Multifunctional peptide and its application in anti-inflammatory, antibacterial, mucosal repair and intestinal secretion promotion

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a multifunctional peptide and application thereof in anti-inflammation, antibiosis, mucosa repair and intestinal secretion promotion. Specifically, the present application designs a polypeptide and analogs with both guanylate cyclase C receptor (GUCY2C) agonist activity and anti-inflammatory, antibacterial and mucosa repair functions. The polypeptide can not only bind with GUCY2C, stimulate the production of intracellular cyclic guanosine monophosphate (cGMP), promote intestinal fluid secretion, but also inhibit the release of pro-inflammatory factors, reduce inflammatory response, promote the repair of intestinal mucosa damaged, and inhibit the growth of opportunistic pathogenic bacteria, accelerate the recovery of intestinal homeostasis after intestinal preparation. While effectively promoting intestinal cleaning, the polypeptide does not cause intestinal inflammation and mucosa damage, has no hemolytic toxicity and cytotoxicity, is high in safety, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a multifunctional peptide and its application in anti-inflammatory, antibacterial, mucosal repair and intestinal secretion promotion. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, and colonoscopy screening is the most effective means of reducing its incidence and mortality. The rising incidence of diseases such as inflammatory bowel disease has also led to a continuous increase in the demand for colonoscopy. Whether for endoscopic diagnosis and treatment or abdominal and pelvic surgery, high-quality bowel preparation is a crucial prerequisite.

[0003] Currently, commonly used bowel preparation methods in clinical practice suffer from problems such as poor patient tolerance, low compliance, and inadequate preparation. Heat-stable enterotoxins (STa), secreted by *E. coli*, can activate guanylate cyclase C receptors (GUCY2C), promoting intestinal secretion and potentially achieving bowel preparation. However, their widespread activation of GUCY2C may induce intestinal inflammation and mucosal damage. Existing GUCY2C agonists, such as STa core analogs (e.g., linaclotide), can also induce inflammatory responses and lead to mucosal damage at high doses. While peptide fragments with anti-inflammatory, antibacterial, and mucosal repair functions (e.g., KPV, KPT) can combat intestinal inflammation and promote mucosal repair, they lack the effect of promoting bowel cleansing. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided a multifunctional peptide and its applications in anti-inflammatory, antibacterial, mucosal repair, and intestinal secretion promotion. Specifically, this invention designs a polypeptide and analogue that possesses both guanylate cyclase C receptor (GUCY2C) agonist activity and anti-inflammatory, antibacterial, and mucosal repair functions. These polypeptides can bind to the GUCY2C receptor, stimulating the production of intracellular cyclic guanosine monophosphate (cGMP) and promoting intestinal fluid secretion; they can also inhibit the release of pro-inflammatory factors, reduce inflammatory responses, promote the repair of damaged intestinal mucosa, inhibit the growth of opportunistic pathogens, and accelerate the restoration of intestinal homeostasis after intestinal preparation. Based on the above research results, this invention is thus completed.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0006] A first aspect of the present invention provides a multifunctional peptide, the multifunctional peptide comprising:

[0007] A first peptide with guanylate cyclase C (GUCY2C) agonist activity; and a second peptide or compound with anti-inflammatory, antibacterial, or mucosal repair activity;

[0008] The first peptide segment and the second peptide segment or compound are covalently linked by a linker. The linker can be a cleavable linker or an incleavable linker.

[0009] In some embodiments, the cleavable linker is a digestive enzyme-sensitive amino acid or peptide chain (e.g., pepsin, chymotrypsin, trypsin, and elastase).

[0010] The linker can be any combination of amino acids readily cleaved by pepsin, such as Leu, Phe, Trp, and Tyr; or any combination of amino acids readily cleaved by trypsin, such as Lys and Arg; or any combination of amino acids readily cleaved by chymotrypsin, such as Phe, Tyr, and Trp; or any combination of amino acids readily cleaved by elastase, such as Ala, Gly, Ser, and Val. Alternatively, it can be a single amino acid readily cleaved by digestive enzymes as mentioned above.

[0011] In some embodiments, the insoluble linker is an enzyme-insensitive amino acid (such as proline).

[0012] In some embodiments, the multifunctional peptide comprises six cysteine ​​residues, which appear in order from the N-terminus to the C-terminus, with the first cysteine ​​pairing with the fourth cysteine ​​to form a disulfide bond, the second pairing with the fifth, and the third pairing with the sixth, forming a cyclic peptide structure with three disulfide bonds.

[0013] In some embodiments, the multifunctional peptide comprises four cysteine ​​residues, which appear in order from the N-terminus to the C-terminus, with the first cysteine ​​pairing with the third cysteine, the second with the fourth, to form a cyclic peptide structure with two disulfide bonds.

[0014] In some embodiments, the N-terminus and / or C-terminus of the multifunctional peptide are modified, the modification being selected from formylation, acetylation, cardamomylation, bioacylation, amidation, stearylation, palmitoylation, succinylation, farnesylation, sulfonation, cyclization, or phosphorylation.

[0015] In some embodiments, one or more disulfide bonds in the polypeptide of the present invention may be replaced by other covalent crosslinking methods, such as: a thioamide linker (-CH2CH(S)HNHCH2- or -CH2NHCH(S)CH2-), a thioether linker (-CH2CH2SCH2- or -CH2SCH2CH2-), an amine linker (-CH2CH2NHCH2- or -CH2NHCH2CH2-), an alkenyl linker (-CH2CH=CHCH2-), an alkyl linker (-CH2CH2CH2CH2-), an ether linker (-CH2CH2OCH2- or -CH2OCH2CH2-), an amide linker, an ester linker, a thioester linker, a lactam bridge, a carbamoyl linker, a urea linker, a thiourea linker, or a phosphate ester linker.

[0016] In some embodiments, the multifunctional peptide comprises non-natural amino acids or D-amino acids.

[0017] In some embodiments, the amino acid sequence of the multifunctional peptide is as follows:

[0018] G3P2501: KPVYYCCEYCCNPACTGCY (SEQ ID No: 1);

[0019] G3P2502:KPVFYCCEYCCNPACTGCY (SEQ ID No: 2);

[0020] G3P2503: KPVRKCCELCCNPACTGCY (SEQ ID No: 3);

[0021] G3P2504: KPVSGCCELCCNPACTGCY (SEQ ID No: 4);

[0022] G3P2505: KPVYFYCCELCCNPACTGCY (SEQ ID No: 5);

[0023] G3P2506: KPVYCCEYCCNPACTGCY (SEQ ID No: 6);

[0024] G3P2601: CCEYCCNPACTGCYYFKPV (SEQ ID No: 7);

[0025] G3P2602: CCELCCNPACTGCYKPV (SEQ ID No: 8);

[0026] G3P2701:KPTYLCCELCCNPACTGCY(SEQ ID No:9);

[0027] G3P2702:KPTACCELCCNPACTGCY(SEQ ID No:10);

[0028] G3P2703:KPTAGSCCELCCNPACTGCY(SEQ ID No:11);

[0029] G3P2801:CCEYCCNPACTGCYFFKPT(SEQ ID No:12);

[0030] G3P2802:CCELCCNPACTGCYRKPT(SEQ ID No:13);

[0031] G3P2901:LLLEWFCCEYCCNPACTGCY(SEQ ID No:14);

[0032] G3P3501:KPVFNTFYCCEYCCNPACAGCY(SEQ ID No:15);

[0033] G3P3601:NTFYCCELCCNPACAGCYVYKPV(SEQ ID No:16);

[0034] G3P3701:KPTKYNTFYCCEYCCNPACAGCY(SEQ ID No:17);

[0035] G3P3702:KPTLNTFYCCELCCNPACAGCY(SEQ ID No:18);

[0036] G3P3703:KPTFWYNTFYCCEYCCNPACAGCY(SEQ ID No:19);

[0037] G3P3801:NTFYCCELCCNPACAGCYFKPT(SEQ ID No:20);

[0038] G3P4501:KPVYFNDECELCVNVACTGCL(SEQ ID No:21);

[0039] G3P4601:NDECELCVNVACTGCLWYKPV(SEQ ID No:22);

[0040] G3P4701: KPTAYNDDCELCVNVACTGCL (SEQ ID No: 23);

[0041] G3P4801: NDECELCVNVACTGCLFKPT (SEQ ID No: 24);

[0042] G3P5501: KPVYANSSNYCCEYCCNPACTGCY (SEQ ID No: 25);

[0043] G3P5601: NSSNYCCEYCCNPACTGCYVYKPV (SEQ ID No: 26);

[0044] G3P5602: NSSNYCCELCCNPACTGCYRKPV (SEQ ID No: 27);

[0045] G3P5603: NSSNYCCEYCCNPACTGCYGKPT (SEQ ID No: 28);

[0046] G3P6003: KPVPCCELCCNPACTGCY (SEQ ID No: 29).

[0047] In addition, it should be noted that the motif (motif) with anti-inflammatory and mucosal repair functions in the multifunctional peptides of the present invention can exert anti-inflammatory, antibacterial and repair functions of damaged mucosa in the form of heterozygous peptides, but can also be combined with STa (and its derivatives) and endogenous GUCY2C agonists (and derivatives). The motifs for anti-inflammatory, antibacterial and mucosal repair functions in the polypeptides of the invention may, but are not limited to, KPV, KPT, LLLE, TFF (trifoliate factor) and collagen peptides.

[0048] A second aspect of the invention provides a pharmaceutical composition comprising a multifunctional peptide or a medicinal salt thereof as described in the first aspect above, and a pharmaceutically acceptable carrier or excipient.

[0049] In some embodiments, the pharmaceutical composition further comprises an electrolyte used to supplement potassium, sodium, chloride, calcium and / or magnesium ions.

[0050] In some embodiments, the pharmaceutical composition further comprises excipients selected from the group consisting of defoaming agents, gas adsorbents, digestive enzyme formulations, microecological formulations, or combinations thereof.

[0051] A third aspect of the invention provides the use of the multifunctional peptide described in the first aspect above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in the second aspect above, in the preparation of a medicament for bowel preparation.

[0052] The drug has anti-inflammatory, antibacterial, mucosal repair, and intestinal secretion-promoting effects.

[0053] In some implementations, the bowel preparation is used before endoscopic diagnosis or abdominopelvic surgery.

[0054] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects:

[0055] The multifunctional peptide provided by the above technical solution achieves the dual function of promoting intestinal cleansing and protecting and repairing the intestinal mucosa by fusing GUCY2C agonists with anti-inflammatory, antibacterial and mucosal repair functional peptides, overcoming the defects of inflammation and mucosal damage that may be caused by traditional GUCY2C agonists when used alone.

[0056] The multifunctional peptides provided by the above technical solution can be targeted and cleaved by digestive tract endopeptides in vivo, releasing two functional peptide segments that synergistically exert secretion-promoting and anti-inflammatory and repairing effects in the intestine, achieving sequential synergy in time and space.

[0057] Experiments have shown that the multifunctional peptides of this invention effectively promote intestinal cleansing without causing intestinal inflammation or mucosal damage, and are free from hemolytic toxicity and cytotoxicity, demonstrating high safety and promising clinical application prospects. Attached Figure Description

[0058] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0059] Figure 1 These are the results of high performance liquid chromatography and mass spectrometry analysis of G3P2501; where A is the high performance liquid chromatogram and B is the mass spectrum.

[0060] Figure 2 This is the analysis result of G3P2501 being digested by pepsin in gastric juice into STa core analogues and KPV.

[0061] Figure 3 This is the analysis result of G3P2501 being digested in intestinal fluid into STa core analogues and KPV.

[0062] Figure 4 The results are the analysis of the ability of G3P2501 and G3P2502 to stimulate the synthesis of cyclic guanosine monophosphate (cGMP) in T84 cells.

[0063] Figure 5 The results are from the analysis of the ability of G3P2501 and G3P2504 to antagonize the production of the inflammatory factor IL-8.

[0064] Figure 6 The antibacterial effect of G3P2501 and G3P2503 after co-incubation with Escherichia coli, a common opportunistic pathogen in the intestine.

[0065] Figure 7 This refers to the roles of G3P2501 and G3P2504 in repairing epithelial barrier damage mimicking inflammation; where A represents the relative expression level of the tight junction protein Occludin, and B represents the relative expression level of the tight junction protein ZO-1.

[0066] Figure 8 These are the hemolytic toxicity and cytotoxicity data for G3P2501 erythrocytes; where A represents the hemolysis rate and B represents the cell viability.

[0067] Figure 9 This is the dose-response relationship between the severity of diarrhea and the drug dosage in animal studies of STa core analogues.

[0068] Figure 10 The images show the pathological changes that cause intestinal inflammation and mucosal damage in animal studies of STa core analogs, with a scale bar of 100 μM.

[0069] Figure 11 This is a statistical analysis of the pathological changes in intestinal mucosal damage caused by STa core analogs in animal experiments; where A represents the statistical changes in the number of goblet cells and B represents the statistical changes in the number of lymphocytes.

[0070] Figure 12 These are pathological images of animals with diarrhea but no mucosal damage in the G3P2501 animal experiment; among them, A is a representative image of pathological HE staining and immunohistochemistry of each group, with a scale bar of 100 μM, B is the statistical change of the immunohistochemical tight junction protein ZO-1 score of each group, and C is the statistical change of the immunohistochemical tight junction protein Occludin score of each group.

[0071] Figure 13 This is a comparison of changes in serum inflammatory factor levels after the application of G3P2501 and STa core analogs; where A represents the statistical changes in diamine oxidase in each group, and B represents the statistical changes in inflammatory factor TNF-α in each group.

[0072] Figure 14 This represents the dose-response relationship between the severity of diarrhea and stool volume after G3P2501 application and the dosage of the applied peptide; where A represents the statistical change in diarrhea scores for each group, and B represents the statistical change in stool volume for each group.

[0073] Figure 15This is a diagram showing the effect of colonoscopy when the G3P2501 is used for bowel preparation, demonstrating the achievement of bowel preparation requirements.

[0074] Figure 16 This is the result of the bowel preparation score under endoscopy when using G3P2501 for bowel preparation. Detailed Implementation

[0075] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, 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 invention pertains.

[0076] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0077] The multifunctional peptides of this invention can be prepared by chemical synthesis or biosynthesis.

[0078] 1. Chemical Synthesis

[0079] Peptides can be synthesized using a variety of methods, including liquid-phase and solid-phase synthesis. Liquid-phase synthesis utilizes various coupling methods (such as cross-coupling reactions and novel reductive couplings) and protecting groups (such as hydroxyl, amino, and carboxyl protecting groups). In solid-phase synthesis, standardized protecting groups such as 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl are chosen, or other protecting groups such as triphenylmethyl, acetamidomethyl, tert-butylthiol, or 4-methoxytriphenylmethyl can be selected to prevent side reactions and ensure proper folding.

[0080] (1) The amino acid sequence of the polypeptide of the present invention is rich in cysteine ​​(Cys) residues, which are crucial for forming a stable three-dimensional polypeptide structure. In the polypeptide of the present invention, six cysteine ​​residues form a unique rigid conformation by forming three pairs of intramolecular disulfide bonds. The three pairs of disulfide bonds are connected in a 1-4 / 2-5 / 3-6 pattern, that is, the first cysteine ​​is paired with the fourth cysteine, the second with the fifth, and the third with the sixth. The multifunctional peptide of the present invention can also be similar to endogenous GUCY2C agonists (such as guanosine and uroguanosine) and their derivatives, where four cysteine ​​residues form a rigid conformation by forming two intramolecular disulfide bonds, and the two pairs of disulfide bonds are connected in a 1-3 / 2-4 pattern, that is, the first cysteine ​​is paired with the third cysteine, and the second with the fourth. This unique disulfide bond arrangement locks the topological structure of the polypeptide of the present invention, enabling it to bind precisely to GUCY2C and exert an intestinal secretion-promoting effect for intestinal preparation.

[0081] (2) The peptide is synthesized by the regioselective peptide synthesis method of the present invention. Different cysteine ​​thiol groups are differentially protected by using orthogonal protecting groups. Then, the protecting groups are removed stepwise and selectively and oxidized, thereby guiding the disulfide bonds to form in a preset order and position, thereby improving the yield and purity of the peptide.

[0082] (3) The oxidants required for the peptides of the present invention include, but are not limited to, N-chlorosuccinimide (NCS), dithiamine (DSF) and hemin chloride. When preparing the peptides, it is necessary to optimize the concentration, reaction time, temperature and solvent system of each oxidant in detail to find the best reaction window, maximize the yield of the target product and suppress the generation of by-products.

[0083] (4) Since the peptides of the present invention contain complex molecules with multiple disulfide bonds, their crude products usually contain a large number of mismatch isomers, incompletely reacted linear peptides and by-reaction products, and need to be separated and purified by reversed-phase high-performance liquid chromatography, but not limited to reversed-phase high-performance liquid chromatography.

[0084] 2. Biosynthesis

[0085] Using gene recombination technology, an artificially designed gene capable of expressing the amino acid sequence of the peptide of this invention is spliced ​​with a related protein fusion tag gene and introduced into host bacteria (such as E. coli and yeast) to construct engineered bacteria. Subsequently, large-scale culture is carried out in a fermenter with strictly controlled dissolved oxygen (30%-40%) and pH (e.g., 7.0), and an inducer is added to promote the efficient expression of the fusion protein composed of the fusion tag and the peptide of this invention by the engineered bacteria. After fermentation, the fusion protein is harvested by centrifugation and cell disruption, and then the linear precursor peptide chain of the peptide of this invention is released by cleavage with a specific protease. Finally, under suitable oxidation conditions, the sulfhydryl groups in the linear peptide are correctly paired to form three pairs of key disulfide bonds (six cysteine ​​residues linked in a 1-4 / 2-5 / 3-6 pattern) or two pairs of key disulfide bonds (four cysteine ​​residues linked in a 1-3 / 2-4 pattern). Purification is then performed using reversed-phase high-performance liquid chromatography and other steps to finally obtain a high-purity active product.

[0086] Genetic constructs and methods suitable for producing immature and mature forms of the peptides and variants described in this invention in protein expression systems (other than bacteria) and well known to those skilled in the art can also be used to produce peptides in biological systems.

[0087] Peptides can be prepared, isolated, or used either as a free base or as a pharmaceutically acceptable salt. Examples of salts include, but are not limited to, acetates, chlorides, sulfates, and phosphates of peptides.

[0088] Composition of peptides and GC-C receptor agonists

[0089] On one hand, the present invention provides compositions comprising peptides, alone or in combination, which can be mixed with any pharmaceutically acceptable carrier or medium. In some embodiments, the peptide or a pharmaceutically acceptable salt thereof may be formulated into a pharmaceutical composition. In other embodiments, it may also be formulated as a non-pharmaceutical composition. The peptide may be used in combination with substances that do not cause adverse, allergic, or other adverse reactions in patients. The carrier or medium used may comprise solvents, dispersants, coating materials, absorption enhancers, controlled-release agents, and one or more inert excipients, such as starch, polyols, granulating agents, microcrystalline cellulose, etc., and may also include diluents, lubricants, binders, disintegrants, etc. If necessary, conventional aqueous or non-aqueous processes may be used to coat tablets of the composition.

[0090] Pharmaceutically acceptable carriers, inert carriers, and other excipients used in the above-mentioned components include, but are not limited to: binders, fillers, disintegrants, lubricants, antimicrobial agents, and coating materials.

[0091] As used in this invention, the term "binder" refers to a substance that imparts cohesive force to powders or granules through intermolecular forces or physical entanglement during formulation processing, thereby ensuring that the powder or granules are formed into a solid dosage form (such as tablets or granules) with sufficient mechanical strength. Binders are extremely diverse and include, but are not limited to: cellulose and its derivatives: such as microcrystalline cellulose (which also acts as a filler), hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, and sodium carboxymethyl cellulose. Synthetic or semi-synthetic polymers: such as povidone (PVP), copovidone, polyvinyl alcohol, and polyethylene glycol (molecular weight range 4000-6000). Natural polymers and their derivatives: such as pregelatinized starch, various starch pastes (derived from corn, potatoes, wheat, etc.), gelatin, gum arabic, tragacanth gum, alginate, and chitosan. Sugars and syrups: such as sucrose, glucose syrup, maltodextrin, and pectin. Other functionalized materials: such as cross-linked polymers that partially also have disintegrant properties, and surface-modified composite binder systems.

[0092] As used in this invention, the term "filler" (also known as a diluent) refers to an inert or functional substance primarily used to increase the volume and mass of a formulation to ensure accurate dosing, improve processing performance, or regulate release behavior. Such systems include, but are not limited to: sugars and sugar alcohols: such as lactose (monohydrate, anhydrous, and spray-dried), sucrose, mannitol, sorbitol, xylitol, and maltose. Cellulose materials: such as microcrystalline cellulose of various grades, powdered cellulose, and functional variants such as silicified microcrystalline cellulose. Inorganic salts and minerals: such as calcium hydrogen phosphate, calcium sulfate, calcium carbonate, magnesium carbonate, and magnesium oxide. Starches and their processed products: such as corn starch, potato starch, and pregelatinized starch. Co-treated or compound fillers: such as directly compressible complexes formed by co-treating lactose with polymers (e.g., Ludipress®).

[0093] As used in this invention, the term "disintegrant" refers to a substance that, through mechanisms such as water absorption and swelling, gas production, or capillary action, causes a solid dosage form to rapidly break down or depolymerize upon contact with an aqueous medium, thereby accelerating the dissolution and release of the active ingredient. Detailed scientific classifications and examples include: superdisintegrants: such as crospovidone, crospovidone sodium carboxymethyl cellulose, and crospovidone sodium carboxymethyl starch.

[0094] Cellulose disintegrants: such as low-substituted hydroxypropyl cellulose and calcium carboxymethyl cellulose. Traditional starches: such as corn starch, potato starch, and pregelatinized starch. Other natural and synthetic polymers: such as alginate and certain hydrophilic gel polymers.

[0095] Effervescent disintegration systems: such as combinations of sodium bicarbonate and organic acids (e.g., citric acid, tartaric acid). Multifunctional excipients: such as microcrystalline cellulose, which combines filling, binding, and disintegration properties.

[0096] As used in this invention, the term "lubricant" system is used to reduce friction between powders or particles and between powders or particles and the metal surfaces of equipment, ensuring smooth processes such as tableting and filling, and preventing sticking and cracking. It fully encompasses the following categories: metal stearates: e.g., magnesium stearate, calcium stearate, zinc stearate. Fatty acids and their derivatives: e.g., stearic acid, palmitic acid, glyceryl monostearate, hydrogenated vegetable oils. Inorganic powders: e.g., talc, colloidal silica (gas phase / precipitation method), magnesium silicate. Polymer waxes and polymers: e.g., polyethylene glycol (specific molecular weight), polytetrafluoroethylene micropowder. Surfactants: e.g., sodium (magnesium) dodecyl sulfate, poloxamer. Amino acids: e.g., leucine.

[0097] As used herein, the term "antimicrobial agent" refers to a substance used to inhibit or prevent the growth of microorganisms (including bacteria and fungi) in a formulation to ensure the microbiological stability of the product during its shelf life. Its complete spectrum includes, but is not limited to: organic acids and their salts: for example, benzoic acid / sodium benzoate, sorbic acid / potassium sorbate, sodium dehydroacetate, calcium propionate; parabens: for example, methylparaben, ethylparaben, propylparaben, butylparaben, and mixtures thereof; quaternary ammonium compounds: for example, benzalkonium chloride, benzalkonium bromide; alcohol preservatives: for example, benzyl alcohol, chlorobutanol, phenethyl alcohol; phenolic compounds: for example, chlorocresol, phenol.

[0098] Mercury-containing preservatives: such as thimerosal (suitable for certain dosage forms). Other synthetic antibacterial agents: such as chlorhexidine gluconate (Hibitane), biguanide derivatives.

[0099] As used in this invention, the term "coating system" is a complex, multi-component system used to form a functional film on the surface of a solid unit formulation to achieve purposes such as masking taste, moisture protection, light protection, improved appearance, controlled drug release, or site-specific release (e.g., enteric coating). Its complete composition includes, but is not limited to: film-forming polymers: cellulose derivatives: such as hydroxypropyl methylcellulose, ethyl cellulose (aqueous dispersion or organic solvent type), cellulose acetate, hydroxypropyl methylcellulose phthalate. Acrylic resins: such as methacrylic acid copolymers with different pH dependencies (e.g., Eudragit® series). Vinyl polymers: such as polyvinyl alcohol, povidone. Natural film-forming materials: such as shellac, zein, chitosan. Plasticizers: used to increase the flexibility of the polymer film, such as triethyl citrate, diethyl phthalate, polyethylene glycol, glycerin, castor oil. Opacifiers and colorants: such as titanium dioxide, inorganic pigments such as iron oxide red / yellow / black, and various approved food colorings. Anti-blocking agents and gloss agents: such as talc, magnesium stearate, and carnauba wax, are used to prevent sticking during the coating process and improve the appearance. Solvents or dispersion media: such as water, ethanol, isopropanol, acetone, or mixtures thereof. Modern coating technologies also widely use aqueous dispersions.

[0100] As used in this invention, the term "stabilizer" refers to a functional excipient added to a pharmaceutical formulation to improve and maintain the chemical, physical, and biological stability of a drug (especially biomolecules such as peptides and proteins) throughout its shelf life. Its mechanisms of action include, but are not limited to: inhibiting chemical degradation (such as oxidation, hydrolysis, and deamidation), preventing physical aggregation or precipitation, maintaining conformational stability, and regulating osmotic pressure and pH. Based on its mechanisms of action and chemical properties, the stabilizer system comprehensively covers the following categories:

[0101] 1. Antioxidants: Used to prevent the degradation of active ingredients due to oxidation reactions, they are divided into two categories: water-soluble and oil-soluble. Water-soluble antioxidants include: ascorbic acid (vitamin C) and its salt (sodium ascorbate), sodium bisulfite, sodium metabisulfite, sodium thiosulfate, cysteine, methionine, glutathione, thioglycerol, and propyl gallate (water-dispersible). Oil-soluble antioxidants include: tert-butyl-p-hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, vitamin E (α-tocopherol) and its derivatives (tocopheryl acetate), and ascorbyl palmitate.

[0102] 2. Chelating agents: These bind trace amounts of metal ions (such as Fe²⁺) that may catalyze oxidation reactions in the complexing agent. + Cu² + Organic acids and their salts indirectly play a stabilizing role, such as disodium or calcium sodium salts of ethylenediaminetetraacetic acid (EDTA), citric acid and its salts, tartaric acid and its salts, succinic acid, malic acid, and phytic acid. Nitrogen-containing ligands, such as nitrotriacetic acid (NTA) and diethylenetriaminepentaacetic acid (DTPA).

[0103] 3. Buffers and pH Adjusters: Used to establish and maintain an optimal pH environment for drug stability, preventing hydrolysis or conformational changes due to pH shifts. Inorganic salt buffer pairs: e.g., phosphate buffer systems (sodium dihydrogen phosphate / disodium hydrogen phosphate, potassium dihydrogen phosphate / disodium hydrogen phosphate), carbonate buffer systems (sodium bicarbonate / sodium carbonate). Organic acid buffer pairs: e.g., acetate buffer (acetic acid / sodium acetate), citrate buffer (citric acid / sodium citrate), succinate buffer, histidine buffer, tris(hydroxymethyl)aminomethane (Tris) buffer. Acid-base adjusters: e.g., hydrochloric acid, sodium hydroxide, sodium bicarbonate, acetic acid, citric acid.

[0104] 4. Surfactants: These enhance physical stability by reducing interfacial tension, inhibiting protein adsorption and aggregation at the interface, and through micellar encapsulation. Nonionic surfactants include polysorbates (Tween 20, Tween 40, Tween 80), poloxamers (such as poloxamer 188, poloxamer 407), Span derivatives (sorbitan esters), polyoxyethylene castor oil derivatives (such as Cremophor® EL, RH 40), Brij® series, and Triton X-100. Ionic surfactants include sodium dodecyl sulfate (SDS) and cholates (such as sodium cholate, sodium deoxycholate).

[0105] 5. Carbohydrates and Polyols: As osmotic pressure regulators and protein stabilizers, they mainly stabilize the native conformation of proteins through a "preferential repulsion" mechanism and glass fixation. Carbohydrates include sucrose, trehalose, mannitol, sorbitol, lactose, glucose, and maltose. Polyols include glycerol (glycerol), propylene glycol, and polyethylene glycol (low molecular weight).

[0106] 6. Amino acids and their derivatives: These can act as stabilizers, buffer components, or antioxidants, stabilizing protein structure through various intermolecular forces: leucine; neutral amino acids: such as glycine, proline, arginine, and histidine (which also have buffering capacity); sulfur-containing amino acids: such as methionine (which also has antioxidant properties) and cysteine.

[0107] 7. Polymers and high molecular weight stabilizers: These prevent particle aggregation or protein denaturation through steric hindrance or the formation of a protective layer. Examples of natural polymers include human serum albumin (HSA), hydroxyethyl starch, hyaluronic acid, and chondroitin sulfate. Examples of synthetic polymers include polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG, high molecular weight).

[0108] 8. Other specialized stabilizers: Protease inhibitors: used to inhibit potential protease activity (especially in biological extracts), such as PMSF, aprotinin, and EDTA (which inhibits metalloproteinases by chelating metal ions). Anti-adsorption agents: for example, small amounts of surfactants (such as polysorbate 80) added to high-concentration protein formulations can reduce adsorption loss of the drug on container and tubing surfaces.

[0109] In various embodiments, the peptide or a pharmaceutically acceptable salt thereof can be used for colon cleansing. In some embodiments, the peptide or a pharmaceutically acceptable salt thereof can be used as a method for cleansing the colon prior to colonoscopy or surgery. In other embodiments, the peptide can be used to prepare a subject for colonoscopy. In some embodiments, the peptide or a pharmaceutically acceptable salt can be used to prepare a subject for surgery, such as intestinal surgery.

[0110] In one embodiment, a method is provided for cleaning the colon of a subject preparing for a colonoscopy procedure, the method comprising administering an effective dose of a colonic cleansing composition to the subject, wherein the colonic cleansing composition comprises a pharmaceutically acceptable excipient, diluent, or carrier, and a peptide or a pharmaceutically acceptable salt thereof, the peptide comprising six cysteine ​​residues appearing in order from the N-terminus to the C-terminus, the first cysteine ​​pairing with the fourth cysteine ​​to form a disulfide bond, the second pairing with the fifth, and the third pairing with the sixth, thereby forming a cyclic peptide structure having three disulfide bonds.

[0111] The peptide or a pharmaceutically acceptable salt thereof comprises the following amino acid sequence:

[0112] G3P2501: KPVYYCCEYCCNPACTGCY (SEQ ID No: 1);

[0113] G3P2502:KPVFYCCEYCCNPACTGCY (SEQ ID No: 2);

[0114] G3P2503: KPVRKCCELCCNPACTGCY (SEQ ID No: 3);

[0115] G3P2504: KPVSGCCELCCNPACTGCY (SEQ ID No: 4);

[0116] G3P2505: KPVYFYCCELCCNPACTGCY (SEQ ID No: 5);

[0117] G3P2506: KPVYCCEYCCNPACTGCY (SEQ ID No: 6);

[0118] G3P2601: CCEYCCNPACTGCYYFKPV (SEQ ID No: 7);

[0119] G3P2602: CCELCCNPACTGCYKPV (SEQ ID No: 8);

[0120] G3P2701: KPTYLCCELCCNPACTGCY (SEQ ID No: 9);

[0121] G3P2702: KPTACCELCCNPACTGCY (SEQ ID No: 10);

[0122] G3P2703: KPTAGSCCELCCNPACTGCY (SEQ ID No: 11);

[0123] G3P2801:CCEYCCNPACTGCYFFKPT(SEQ ID No:12);

[0124] G3P2802:CCELCCNPACTGCYRKPT(SEQ ID No:13);

[0125] G3P2901:LLLEWFCCEYCCNPACTGCY(SEQ ID No:14);

[0126] G3P3501:KPVFNTFYCCEYCCNPACAGCY(SEQ ID No:15);

[0127] G3P3601:NTFYCCELCCNPACAGCYVYKPV(SEQ ID No:16);

[0128] G3P3701:KPTKYNTFYCCEYCCNPACAGCY(SEQ ID No:17);

[0129] G3P3702:KPTLNTFYCCELCCNPACAGCY(SEQ ID No:18);

[0130] G3P3703:KPTFWYNTFYCCEYCCNPACAGCY(SEQ ID No:19);

[0131] G3P3801:NTFYCCELCCNPACAGCYFKPT(SEQ ID No:20);

[0132] G3P4501:KPVYFNDECELCVNVACTGCL(SEQ ID No:21);

[0133] G3P4601:NDECELCVNVACTGCLWYKPV(SEQ ID No:22);

[0134] G3P4701:KPTAYNDDCELCVNVACTGCL(SEQ ID No:23);

[0135] G3P4801:NDECELCVNVACTGCLFKPT(SEQ ID No:24);

[0136] G3P5501:KPVYANSSNYCCEYCCNPACTGCY(SEQ ID No:25);

[0137] G3P5601: NSSNYCCEYCCNPACTGCYVYKPV (SEQ ID No: 26);

[0138] G3P5602: NSSNYCCELCCNPACTGCYRKPV (SEQ ID No: 27);

[0139] G3P5603: NSSNYCCEYCCNPACTGCYGKPT (SEQ ID No: 28);

[0140] G3P6003: KPVPCCELCCNPACTGCY (SEQ ID No: 29).

[0141] In some embodiments, the peptides and pharmaceutically acceptable salts of the present invention can be used for bowel preparation treatment prior to colonoscopy.

[0142] Specifically, the peptide or its salt can be used in colon cleansing methods, for example, by administering an effective first dose between 300 μg and 1000 mg, followed by an effective second dose between 300 μg and 1000 mg the following morning, to achieve basic colon cleansing. In other embodiments, the peptide or its salt can be administered as a single dose between 300 μg and 1000 mg.

[0143] The peptides and their pharmaceutically acceptable salts can be used alone or in combination to treat, prevent, or alleviate visceral pain associated with gastrointestinal symptoms. Furthermore, the peptides and salts can be used in combination with other drugs, such as anti-inflammatory peptides, analgesic peptides, analgesics, intestinal guanylate cyclase stimulants, or other compounds. The anti-inflammatory peptides, analgesic peptides, or compounds can be covalently linked to the peptides of the present invention, or used as independent drugs simultaneously or sequentially with the peptides of the present invention in combination therapy. In the treatment of gastrointestinal diseases, they can also be used in combination with antidepressants, prokinetic agents, antiemetics, antibiotics, proton pump inhibitors, aldosterone inhibitors, acid antagonists, PDE5 inhibitors, ODC inhibitors, GABA-B agonists, bile acid sequestrants, COX-2 inhibitors, NSAIDs, corticosteroids, opioids, adrenergic receptor agonists, anticholinergics, tricyclic antidepressants, and mucosal protectants. Another type of therapeutic substance includes, but is not limited to, peptides with anti-inflammatory and mucosal repair functions such as KPV, KPT, LLLE, TFF, collagen peptides, and other peptides with anti-inflammatory, antibacterial, and / or mucosal repair functions; mucosal protectants such as teprenone, rebamipide, isoladine, and zinc peptidase; intestinal hormones and signaling molecules such as glucagon-like peptide-2 (GLP-2) and ghrelin; growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, and keratinocyte growth factor; amino acids and their derivatives such as glutamine, L-arginine, histidine, and cysteine; and other substances such as tannic acid protein, lactoferrin, short-chain fatty acids, curcumin, resveratrol, quercetin, mannose and its derivatives, hyaluronic acid, and heat shock proteins.

[0144] For enhancing bowel preparation and stabilizing the intestinal microecology, the peptides and salts can also be used in combination with other drugs, including antifoaming agents such as simethicone and dimethicone, which can remove air bubbles in the intestines during bowel preparation and improve the quality of bowel preparation. Other drugs that can alleviate bloating and discomfort during bowel preparation include: antifoaming agents and gas absorbents such as simethicone, dimethicone, and activated charcoal; digestive enzyme preparations such as enteric-coated pancreatic enzyme preparations, Aspergillus oryzae pancreatic enzyme tablets, and compound digestive enzyme preparations; and microecological preparations such as triple / quadrivalent live Bifidobacterium, dual live Clostridium butyricum, and live Bacillus licheniformis.

[0145] Other treatment combinations include celecoxib and other nonsteroidal anti-inflammatory drugs (such as sulindac and etoricoxib), phosphodiesterase inhibitors and ornithine decarboxylase inhibitors (such as DFMO) for sporadic colonic polyps and Lynch syndrome, mesalazine or 5-aminosalicylic acid and their analogues, budesonide for inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), and opioids, tramadol and its isoform analogues, eshadrol, etc. for chronic pain (including cancer pain).

[0146] The peptides and salts may also form pharmaceutical compositions with other drugs or compounds suitable for bowel preparation purposes. To achieve safe and effective bowel cleansing, the pharmaceutical compositions further contain electrolytes that may be lost during the bowel preparation process, primarily potassium ions (K). + Sodium ions (Na) + ), chloride ions (Cl) - ), calcium ions (Ca²) + ) and magnesium ions (Mg²) + These electrolytes can be provided in their respective pharmaceutically acceptable salt forms, including but not limited to sodium chloride, potassium chloride, sodium citrate, and potassium citrate. For example, each sachet of this powder can be used to prepare a 500 ml oral solution, with the following specific ingredients: 1.90 g sodium chloride, 1.02 g potassium citrate, 0.24 g sodium citrate, and 6.75 g glucose. During preparation, the above ingredients are accurately weighed, uniformly mixed, and then packaged into aluminum-plastic composite film bags. This formula provides the sodium, potassium, and chloride ions required during bowel preparation, and uses citrate to adjust osmotic pressure and pH, supplemented by glucose to promote water absorption, achieving an overall isotonic effect to reduce gastrointestinal irritation. Before use, dissolve one sachet of powder completely in approximately 20-500 ml of warm water. It is suitable for use in combination with GUCY2C agonists or alone for electrolyte supplementation during bowel preparation. Quality testing is conducted in accordance with relevant standards in the Chinese Pharmacopoeia, including properties, identification, content determination, and microbial limits. The composition of the present invention can effectively maintain the body's electrolyte balance while exerting the secretion-promoting effect mediated by GUCY2C and promoting intestinal emptying, thereby improving the safety and tolerability of the intestinal preparation process.

[0147] In some embodiments, analgesics suitable for use in combination with the peptides of the present invention include calcium channel blockers (such as phenytoin sodium), 5HT receptor antagonists (such as antagonists acting on 5HT3, 5HT4 and 5HT1), 5HT4 agonists (such as tegaserod, mosapride, metoclopramide, cisapride, lenzapride, and benzimidazolidinone derivatives such as BIMU1, BIMU8 and risapride), and 5HT1 agonists (such as sumatriptan and buspirone).

[0148] In combination therapy, multiple active ingredients can be administered in one or more of the following ways: by separately formulating the peptide of the present invention or a pharmaceutically acceptable salt thereof with another therapeutic peptide or compound and administering them sequentially or simultaneously; or by co-formulating them into a single formulation for administration. Furthermore, the combination therapy may also include: formulating two active ingredients into a composition and using it in combination with a separate formulation containing a third active ingredient.

[0149] The timing of administration of the multiple active ingredients can be the same or different. For example, the first active ingredient (or combination thereof) may be administered minutes, hours, days, or weeks before the second active ingredient (or combination thereof). Specifically, the active ingredients may be administered separately at intervals of minutes, 1 hour to 24 hours, 1 day to 14 days, or 2 weeks to 10 weeks. Longer dosing intervals may also be used depending on the circumstances. Although it is desirable in most cases for the multiple active ingredients used in combination therapy to be present in the patient simultaneously, this condition is not mandatory.

[0150] dose

[0151] General principles and influencing factors

[0152] In pharmaceutical compositions, the dosage level of the active ingredient should be varied to achieve an effective concentration of the compound within the body (especially at sites of inflammation or near disease areas) to produce the desired therapeutic effect. Typically, the initial dose should be lower than that required to achieve the desired therapeutic effect, and then gradually increased until the expected effect is reached. Specific individualized dosages depend on a variety of factors, including: the patient's weight, overall health condition, diet, medical history, route and timing of administration, whether it is used in combination with one or more other medications, and the severity of the disease.

[0153] Dosage range and administration regimen

[0154] The effective dose range of the composition is typically from about 1 microgram to about 100 milligrams per kilogram of body weight, preferably from about 10 micrograms to 100 milligrams per kilogram of body weight. Guanylate cyclase receptor agonists can be administered orally, systemically, or locally, in dosage forms including inhalers, injections, tablets, capsules, granules, powders, suspensions, topical ointments and lotions, transdermal preparations, and other known peptide preparations and PEGylated peptide analogs. The agonist can be administered as a single active ingredient or in combination with other drugs such as cGMP-dependent phosphodiesterase inhibitors and anti-inflammatory drugs. In practical applications, the daily dose of the intestinal guanylate cyclase agonist of the present invention is typically in the range of about 0.001 mg to about 10,000 mg (i.e., about 0.001 / 75 mg / kg to about 10,000 / 75 mg / kg), preferably in the range of about 0.005 mg to about 1,000 mg (i.e., about 0.005 / 75 mg / kg to about 1,000 / 75 mg / kg). The total daily dose may be administered as a single dose or in divided doses. In some embodiments, the composition containing the peptide described in the present invention is provided in divided doses. The divided doses are administered the night before colonoscopy and on the day of colonoscopy. In other embodiments, the dose is provided as a single dose the night before or during colonoscopy, or on the day of colonoscopy.

[0155] Development of personalized dosing regimens

[0156] Dosing regimens used for the prevention, treatment, alleviation, or improvement of medical conditions, or in combination with the compositions of the present invention, should be selected based on a variety of factors. These factors include, but are not limited to: the subject's type, age, weight, sex, diet and medical condition, severity of disease, route of administration, pharmacological considerations (such as the activity, efficacy, pharmacokinetic, and toxicological characteristics of the specific inhibitor used), and whether a drug delivery system is used or whether it is administered in combination with other active ingredients. Therefore, the dosing regimens used in practice may vary considerably and may exceed the preferred scope described in this invention.

[0157] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Experimental conditions not specifically specified in the examples are generally performed under conventional conditions or as recommended by the sales company; unless otherwise specified in the present invention, these conditions are commercially available.

[0158] Example 1: Synthesis and purification of G3P

[0159] The peptides were synthesized using a solid-phase peptide synthesis method by a commercial peptide synthesis company. Fmoc-Tyr(tBu)-CTC resin was used as the solid support. Each extension cycle began with the removal of the Fmoc protecting groups using a DMF solution containing 20% ​​piperidine, followed by alternating additions of DMF and isopropanol to induce swelling and shrinkage washing of the solid support. The peptide chains were assembled stepwise from the C-terminus to the N-terminus. Proper disulfide bond orientation was achieved by differentially protecting cysteine ​​residues with Acm, Trt, or Mob groups. When D-amino acid substitutions were introduced, the corresponding protected D-type derivatives were directly inserted into the designated sites using the same activation strategy.

[0160] After the complete peptide synthesis was completed, the resin was treated with a lysis buffer (TFA:water:triisopropylsilane = 8.5:0.75:0.75, mL / g resin) at room temperature for 2 hours to complete the cleavage of the peptide chain from the solid support and the removal of some protecting groups. After filtering out the resin, the crude peptide was precipitated and collected in cold diethyl ether.

[0161] The construction of disulfide bonds followed a stepwise approach: First, the crude peptide was dissolved in an aqueous solution containing NH4OH and the pH was adjusted to 9.0. After complete dissolution, it was titrated with H2O2 for oxidation. Taking a molecule containing three pairs of disulfide bonds as an example, the first pair of disulfide bonds was formed between the 2nd and 5th Cys positions. The resulting monocyclic intermediate was purified by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0162] The purified product was then treated in iodine solution to remove Acm and initiate the formation of a second disulfide bond between the 3rd and 6th Cys sites. For enterotoxin molecules, the bicyclic peptide was reacted in a TFA solution containing 10% DMSO and 5% anisole thioether for 2 hours to release the Mob-protected third site.

[0163] All target products were purified by a two-step RP-HPLC process: the first step used a TEAP buffer system (water / acetonitrile) for separation, and the second step used a TFA system (water / acetonitrile) for further purification. The high-purity fractions were combined and freeze-dried. The final product was converted to acetate form, which can be achieved by ion exchange or RP-HPLC.

[0164] As an alternative route, enterotoxin analogs can also be constructed by using Fmoc-Cys (Trt) or Boc-Cys (MeB) to build linear precursors, followed by random oxidation, and the introduction of redox pairs to promote disulfide bond exchange.

[0165] Side chain modification was performed using solid-phase orthogonal synthesis.

[0166] The peptide fraction of G3P2501 (SEQ ID No:1) was analyzed using high performance liquid chromatography and mass spectrometry. Figure 1 Tables A through B demonstrate the high-performance liquid chromatography (HPLC) and mass spectrometry characterization of the synthesized and purified G3P peptide. Quantitative analysis showed that the purity of G3P2501 was >95%.

[0167] Example 2: In vitro protein hydrolysis stability test using artificial gastric juice (SGF)

[0168] To evaluate the stability of peptide G3P2501 in a simulated gastric juice environment, this study designed an in vitro artificial gastric juice (SGF) digestion experiment. The specific procedures were as follows: Commercially available simulated gastric juice (Yuanye, catalog number R41110) was purchased. The reagents followed the Federal Reserve's formulation and mainly consisted of potassium chloride, calcium chloride, ammonium chloride, sodium dihydrogen phosphate, glucose, glucuronic acid, glucosamine hydrochloride, sodium chloride, urea, BSA, pepsin, and mucin, with a pH of 1.3. All reagents were prepared on the day of the experiment, and the pH was precisely adjusted to 1.3 ± 0.1 using hydrochloric acid or NaOH, followed by sterilization through a 0.22-micron filter. In the experiment, G3P2501 was added to the SGF at an initial concentration of 5 mg / mL and incubated at 37°C for 0, 15, 30, 60, and 120 minutes, with three parallel samples at each time point. After incubation, digestion was terminated by neutralization with sodium carbonate, and the samples were collected by centrifugation and stored at -80°C for subsequent analysis.

[0169] Chromatography-Mass Spectrometry (HPLC) Analysis: High-performance liquid chromatography (HPLC) conditions were used to separate samples using a Shimadzu Nexera X2 LC-30AD ultra-high-performance liquid chromatography (UHPLC) system. Mobile phase: Solution A was water, and solution B was acetonitrile containing 0.1% formic acid. Samples were placed in an autosampler at 4℃, column temperature 40℃, flow rate 300 μL / min, and injection volume 1 μL. The relevant HPLC gradients were as follows: 0-0.5 min, solution B maintained at 5%; 0.5-3 min, solution B linearly changed from 5% to 90%; 3-6.5 min, solution B maintained at 90%; 6.5-7 min, solution B linearly changed from 90% to 5%; 7-10 min, solution B maintained at 5%. Mass spectrometry analysis was performed using an AB SCIEX 5500 QTRAP mass spectrometer in positive ion mode. The 5500 QTRAP ESI source conditions were as follows: source temperature 550℃; ion source gas 1 (GS1): 55; ion source gas 2 (GS2): 55; curtain gas (CUR): 35; ion spray voltage (IS) 5500 V. Analyst 1.6.3 software was used to extract chromatographic peak areas and retention times. Based on the retention times and peak shapes of the standards, all samples were analyzed to obtain the analytical results for the analytes in all samples. Figure 2 This study demonstrates the cleavage of the linker of the G3P2501 heteropeptide in gastric juice by gastric digestive enzymes, and the changes in the detection of G3P2501, intestinal secretagogue peptide (STa core analog CCEYCCNPACTGCY), and peptides with anti-inflammatory, antibacterial, and mucosal repair functions (KPV) over time. The results indicate that G3P2501 can be targeted and cleaved by pepsin in the stomach, and G3P2501, the STa core analog CCEYCCNPACTGCY, and KPV were detected in gastric juice. Summary data for other variants are detailed in Table 1.

[0170] Table 1. Stability data of G3P variant in simulated gastric fluid.

[0171]

[0172] In this context, "-" indicates that it is not detectable, and "+" indicates that it is detectable. The sequence includes STh, STp, uroguanosine analogue, STa core, and STa core analogue.

[0173] Example 3: In vitro protein hydrolysis stability test using artificial intestinal fluid (SIF)

[0174] This experiment aimed to evaluate the proteolytic stability of G3P2501 in a simulated intestinal environment. Commercially available simulated intestinal fluid (Yuanye, catalog number R22156) was purchased and modified with chymotrypsin (0.2 mg / mL, activity ≥1200 U / mg) and elastase (0.05 mg / mL, activity ≥30 U / mg). The modified simulated intestinal fluid mainly contained NaCl, KH2PO4, CaCl2, KCl, trypsin, chymotrypsin, and elastase. Before use, the pH of the solution was adjusted to 6.8 ± 0.1, and the solution was filtered for sterilization. During the experiment, G3P2501 was added to the SIF at an initial concentration of 5 mg / mL and incubated at 37°C for 0, 15, 30, 60, 120, 240, and 480 minutes. Three replicates were set at each time point. After incubation, digestion was terminated with trifluoroacetic acid, and the samples were collected by centrifugation and stored at -80°C for subsequent analysis. The conditions for liquid chromatography and mass spectrometry are the same as before. Figure 3 This study demonstrates the cleavage of the linker of the G3P2501 heterozygous peptide by intestinal digestive enzymes, and the changes in the detection of G3P2501, intestinal secretagogue peptide (STa core analog CCEYCCNPACTGCY), and peptides with anti-inflammatory, antibacterial, and mucosal repair functions (KPV) over time. The results indicate that the G3P2501 peptide can be targeted and cleaved by intestinal digestive enzymes in the intestine, and G3P2501, the STa core analog CCEYCCNPACTGCY, and KPV were detected in the intestinal fluid. Data for other variants are detailed in Table 2.

[0175] Table 2. Stability data of G3P variant in artificial intestinal fluid.

[0176]

[0177] In this context, "-" indicates that it is not detectable, and "+" indicates that it is detectable. The sequence includes STh, STp, uroguanosine analogue, STa core, and STa core analogue.

[0178] Example 4: Experiment on the stimulation effect of cyclic guanosine monophosphate (cGMP)

[0179] This experiment aimed to evaluate the ability of G3P peptides to bind to and activate the intestinal guanylate cyclase-C (GC-C) receptor using the human T84 colon cancer cell line. Human epithelial colorectal adenocarcinoma T84 cells (ATCC) were routinely cultured in DMEM / Ham's F-12 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine, and incubated at 37°C with 5% CO2. cGMP accumulation was determined strictly according to the manufacturer's instructions (cGMP HTRF assay kit, Cisbio International). The procedure was briefly as follows: 20,000 cells per well (cells suspended in DMEM / F12 medium containing 0.5 mM IBMX) of progressively increasing concentrations of the G3P peptide series were added to white 384-well Corning plates. The plates were incubated at 37°C with 5% CO2 for 30 minutes. Subsequently, cells were lysed by adding HTRF detection reagents (i.e., anti-cGMP-europium cavitation antibody and d2-labeled cGMP analogue diluted in lysis buffer) (using the CGMP HTRF kit, Cisbio International), and incubated at 25°C for 1 hour. Finally, the emission signals at 620 nm and 665 nm were measured using a BioTex multiplate reader under 330 nm excitation light.

[0180] like Figure 4 As shown, G3P2501 and G3P2502 stimulate the synthesis of cyclic guanosine monophosphate (cGMP) in T84 cells. STa core analogue (CCEYCCNPACTGCY) EC 50 The measured value was 2.097. 10 -8 M, G3P2501 and G3P2502 are 3.995 respectively. 10 - 8 M and 1.355 10 - 7 M. The results showed that G3P2501 was non-inferior to STa core analogs in stimulating the synthesis of cyclic guanosine monophosphate (cGMP) in T84 cells. Data for other variants are detailed in Table 3.

[0181] Table 3. Activity data of G3P variants

[0182]

[0183] Example 5: Evaluation of anti-inflammatory effects of a cell model simulating inflammatory stimulation

[0184] This experiment aimed to evaluate the anti-inflammatory capacity of G3P peptides using the human Caco-2 colonic epithelial cell line. An inflammation model of Caco-2 cells was induced using the cytokine IL-1β. IL-1β at a stimulation concentration of 2 ng / mL was used to induce inflammation. After culturing Caco-2 cells in six-well plates for 48 h, 2 ng / mL IL-1β or 2 ng / mL IL-1β and different concentrations of peptides were added, and the cells were incubated for further growth. Cell supernatants were collected, centrifuged at 5000g and 4℃, and aliquoted and frozen for later use. The levels of inflammatory factors were detected using a Huaan Biotechnology ELISA kit. Figure 5 The ability of G3P2501 and G3P2504 to antagonize the inflammatory factor IL-8 after application was demonstrated, proving that G3P2501 and G3P2504 have anti-inflammatory functions. Data for other variants are detailed in Table 4.

[0185] Table 4. In vitro anti-inflammatory activity data of G3P variants

[0186]

[0187] The anti-inflammatory activity was demonstrated by the expression level of the inflammatory cytokine IL-8. "P < 0.05 compared to the control group that received only IL-1β" indicates that the difference was statistically significant.

[0188] Example 6: Evaluation of the in vitro antibacterial effect of G3P

[0189] This experiment aimed to evaluate the antibacterial activity of G3P peptides using *Escherichia coli*, a common opportunistic pathogen. *Escherichia coli* (final concentration 1×10⁻⁶) was used. 4 Cells / mL were co-incubated with G3P peptide (100 μg / mL) or phosphate-buffered saline (PBS) at 37°C for 2 hours. The cells / mL were then centrifuged (13,000 g, 10 min) and resuspended in PBS. Aliquots (100 μL) of each suspension were plated on NB agar plates and incubated at 37°C for 24 hours. The final number of colonies (CFU) was counted and expressed as a percentage relative to the PBS control group. Figure 6 The antibacterial effects of G3P2501 and G3P2503 after co-incubation with bacteria were demonstrated. After 2 hours of co-incubation with *Escherichia coli*, a common opportunistic pathogen in the intestine, the bacterial load decreased to varying degrees. These results indicate that the G3P peptides possess antibacterial activity against common opportunistic pathogens. Data for other variants are detailed in Table 5.

[0190] Table 5. In vitro antibacterial activity data of G3P variant.

[0191]

[0192] Example 7: Evaluation of G3P's in vitro mucosal repair-promoting efficacy

[0193] To evaluate the mucosal repair efficacy of G3P2501, we used the expression level of tight junction proteins as the evaluation index. Caco-2 cell lines stimulated with the inflammatory factor IL-1β for 24 h served as a positive control in an inflammatory injury model, with a drug concentration of 10 μg / mL. PBS was used as a negative control to simulate drug administration. First, total RNA was extracted from Caco-2 cells and reverse transcribed into cDNA. For gene expression analysis of ZO-1 and Occludin, we used real-time quantitative PCR using the SYBR Green assay. Specific primer sequences were designed, and GAPDH was used as an internal reference gene for standardization. The PCR reaction system was prepared strictly according to the kit instructions and amplification was performed on a real-time quantitative PCR instrument. The reaction procedure included pre-denaturation, followed by 40 cycles of denaturation, annealing, and extension. Each sample was tested in triplicate to ensure reproducibility. The final result was obtained by passing the assay through two wells. （-ΔΔCT） The method calculates the relative expression level of the target gene. Figure 7 Tables A through B show the changes in the transcriptional levels of the cell barrier tight junction proteins ZO-1 and Occludin after applying G3P2501 and G3P2504 in an inflammatory cytokine injury model, demonstrating that G3P2501 and G3P2504 can repair the epithelial barrier damaged in an inflammatory simulation. Data for other variants are detailed in Table 6.

[0194] Table 6. Data on the in vitro mucosal repair effects of G3P variants.

[0195]

[0196] Among them, the mucosal repair effect is expressed as the relative mRNA expression level of Occludin. "This indicates that compared with the positive control, P<0.05;" "This indicates that compared with the positive control, P<0.01."

[0197] Example 8: In vitro toxicity evaluation of G3P

[0198] To assess the blood compatibility and in vitro biosafety of specific peptides, we systematically determined their hemolytic activity and cytotoxicity to normal mammalian cells. For the hemolysis assay, anticoagulated whole blood from healthy humans was centrifuged and washed to obtain a 4% red blood cell suspension. Different concentrations of peptide solutions were incubated with an equal volume of red blood cell suspension at 37°C for 1 hour, with physiological saline and deionized water serving as negative and positive controls, respectively. After incubation, the supernatant was collected by centrifugation and measured at 570 nm using a microplate reader. The hemolysis rate was calculated as the ratio of the absorbance of the sample to the positive control. For the cytotoxicity assay, human intestinal epithelial cells (Caco-2 cell line) were used. Cells were seeded at an appropriate density in 96-well plates and grown adherently under standard culture conditions. The medium was then replaced with fresh medium containing a series of peptide concentrations, and cultured for another 24 hours. A control group without peptides and a blank group containing only medium were also included. After treatment, cell viability was assessed using the CCK-8 assay: CCK-8 reagent was added to each well, and after incubation for a certain period, absorbance was measured at 450 nm. Cell viability was expressed as a percentage of absorbance relative to the control group. All experiments were independently repeated at least three times, and data are presented as mean ± standard deviation. Figure 8 Tables A through B present the hemolytic toxicity and cytotoxicity data of G3P2501, demonstrating that G3P2501 has no hemolytic toxicity or cytotoxicity.

[0199] Example 9: In vivo intestinal mucosal toxicity evaluation of G3P

[0200] To assess whether G3P2501, when exerting its intestinal secretory function, causes intestinal mucosal damage, Bama pigs were orally administered G3P2501 at dose gradients of 10 mg / kg and 20 mg / kg, with a STa core analog serving as a positive control at dose gradients of 1 mg / kg, 10 mg / kg, and 20 mg / kg to determine the degree of diarrheal response. Simultaneously, to compare the difference in intestinal mucosal damage between concurrent administration of the motif and STa core analog versus G3P2501 alone, a mixture group was established. Serum was collected and plasma DAO activity was measured using the Solarbio DAO kit. Serum TNF-α levels were measured using a commercially available R&D porcine TNF-α ELISA kit. After sacrifice, duodenal, jejunal, ileal, and colonic specimens were aseptically collected and dissected. The specimens were washed with PBS and then fixed in 4% (w / v) paraformaldehyde solution for further pathological staining analysis. To detect the expression and distribution of tight junction proteins Occludin and ZO-1 in porcine colonic intestinal tissue, we used immunohistochemistry.

[0201] Figure 9This study describes the effect of STa core analogues on excessive activation of GC-C receptors and their promotion of intestinal secretion. When pigs were orally administered high doses of STa core analogues, the STa core analogues activated GUCY2C receptors, leading to watery diarrhea. The severity of diarrhea was dose-dependent.

[0202] In animal studies, the application of high doses of STa core analogues can promote excessive activation of GUCY2C, leading to intestinal inflammation and mucosal damage. Figure 10 The study demonstrates the changes in HE staining of different parts of the digestive tract after applying different concentrations of STa core analogues. Figure 11 Figures A through B show the changes in the number of colonic goblet cells and lymphocytes after the application of STa core analogues. The results show that the degree of intestinal mucosal damage is related to the dose of the GUCY2C agonist used, and the inflammatory damage worsens with increasing dose.

[0203] Figure 12 Tables A to C show the changes in colonic mucosal inflammation and edema, and decreased levels of the cell barrier tight junction proteins ZO-1 and Occludin in pathological sections when pigs received a high dose of STa core analogue (20 mg / kg) via oral gavage, resulting in watery diarrhea. When the same dose of G3P2501 (20 mg / kg) induced watery stool, no inflammation or edema was observed in the colonic mucosa, and ZO-1 and Occludin levels remained unchanged. Administration of the motif in a mixture with the STa core analogue provided partial protection while still resulting in mucosal damage; the protective effect was significantly weaker than that of G3P2501.

[0204] Serum DAO represents changes in the integrity and damage of the intestinal mechanical barrier, while serum TNF-α represents changes in inflammatory factors. Figure 13 Figures A through B show that serum DAO significantly increased after administration of 20 mg / kg of STa analogs, but no significant change in serum DAO was observed after administration of the same doses of G3P2501 or G3P2602 (20 mg / kg). Similarly, serum TNF-α was significantly increased compared to the control group after administration of STa core analogs, while no significant change in serum TNF-α levels was observed after administration of G3P2501. Animal studies demonstrate that high doses of STa core analogs can promote excessive activation of GUCY2C, leading to watery diarrhea and simultaneously promoting intestinal inflammation and mucosal damage. However, the same doses of G3P2501 or G3P2504, while also promoting excessive activation of GUCY2C and causing watery diarrhea, did not lead to intestinal inflammation and mucosal damage. G3P4501 to G3P4801 were administered at 200 mg / kg, and the remaining variants were administered at 20 mg / kg. Detailed data are shown in Table 7.

[0205] Table 7. Data on mucosal repair effects of G3P variants in vivo.

[0206]

[0207] Wherein, "+" indicates a significant upregulation compared to the STa core analog group (P<0.05 is significant), " "This indicates a significant downregulation of expression levels compared to the STa core analog group (P<0.05 is considered significant).

[0208] Example 10: Evaluation of the in vivo intestinal cleansing effect of G3P

[0209] To evaluate the cleansing effect of G3P2501's intestinal secretion stimulation function, Bama pigs were orally fed G3P2501 at 20 mg / kg, with PBS as a negative control and STa core analogues at 1 mg / kg and 20 mg / kg as positive controls. Pigs were fasted for 2 hours before feeding, and fecal volume, diarrhea severity, and endoscopic bowel preparation level were recorded. Endoscopic equipment was provided by Pentax Medical. The diarrhea scoring system for piglets was divided into five levels: 1 point indicates hard, dry feces (normal); 2 points indicates no diarrhea; 3 points indicates soft, partially formed feces (mild diarrhea); 4 points indicates unformed, loose feces (moderate diarrhea); and 5 points indicates watery feces (severe diarrhea). Endoscopic cleanliness rating: Grade 1, Poor, large amount of solid excrement, cannot be aspirated (1 point); Grade 2, Fair, moderate amount of thick liquid semi-solid fecal matter, can be aspirated (2 points); Grade 3, Good, small thin liquid excrement, easy to aspirate (3 points); Grade 4, Excellent, no or almost no feces (4 points). Figure 14 Figures A through B show the changes in the degree of diarrhea and stool volume after the application of G3P2501. The degree of diarrhea and stool volume are related to the dosage of the applied peptide. Figure 15 This is an endoscopic image. G3P is used for bowel preparation. When pigs are orally administered G3P2501 at a dose of 20 mg / kg, the bowel cleanliness is high, and colonoscopy shows that the bowel preparation requirements have been met. Figure 16 This table shows the endoscopic bowel preparation score when G3P2501 was administered at a dose of 20 mg / kg. The results showed a high endoscopic bowel cleanliness score. G3P4501-4801 was administered at 200 mg / kg, and other variants were administered at 20 mg / kg. See Table 8 for detailed data.

[0210] Table 8. Intestinal lumen cleanliness data of G3P variants

[0211]

[0212] Among them, "excellent" means that the endoscopic bowel cleanliness score is 3-4 points (inclusive), "fair" means that the endoscopic bowel cleanliness score is 2-3 points (exclusive), and "poor" means that the endoscopic bowel cleanliness score is less than or equal to 2 points.

[0213] Matters not covered in this invention are common knowledge.

[0214] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multifunctional peptide with anti-inflammatory, antibacterial, mucosal repair, and intestinal secretion-promoting functions, characterized in that, The amino acid sequence of the multifunctional peptide is selected from any one of SEQ ID NO:1-21 and SEQ ID NO:24-28.

2. A pharmaceutical composition, characterized in that, It comprises the multifunctional peptide of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition further comprises an electrolyte for replenishing potassium, sodium, chloride, calcium, and / or magnesium ions.

4. Use of the multifunctional peptide of claim 1 or the pharmaceutical composition of any one of claims 2-3 in the preparation of a medicament for bowel preparation; said bowel preparation is for use before endoscopic diagnosis or abdominopelvic surgery.

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