid sequence of the GLP-2 derivative M3 disclosed herein is shown in SEQ ID NO: 4, and the epsilon amino group at the amino acid residue K at position 24 is bonded to (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H(18-{[(23S)-23-carboxy-2,11,20-trioxo-10,19-diaza-4,7,13,16-tetraoxatricos-23-yl]amino}-18-oxooctadecanoic acid, i.e., 18-{[(23S)-23-carboxy-2,11,20-trioxyylidene-10,19-diaza-4,7,13,16-tetraoxatriacosan-23-yl]amino}-18-oxyylideneoctadecanoic acid); the structural formula of (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H is shown in formula (I) herein.

It should be understood that the GLP-2 derivatives of the present disclosure can also be provided in the form of salts. Pharmaceutically acceptable salts include salts in the form of anions and salts in the form of cations. Some examples of salts in the form of anions include hydrochlorides, citrates, chloride salts, and acetates. Preferably, the salt is acetate. Some examples of salts in the form of cations include salts in which the cation is selected from the group consisting of alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium), and the like.

In addition, the GLP-2 derivatives of the present disclosure can also form coordination complexes with metal ions (such as Mn2 ⁺ and Zn2 ⁺ ), thereby existing in the form of complexes. Since the GLP-2 derivatives of the present disclosure have hydroxyl groups or carboxylic acids, the derivatives can also react with suitable carboxylic acids or alcohols to form esters, thereby existing in the form of esters. The GLP-2 derivatives of the present disclosure can also exist in the form of prodrugs, which can be converted into one of the parent compounds in vivo or in vitro. Generally, at least one biological activity of the GLP-2 derivative will be reduced in the prodrug form and can be activated by conversion of the prodrug to release the GLP-2 derivative or its metabolites. Some examples of prodrugs include the use of protecting groups, which can be removed in situ to release the active compound or used to inhibit the clearance of the drug in the body.

Synthetic GLP-2 derivatives

The GLP-2 derivatives disclosed herein can be prepared using chemical and/or biological methods. Chemical methods are preferred, for example, liquid or solid phase peptide synthesis methods can be used to synthesize the GLP-2 derivatives disclosed herein. Biological methods include molecular biology methods and cell biology methods.

The method for preparing the GLP-2 derivative of the present disclosure may include the following steps:

Synthesize the GLP-2 derivative disclosed herein by liquid-phase or solid-phase peptide synthesis, stepwise or by fragment assembly according to the peptide sequence; or

A nucleic acid construct encoding a polypeptide sequence of a GLP-2 derivative is transferred into a host cell, and then after culturing under certain conditions for a period of time, a polypeptide product of the GLP-2 derivative is obtained from the host cell culture, for example, by expressing the polypeptide from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants, or animals) using recombinant technology, and then modifying the polypeptide with fatty acid side chains to obtain the GLP-2 derivative of the present disclosure; or

The GLP-2 derivative of the present disclosure is obtained by using a nucleic acid construct encoding the polypeptide sequence of the glucagon-like peptide 2 derivative to express the polypeptide product of the GLP-2 derivative in a cell-free system, and then modifying the polypeptide with fatty acid side chains.

In some embodiments, the GLP-2 derivatives of the present disclosure are prepared by solid-phase peptide synthesis on a suitable resin. Solid-phase peptide synthesis procedures are well known in the art, for example, by attaching an N-terminally protected amino acid and its carboxyl terminus to an inert solid support carrying a cleavable linker to initiate solid-phase synthesis. The solid support can be any polymer that allows for coupling of the initial amino acid, such as Fmoc-Asp(OtBu)-Wang Resin resin. In some embodiments, using Fmoc-Asp(OtBu)-Wang Resin as the starting material, amino acids with Fmoc-protecting groups are sequentially connected according to the solid-phase synthesis method to obtain a protected linear peptide resin, during which the Fmoc-protecting groups are sequentially removed. The peptide grafting reaction is carried out using TBTU as a condensing agent to obtain a protected linear peptide resin. The peptide is then cleaved from the resin, and the side chain protecting groups and coupling building blocks are then simultaneously removed to obtain a fatty acid-modified peptide resin. The peptide is then cleaved from the resin and separated and purified by a chromatographic column, followed by freeze-drying to obtain a powdered purified peptide derivative.

In some embodiments, the polypeptide sequence of the GLP-2 derivative of the present disclosure is prepared using recombinant technology. In this case, the present disclosure also provides a nucleic acid molecule encoding the polypeptide sequence of the GLP-2 derivative of the present disclosure, the nucleotide sequence of which can be a codon-optimized sequence according to the host to be transferred; the nucleic acid molecule can be a DNA fragment or an RNA fragment, which can usually be obtained by amplification using a PCR instrument or artificial synthesis.

In some embodiments, the polypeptide sequences of the GLP-2 derivatives disclosed herein are prepared using recombinant technology. In such cases, the present disclosure also provides a recombinant vector comprising the aforementioned nucleic acid molecule; the recombinant vector includes a cloning vector for replicating the relevant sequence and an expression vector for expressing the relevant gene. The vector can be any vector commonly used in the art, such as a plasmid, phage, cosmid, minichromosome, or virus. In addition to the nucleic acid encoding the polypeptide sequence of the GLP-2 derivative described above, the expression vector can include not only a promoter for initiating transcription of the gene encoding the polypeptide sequence, but also a signal peptide sequence, a terminator for terminating transcription of the gene encoding the polypeptide sequence, and an enhancer sequence.

The method for constructing a recombinant expression vector can be any known method. The promoter described above, the nucleic acid encoding the gene for the polypeptide sequence, and other DNA segments (e.g., terminators, enhancers) if present, can be introduced into a suitable selected vector as a basis in a predetermined order. For example, a recombinant vector can be constructed by using restriction endonucleases and ligases, etc.

In some embodiments, the polypeptide sequences of the GLP-2 derivatives disclosed herein are prepared using recombinant technology. In such cases, the present disclosure further provides a recombinant cell comprising the aforementioned recombinant vector, wherein the recombinant cell expresses the polypeptide sequences of the GLP-2 derivatives disclosed herein with or without induction. In some embodiments, the method for constructing the recombinant cell comprises the following: transforming the recombinant expression vector into an expression host cell, culturing the cell, and inducing expression (if necessary) with the addition of an inducer to obtain the polypeptide sequences of the GLP-2 derivatives disclosed herein. Furthermore, the expression host cell is a prokaryotic or eukaryotic cell, such as Escherichia coli, yeast, plant cells, animal cells, and the like.

More specifically, the method for constructing the above-mentioned recombinant cell comprises the following steps:

(1) Amplification of genes encoding polypeptide sequences of GLP-2 derivatives;

(2) Construction of recombinant expression vector;

(3) The recombinant expression vector is transformed or transfected into the expression host cell;

(4) Optionally screen to obtain positive clones.

The polypeptides of the present disclosure may be secreted outside the cell or expressed on the cell surface or inside the cell.

In some embodiments, the preparation of the polypeptide sequence of the GLP-2 derivative of the present disclosure using recombinant technology comprises the following steps:

(1) culturing the recombinant cells and adding an inducer to induce (if necessary) the expression of the polypeptide sequence of the GLP-2 derivative disclosed herein to obtain a cell culture;

(2) Optionally, the polypeptide sequence of the GLP-2 derivative disclosed herein is isolated and purified from the cell culture (eg, cells, cell culture supernatant).

Biological activity

The GLP-2 derivatives disclosed herein have activation activity on the receptor GLP-2R and are GLP-2R agonists.

In this disclosure, _EC50 values are used as a numerical measure of the potency of an agonist for a given receptor (i.e., GLP-2R). The _EC50 value refers to the concentration that elicits 50% of the maximal effect. In the same assay for a specific receptor, a compound with a low _EC50 value can be considered to have a higher potency at the receptor.

The present disclosure tested the GLP-2R agonist activity of GLP-2 derivatives and found that in human GLP-2R reporter gene cell assays and rat GLP-2R reporter gene cell assays, the activities of M2 and M3 were at least 3 times that of the control Apraglutide.

Furthermore, through rat pharmacokinetic experiments and beagle dog pharmacokinetic experiments, it was found that compared with apraglutide, M2 of the present disclosure has a significantly prolonged half-life and significantly increased plasma exposure. Specifically, in the rat pharmacokinetic experiment, the half-life of apraglutide and M2 was 7.3h vs. 10.3h, and the plasma exposure was 6620.0h*nmol/L vs. 13265.1h*nmol/L; in the beagle dog pharmacokinetic experiment, the half-life of apraglutide and M2 was 17.7h vs. 54.8h, and the plasma exposure was 4854.6h*nmol/L vs. 25116.8h*nmol/L.

Furthermore, the efficacy of the small intestinal growth promoting drug was evaluated in a small intestinal growth model in rats. The results showed that the M2 disclosed herein can increase the small intestinal weight and small intestinal thickness coefficient in a dose-dependent manner, and its efficacy is significantly higher than that of Apraglutide. The efficacy of 10 nmol/kg M2 is between 50 nmol/kg and 100 nmol/kg Apraglutide.

The GLP-2 derivative disclosed herein was prepared into oral tablets and administered to male beagle dogs at a dose of one tablet once a day for five consecutive days. It was found that 7 mg M2 reached a maximum plasma exposure of 62.4 nM on the fourth day, while the steady-state concentration of 7 mg semaglutide (Rybelsus), a glucagon-like peptide 1 (GLP-1) receptor agonist, in humans was approximately 7.5 nM. This shows that the oral bioavailability of M2 is much higher than that of Rybelsus.

Pharmaceutical compositions of GLP-2 derivatives

The GLP-2 derivative or pharmaceutically acceptable salt thereof disclosed herein can be formulated into a pharmaceutical composition, wherein the GLP-2 derivative or pharmaceutically acceptable salt thereof is present in a therapeutically effective amount.

The pharmaceutical compositions described herein may be orally deliverable compositions.

The pharmaceutical composition or oral delivery composition described herein contains, in addition to the active ingredient GLP-2 derivative or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable excipient. Those skilled in the art are familiar with pharmaceutically acceptable excipients, such as non-toxic fillers, stabilizers, diluents, carriers, solvents or other formulation excipients. For example, diluents, excipients, such as microcrystalline cellulose, mannitol, etc.; fillers, such as starch, sucrose, etc.; binders, such as starch, cellulose derivatives, alginates, gelatin and/or polyvinyl pyrrolidone; disintegrants, such as calcium carbonate and/or sodium bicarbonate; absorption enhancers, such as quaternary ammonium compounds; surfactants, such as cetyl alcohol; carriers, solvents, such as water, saline, kaolin, bentonite, etc.; lubricants, such as talc, calcium/magnesium stearate, polyethylene glycol, etc. In some embodiments, the absorption enhancer disclosed in the present invention is an oral absorption enhancer or an oral delivery agent, which should be understood as any component or combination thereof that can improve the oral absorption of polypeptides that is well known to those skilled in the art, such as NAC salt, decanoate, Cu, Zn, Fe ions, reducing agents (such as ascorbic acid, etc.), tetrasodium ethylenediaminetetraacetic acid, sodium phosphate, tris(hydroxymethyl)aminomethane, lysine, etc., or a combination thereof, or an oral delivery agent disclosed in U.S. 8753683B, CN104884078B, US7138546B, etc.; preferably, the oral delivery agent disclosed in the present invention is NAC salt (N-[8-(2-hydroxybenzoyl)amino]octanoate), such as its sodium salt or potassium salt, and further preferably, the NAC salt is PNAC salt, i.e., potassium N-[8-(2-hydroxybenzoyl)amino]octanoate. The NAC salt or PNAC salt of the oral delivery agent described herein should be understood to be any crystalline form that can satisfy the oral delivery form of the composition of the present disclosure.

NAC salt (N-[8-(2-hydroxybenzoyl)amino]caprylate) is disclosed in CN 116327890 B, and its structural formula is shown in formula (II):

NAC salt can be sodium salt (SNAC) or potassium salt (PNAC), preferably PNAC. The structural formula of PNAC is shown in formula (III):

The preparation of NAC and PNAC has been disclosed in CN 116327890 B.

The oral delivery compositions disclosed herein are understood to be in any composition form for oral administration, such as solid, liquid, or semisolid forms. In some embodiments, the oral delivery compositions described herein are in solid form, such as tablets, capsules, granules, pills, and the like. Any solid form and ratio known to those skilled in the art for oral delivery of polypeptide compositions is within the scope of this application.

In some embodiments, the GLP-2 derivative of the present disclosure is prepared into an oral tablet, which contains a polypeptide derivative M2, a delivery agent (PNAC) and a lubricant (magnesium stearate), and the mass ratio of the three can be 7: (200-500): (5-15).

The oral tablets disclosed herein can be prepared by conventional methods, for example, the polypeptide derivative and PNAC are sieved, uniformly mixed with other excipients, and directly compressed into tablets.

The derivatives of the present invention or pharmaceutically acceptable salts thereof can be used alone or in combination with any compound that is beneficial for promoting small intestinal growth, preventing and/or treating obesity, preventing and/or treating gastric and intestinal related disorders (e.g., compounds with anti-inflammatory effects) in a pharmaceutical composition, which is expected to enhance the beneficial therapeutic effects of the derivatives of the present invention.

The pharmaceutical composition of the present disclosure can also be used in combination with one or more other drugs. Such combined use is expected to have a synergistic effect in promoting small intestinal growth, preventing and/or treating obesity, and preventing and/or treating gastric and intestinal related disorders.

Medical conditions

The GLP-2 derivatives of the present disclosure can be used as pharmaceutical agents for the prevention and/or treatment of obesity, the prevention or treatment of gastrointestinal and intestinal disorders (including the upper gastrointestinal tract of the esophagus) by administering an effective amount of a GLP-2 derivative or salt thereof as described herein. Gastric and intestinal disorders include: ulcers of any etiology (e.g., peptic ulcers, drug-induced ulcers, ulcers associated with infections or other pathogens), digestive disorders, malnutrition (e.g., cachexia and anorexia), malabsorption syndromes, short bowel syndromes, blind loop syndromes, inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis), abdominal spongiosa (e.g., caused by gluten-induced enteropathy or celiac disease), tropical spongiosa, hypogammaglobulinemic spongiosa, small intestinal damage and chemotherapy-induced diarrhea/mucositis (CID), irritable bowel syndrome, and graft-versus-host disease.

Among them, short bowel syndrome (SBS), also known as short gut, results from surgical resection, congenital defects, or disease-related intestinal absorption impairment, with patients subsequently unable to maintain a balance of fluids, electrolytes, and nutrients on a regular diet. Although adaptation typically occurs over the two years following resection, SBS patients experience reduced dietary intake and fluid loss.

Example 1: Preparation of polypeptide derivatives

Using solid phase organic synthesis and Fmoc-protected amino acid strategy, peptide derivatives were synthesized, cleaved, and purified to obtain the target product. Taking the peptide derivative M1 in Table 2 as an example, the preparation process is as follows:

1. Solid phase synthesis:

Using Fmoc-Asp(OtBu)-Wang Resin (substitution degree = 0.35 mmol/g) and the Fmoc/tBu process, the amino acids were condensed and connected in sequence from the C-terminus to the N-terminus (from right to left) according to the amino acid sequence of the above-mentioned polypeptide derivatives using the method shown in Table 1 to finally form a linear polypeptide resin.

Table 1 Synthesis procedures

The following amino acids were coupled sequentially:

A-01Fmoc-Thr(tBu)-OH,A-02Fmoc-Ile-OH,A-03Fmoc-Lys(Boc)-OH,A-04Fmoc-Thr(tBu )-OH,A-05Fmoc-Gln(Trt)-OH,A-06Fmoc-Ile-OH,A-07Fmoc-Leu-OH,A-08Fmoc-Trp(Boc) -OH,A-09Fmoc-Asn(Trt)-OH,A-10Fmoc-Ile-OH,A-11Fmoc-Phe-OH,A-12Fmoc-Asp(OtBu) -OH,A-13Fmoc-Arg(Pbf)-OH,A-14Fmoc-Ala-OH,A-15Fmoc-Ala-OH,A-16Fmoc-Leu-OH,A- 17Fmoc-Leu-OH,A-18Fmoc-Asp(OtBu)-OH,A-19Fmoc-Leu-OH,A-20Fmoc-Ile-OH,A-21Fmo c-Thr(tBu)-OH,A-22Fmoc-Asn(Trt)-OH,A-23Fmoc-Met-OH,A-24Fmoc-Lys(Boc)-OH,A-2 5Fmoc-Asp(OtBu)-OH,A-26Fmoc-Ser(tBu)-OH,A-27Fmoc-Phe-OH,A-28Fmoc-Ser(tbu)-O H,A-29Fmoc-Gly-OH,A-30Fmoc-Asp(OtBu)-OH,A-31Fmoc-Gly-OH,A-32Boc-His(Trt)-OH

After the linear polypeptide resin was synthesized, the Dde protection was removed by hydrazine hydrate method, and the following building blocks were coupled in sequence:
B01Fmoc-AEEA-OH,B02Fmoc-AEEA-OH,B03Fmoc-Glu-OtBu,B04 C18 diacid-OtBu

The peptide resin was washed, transferred out and dried to constant weight for cleavage.

2. Peptide resin cleavage:

Preparation of cleavage reagent: Calculate the amount of cleavage reagent (TFA: _H2O :EDT:TIS = 95:1:2:2 (volume ratio)) based on 1g peptide resin to 10ml±2ml of cleavage reagent. Add the required cleavage reagents _H2O , TFA, EDT, and TIS into the cleavage reaction bottle in sequence. Control the cleavage reagent temperature at 0-10°C.

The cleavage reagent was added to the peptide resin with stirring. After the system temperature stabilized, the reaction was stirred at 25-30°C for 2.5 hours. The lysate was filtered and precipitated with 5 times the liquid volume of glacial ether. The precipitate was filtered and washed three times with 3 times the liquid volume of glacial ether. After that, it was dried under reduced pressure at room temperature to obtain a crude solid product.

3. Purification and freeze-drying:

The crude product was finely ground. Purified water was prepared and slowly added to the ground product under stirring. Simultaneously, acetonitrile aqueous solution was added dropwise. After the crude product was completely added and dissolved, it was filtered through a 0.45 μm microporous filter membrane. The crude product was purified using a C-18 preparative column with mobile phases A: 0.1% TFA/ _H2O , B: 0.1% TFA/ACN. Separation and purification were performed at room temperature using an appropriate gradient. The target product was collected, analyzed, tested, and classified. The impurity purity was required to be ≥90%. Unqualified target products were collected and separated and purified again using an appropriate gradient. The qualified main peak was freeze-dried under reduced pressure to obtain a powdered purified polypeptide derivative.

Apraglutide, M2, M3 and M4, the peptide derivatives in Table 2, were synthesized in a similar manner, wherein Apraglutide is a GLP-2 derivative currently under clinical research by VectiveBio.

Table 2 Peptide derivatives

In Table 2, the epsilon amino group of lysine is linked to (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H via an amide bond; (2-(2-(2-Aminoethoxy)ethoxy)acetyl) ₂ -(γGlu)-CO-(CH ₂ ) ₁₆ -CO ₂ H is 18-{[(23S)-23-carboxy-2,11,20-trioxo-10,19-diaza-4,7,13,16-tetraoxatricos-23-yl]amino}-18-oxooctadecanoic acid, namely 18-{[(23S)-23-carboxy-2,11,20-trioxydeoxy-10,19-diaza-4,7,13,16-tetraoxatriacontan-23-yl]amino}-18-oxydecadecanoic acid, whose structural formula is shown in formula (I):

In formula (I),

Part of the connector,

Part of it is fatty acids,

In the connector Part of it is γGlu.

The abbreviations used in the above preparation process have the following meanings:

AA：Amino Acid

AEEA: 2-(2-(2-aminoethoxy)ethoxy)acetic acid

Boc：t-Butyloxy carbonyl, tert-butyloxycarbonyl

ACN: acetonitrile

DMF：N,N-Dimethyl formamide

DIEA：N,N-Diisopropylethylamine

Dde：2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, 2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl

EDT: 1,2-Ethanedithiol

Fmoc：9-fluorenylmethyloxycarbonyl，9-fluorenylmethyloxycarbonyl

OtBu: tert-butyl ester group

Pbf：2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl chloride

Pip：Piperidine, piperidine

TBTU: O-(Benzotriazol-l-yl)-N,N,N',N',-tetramethyluronium Tetrafluoroborate, O-Benzotriazol-N,N,N',N'-tetramethyluronium Tetrafluoroborate

tBu：tertiary butyl

TFA: Trifluoroacetic acid

TIS: Triisopropylsilane, triisopropylsilane

Trt：Triphenylmethyl, triphenylmethyl

γGlu: γ-glutamate

Example 2: Cellular activity detection of GLP-2 derivatives

The activity of GLP-2 derivatives in activating GLP-2R was investigated through human GLP-2R-CRE-Luciferase-HEK293 (human GLP-2R: NCBI Reference Sequence: NM_004246.3) or rat GLP-2R-CRE-Luciferase-HEK293 (rat GLP-2R: NCBI Reference Sequence: NM_021848.2) reporter gene experiments.

Construction of human GLP-2R-CRE-Luciferase-HEK293: HEK293 cells (Cell Resource Center, Basic Medical School, Institute of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, catalog number 1101HUM-PUMC000010) were transfected with the plasmid pGL4.29[luc2P/CRE/Hygro] vector (Promega, catalog number E8471) containing a multi-copy cAMP response element (CRE)-driven luciferase expression cassette and pcDNA3.1(+) (Invitrogen, catalog number V79020) containing the human GLP-2R gene to obtain GLP-2R-CRE-Luciferase-HEK293 transient or stable cell lines containing the luciferin expression plasmid.

Human GLP-2R reporter gene cell test:

In a 96-well plate, 100 μL of human GLP-2R-CRE-Luciferase-HEK293 cells (3 × ¹⁰ cells/mL) cultured in DMEM + 10% FBS was added to each well. After incubation at 37°C, 5% _CO₂ , the old medium was removed. Then, 50 μL of fresh DMEM + 10% FBS was added to the corresponding wells. Ten three-fold dilutions of apraglutide, M1, M2, M3, or M4 were added to the corresponding wells, with each compound having a final concentration of 2500 ng/mL, 833 ng/mL, and 278 ng/mL. After an additional 24 hours of incubation at 37°C, 5% _CO₂ , 100 μL of Bright-Glo reagent (Promega) was added. The plate was gently tapped to mix the solution, and chemiluminescence was read on a microplate reader after 3 minutes. GraphPad Prism 9 was used to generate four-parameter graphs and calculate _EC50 values.

Rat GLP-2R reporter gene cell test:

In a 96-well plate, 30,000 HEK293 cells cultured in DMEM + 10% FBS were added to each well. The next day, 25 ng of pcDNA3.1(+) plasmid containing the rat GLP-2R gene (Invitrogen, catalog number V79020) and 50 ng of pGL4.29[luc2P/CRE/Hygro] vector (Promega, catalog number E8471) were transferred to each well. After incubation for 24 hours at 37°C and 5% _CO2 , the old medium was removed and 50 μL of fresh DMEM + 10% FBS medium was added. Apraglutide, M2, or M3 compound solution with different final concentrations was added to the corresponding wells. Ten three-fold dilutions of each compound were prepared in a descending order of 5000 ng/mL, 1667 ng/mL, and 556 ng/mL. After incubation for another 24 hours in a 37°C, 5% _CO2 incubator, 100 μL of Bright-Glo reagent (Promega) was added. The plate was gently tapped to mix the solution. After 3 minutes, chemiluminescence was read using a microplate reader. Four-parameter graphs were constructed using GraphPad Prism 9, and _EC50 values were calculated.

The results are shown in Table 3.

Table 3 Cell activity test

In Table 3, ND: not measured.

The results showed that in terms of GLP-2R activation activity:

In human GLP-2R cell experiments, M2 and M3 were approximately three times more active than the control molecule, apraglutide, while M1 was approximately half as active, and M4 was nearly 100 times weaker than apraglutide. This suggests that fatty acid modifications at positions 16 and 24 help the molecule maintain high activity, while those at positions 9 and 30 reduce its activity to varying degrees.

In rat GLP-2R cell experiments, both M2 and M3 were more active than the control molecule Apraglutide.

Example 3: Pharmacokinetic Experiment in Rats

This experiment used male SD rats of about 10 weeks old. The experimental animal breeding conditions were room temperature of 20℃～23℃ and relative humidity of 40%～50%. During the animal quarantine period and the experimental process, the feed used was Co60 irradiated experimental growth and breeding mouse feed 1035, and the drinking water was purified water, supplied with drinking bottles, and water was available freely. A single subcutaneous injection of 1 mg/kg dose of the drug molecule (i.e., Apraglutide or M2) (n=3) (solvent was PBS, pH 7.4, drug concentration was 0.2 mg/mL, and administration volume was 5 mL/kg) was administered. Whole blood was collected from the animals before administration (-5 min) and 2h, 4h, 6h, 24h, 48h, 72h, and 96h after administration, and plasma was prepared (EDTA as an anticoagulant). Plasma was stored at -80℃ and used for subsequent analysis. Plasma drug levels were analyzed using LC-MS/MS (Waters ACQUITY I Class Premier UPLC tandem with Sciex 6500+QQQ). The ion-pair mass-to-charge ratio used for apraglutide was 942.2/1178.2, and the ion-pair mass-to-charge ratio used for M2 was 1121.3/1088.3. Experimental data were plotted using GraphPad Prism 10, and the pharmacokinetic curve for rats is shown in Figure 1.

The pharmacokinetic parameters (C _max , T _max , T _1/2 , AUC, MRT) of the drug were calculated using a non-compartmental model. The results are shown in Table 4.

Table 4 Pharmacokinetic parameters

The results showed that in SD rats, compared with Apraglutide, M2 had a longer half-life and higher plasma exposure, further confirming that the introduction of fatty acid side chains into the molecule would significantly improve the pharmacokinetic properties of the molecule.

Example 4: Beagle dog pharmacokinetic study

This study used 8-12 kg male beagle dogs, who were given a single subcutaneous injection of 0.15 mg/kg of the drug molecule (i.e., Apraglutide or M2) (n=2) (the solvent was PBS, pH 7.4, the drug concentration was 0.3 mg/mL, and the single-point injection did not exceed 2 mL). Whole blood was collected from the animals before administration (-10 min) and at 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, 264h, 312h, 360h, 408h, and 456h after administration, and plasma was prepared (sodium heparin was used as an anticoagulant). Plasma was stored at -80°C and used for subsequent analysis. The drug content in plasma was analyzed using LC-MS/MS (Waters ACQUITY I Class Premier UPLC tandem with Sciex 6500+QQQ). The ion-to-mass ratio used for apraglutide was 942.2/1178.2, and the ion-to-mass ratio used for M2 was 1121.3/1088.3. The experimental data were plotted using GraphPad Prism 10, and the pharmacokinetic curve for beagle dogs is shown in Figure 2.

The pharmacokinetic parameters (C _max , T _max , T _1/2 , AUC, MRT) of the drug were calculated using a non-compartmental model. The results are shown in Table 5 .

Table 5 Pharmacokinetic parameters

The results showed that in beagle dogs, M2 had a longer half-life and higher plasma exposure than apraglutide, further confirming that the introduction of fatty acid side chains into the molecule would significantly improve the pharmacokinetic properties of the molecule.

Example 5: Evaluation of GLP-2 derivatives in a rat small intestinal growth model

This study used SD rats (6-8 weeks old, male, weighing approximately 200 g, from Spectrum Biotechnology Co., Ltd., Beijing) as experimental subjects. Animals were randomly divided into groups according to body weight, with 5 animals per group. Vehicle (solvent control, PBS, pH 7.4), apraglutide (10/50/250 nmol/kg), and M2 (10/50/250 nmol/kg) were administered subcutaneously once daily for five consecutive days. After the last dose, all animals were fasted and weighed 24 hours later. The animals were then anesthetized and killed, and the small intestine (from the pylorus to the anterior cecum) was removed and rinsed with saline. After patting dry with gauze, the small intestine length and weight were measured. This was used to evaluate the growth-promoting effects of apraglutide and M2 on the small intestine of SD rats. The thickness coefficient (coefficient of thickness) is the ratio of small intestine weight (g) to small intestine length (cm). Graphs were generated using GraphPad Prism 10. Mean ± standard error (SEM) was used to show the small intestinal weight or thickness coefficient of each group and to compare differences between groups. Statistical differences were analyzed by one-way ANOVA. Compared with the vehicle control group, ** indicates p < 0.01, **** indicates p < 0.0001, and ns indicates no statistical difference.

The small intestinal growth promoting drug efficacy experiment is shown in Figure 3.

The results showed that both apraglutide and M2 increased small intestinal weight and intestinal thickness coefficient in a dose-dependent manner compared to the vehicle control. The efficacy of 10 nmol/kg M2 was intermediate between that of 50 nmol/kg and 250 nmol/kg apraglutide, due to M2's higher GLP-2R activation activity and longer half-life.

Example 6: Pharmacokinetic study of oral GLP-2 derivatives in beagle dogs

Oral tablet preparation:

The contents of some main components in the peptide derivative tablets containing fatty acid side chains are shown in Table 6:

Table 6 Polypeptide derivative tablets

The preparation method of the polypeptide derivative tablet containing fatty acid side chains is as follows: the polypeptide derivative and PNAC (potassium N-[8-(2-hydroxybenzoyl)amino]octanoate) are sieved, mixed evenly with the auxiliary materials, and directly tableted.

Animal experiment protocol:

Male Beagle dogs (9-12 kg) aged 10-15 months were orally administered one drug tablet (M2) once daily for 5 consecutive days (n=5). The first oral administration of the drug was recorded as Day 1, and the last oral administration of the drug was recorded as Day 5. On Day 1, whole blood was collected from the animals before administration (-10 min) and 2h, 4h, and 8h after administration, and plasma was prepared (sodium heparin was used as an anticoagulant). On Days 2-Day 4, whole blood was collected from the animals 2h and 4h after administration, and plasma was prepared (sodium heparin was used as an anticoagulant). On Day 5, whole blood was collected from the animals 2h, 4h, 8h, 24h, 48h, and 72h after administration, and plasma was prepared (sodium heparin was used as an anticoagulant). Plasma was stored at -80°C and used for subsequent analysis. The drug content in plasma was analyzed using LC-MS/MS (Waters ACQUITY I Class Premier UPLC tandem with Sciex 6500+QQQ). The ion-to-mass ratio used for M2 was 1121.3/1088.3. The experimental data were plotted using GraphPad Prism 10, and the pharmacokinetic curve for oral administration to beagle dogs is shown in Figure 4.

The results showed that in the oral experiment in beagle dogs, after 5 consecutive days of administration, 7mg M2 reached a maximum plasma exposure of 62.4nM on the fourth day, while the steady-state concentration of 7mg semaglutide (Rybelsus) in humans was approximately 7.5nM (Assessment Report EMA/95374/2020). It can be seen that the oral bioavailability of M2 is much higher than that of Rybelsus, which means the feasibility of oral administration of M2.

Claims (12)

A glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the glucagon-like peptide 2 derivative is:
HGDGSFSDEMNTILDX ₁₆ LAARDFIX ₂₄ WLIQTKITD, wherein X ₁₆ is selected from K or L, X ₂₄ is selected from N or K, and at least one of X ₁₆ and X ₂₄ is K; The amino acid K residue at position 16 and/or position 24 of the derivative is connected to a fatty acid side chain. The glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein X ₁₆ is K and X ₂₄ is N. The glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein X ₁₆ is L and X ₂₄ is K. The glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that the derivative is linked to the fatty acid side chain via the epsilon amino group of the amino acid K residue at position 16 and/or position 24. The glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that the fatty acid side chain is selected from
One or more of, wherein x is any integer from 4 to 38; Preferably, the fatty acid side chain is selected from:
HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₅ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₇ CO-,
One or more of HOOC(CH ₂ ) ₁₈ CO-, HOOC(CH ₂ ) ₁₉ CO-, HOOC(CH ₂ ) ₂₀ CO-, HOOC(CH ₂ ) ₂₁ CO-, and HOOC(CH ₂ ) ₂₂ CO-. The glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that the fatty acid side chain is connected to the amino acid K residue at position 16 and/or position 24 of the derivative via a linker. The glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: the linker is selected from

One or more of, wherein m is 0, 1, 2 or 3; n is 1 or
2; p is any integer from 1 to 5; Preferably, the connector is:
Where m is 0, 1, 2 or 3, n
is 1; more preferably, wherein m is 1 and n is 1. A method for preparing the glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7; Preferably, the preparation method comprises the steps of preparing the glucagon-like peptide 2 derivative using a chemical method and/or a biological method; Preferably, the chemical method comprises liquid phase or solid phase polypeptide synthesis. A pharmaceutical composition comprising the glucagon-like peptide 2 derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable excipient; Preferably, the pharmaceutical composition is in the form of solid, liquid or semi-solid; Preferably, the pharmaceutical composition is an oral delivery composition; preferably, the oral delivery composition is in the form of solid, liquid, or semi-solid, more preferably solid, and further preferably an oral tablet; Preferably, the oral delivery composition further comprises an oral absorption enhancer, preferably, the oral absorption enhancer is selected from one or more of the following: NAC salt, decanoate, Cu, Zn, Fe ions, reducing agents, tetrasodium ethylenediaminetetraacetic acid, sodium phosphate, tris(hydroxymethyl)aminomethane, lysine; the reducing agent is preferably ascorbic acid; preferably, the oral absorption enhancer is NAC salt (N-[8-(2-hydroxybenzoyl)amino]caprylate), and the NAC salt is preferably one or more of sodium salt (SNAC) and potassium salt (PNAC). Use of the glucagon-like peptide 2 derivative according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9, in the preparation of a medicament for promoting small intestinal growth, preventing and/or treating obesity, or preventing and/or treating gastric and intestinal related disorders; Preferably, the stomach and intestine related disorders are ulcers, digestive disorders, malnutrition, malabsorption syndrome, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, abdominal splenomegaly, tropical splenomegaly, hypogammaglobulinemia splenomegaly, small intestinal damage, chemotherapy-induced diarrhea/mucositis, irritable bowel syndrome, and graft-versus-host disease; preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis; Preferably, the drug is an oral drug; Preferably, the oral medication is in the form of solid, liquid, or semisolid, more preferably solid, and even more preferably oral tablets. The glucagon-like peptide 2 derivative according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9 is used for treatment. A method for promoting small intestinal growth, preventing and/or treating obesity, and preventing and/or treating gastric and intestinal related disorders, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a glucagon-like peptide 2 derivative according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9; Preferably, the stomach and intestine related disorders are ulcers, digestive disorders, malnutrition, malabsorption syndrome, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, abdominal splenosis, tropical splenosis, hypogammaglobulinemia splenosis, small intestinal damage, chemotherapy-induced diarrhea/mucositis, irritable bowel syndrome, graft-versus-host disease; preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.