Butyric acid-coupled polypeptide drugs and their use in the treatment of anti-glomerular basement membrane disease

Abstract

The application discloses a butyric acid coupling-based polypeptide drug and application thereof in anti-glomerular basement membrane disease treatment, the polypeptide drug can significantly reduce kidney injury in an anti-glomerular basement membrane disease model, and the treatment effect is significantly better than that of m-P14. The application provides a brand-new treatment strategy for the technical field of anti-glomerular basement membrane disease treatment, lays a foundation for finally realizing conversion of the therapeutic polypeptide to clinical trials, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to butyrate-conjugated polypeptide drugs and their application in the treatment of glomerular basement membrane disease. Background Technology

[0002] Glomerulonephritis is one of the important causes of chronic kidney disease in my country. Immune inflammation is a core pathogenic mechanism, and developing immunological therapies targeting the underlying cause can slow disease progression and is a specific treatment urgently needed in clinical practice. Among glomerulonephritis, anti-glomerular basement membrane (GBM) disease is a classic model of an important autoimmune kidney disease and also has the worst prognosis. Anti-GBM disease is a severe autoimmune kidney disease. The key steps in its pathogenesis are the exposure of the glomerular basement membrane (GBM) autoantigen α3(IV)NC1 and the production of anti-GBM autoantibodies. After antigen-antibody binding, an immune inflammatory response is mediated, leading to severe kidney damage.

[0003] The inventors' research group spent over 10 years refining the immunopathogenesis of this disease, identifying the pathogenic T-cell antigen epitope α3-P14 and its key amino acids, and establishing an anti-GBM rat model using this epitope, laying the foundation for immunotherapy development. Utilizing the unique structure of the human basement membrane type IV collagen α1-5 chain, the inventors' research group previously used non-pathogenic α1-P14 as a template, replacing the key amino acid WIxLWxGFxF of α3-P14 to design the modified peptide m-P14. In an animal model of anti-GBM nephritis, treatment revealed a significant reduction or complete disappearance of glomerular crescents in rats. The therapeutic peptide exerts its therapeutic effect by intervening in antigen presentation and regulating T-cell differentiation.

[0004] However, classic peptide structures exhibit poor stability against in vivo proteases and are rapidly degraded after entering the body. Furthermore, most bioactive peptides have poor bioavailability and cannot be taken orally, necessitating the development of suitable delivery routes through dosage form modifications. Based on these factors, structural modification and chemical alteration of bioactive peptides are necessary. The objectives of bioactive peptide modification are diverse, primarily including improving the affinity and selectivity of the bioactive peptide with its receptor; enhancing the pharmacokinetic stability of the peptide molecule; reducing degradation or elimination of the bioactive peptide in vivo; improving the membrane permeability of the bioactive peptide; and improving the water solubility of hydrophobic peptides. Designing and synthesizing an effective, structurally optimized conjugated peptide drug is crucial for ultimately translating therapeutic peptides into clinical trials. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a butyrate-conjugated polypeptide drug and its application in the treatment of glomerular basement membrane disease, so as to solve the above-mentioned technical problems existing in the prior art.

[0006] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0007] A first aspect of the present invention provides a butyric acid-based conjugated polypeptide.

[0008] Furthermore, the coupled polypeptide is obtained by modifying the polypeptide sequence with butyric acid or 2-aminobutyric acid;

[0009] The conjugated polypeptide obtained by modifying the polypeptide sequence with butyric acid is: C4H8O2-(K)n-linker-TDIPPCPHGWSSLWKGFSFIMF;

[0010] The conjugated polypeptide obtained by modifying the polypeptide sequence with 2-aminobutyric acid is: C4H9NO2-(K)n-linker-TDIPPCPHGWSSLWKGFSFIMF.

[0011] Furthermore, the linker includes 6-aminohexanoic acid and / or β-alanine.

[0012] Furthermore, the linker is 6-aminohexanoic acid.

[0013] Furthermore, n is selected from any integer value between 3 and 8.

[0014] Furthermore, n = 5.

[0015] In some embodiments, butyric acid (BA) is a straight-chain saturated fatty acid with a molecular weight of 88.11, a chemical formula of C4H8O2, and a melting point of -7.9°C. At room temperature and pressure, butyric acid is a putrid, oily liquid with a foul odor, miscible with water, ethanol, and ether. In specific embodiments of the present invention, butyric acid is also referred to as BA.

[0016] In some embodiments, the 2-aminobutyric acid (2A) is a chemical substance with a molecular weight of 103.1198. It is a colorless monoclinic crystal. It has a melting point of 335°C and begins to sublimate at 280°C. It is readily soluble in water, sparingly soluble in alcohol, and insoluble in ether. Its corresponding chemical formula is C4H9NO2. In specific embodiments of the present invention, the 2-aminobutyric acid is also referred to as 2A.

[0017] In some embodiments, 6-aminohexanoic acid (6-Aminohexanoic acd, Ahx) is a six-carbon linear amino acid analog with one amino group and one carboxyl group. It is commonly used as a linker in polypeptide synthesis to connect different molecular fragments. In specific embodiments of the present invention, 6-aminohexanoic acid is also referred to as Ahx.

[0018] In some embodiments, conjugate peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence homology with the conjugate peptides described above also fall within the scope of protection of this invention.

[0019] Furthermore, the present invention also provides coupling peptide derivatives corresponding to the coupling peptides described above, wherein the coupling peptide derivatives include coupling peptide derivatives obtained by modifying the coupling peptides described above, or coupling peptide derivatives obtained by linking conjugates to the coupling peptides described above.

[0020] Furthermore, the modifications include hydrophobic group modifications, esterification modifications, amidation modifications, glycosylation modifications, acetylation modifications, polyethylene glycol modifications, alkylation modifications, aminoation modifications, carbonylation modifications, phosphorylation modifications, methylation modifications, hydroxylation modifications, carboxylation modifications, sulfation modifications, and / or cyclization modifications.

[0021] Furthermore, the conjugate includes fluorescent dyes, metal ions, radioactive compounds, enzymes, and / or cell-penetrating peptides.

[0022] In some embodiments, the coupled polypeptide derivative of the present invention may be a polypeptide derivative obtained by modifying the N-terminus of the coupled polypeptide described in the first aspect of the present invention with a hydrophobic group. Exemplarily, the hydrophobic group includes, but is not limited to, any one or more combinations of palmitic acid, stearic acid, oleic acid, myristic acid, linoleic acid, cholesterol, and arachidonic acid.

[0023] In some embodiments, the conjugated polypeptide derivative of the present invention may be a polypeptide derivative obtained by linking a cell-penetrating peptide to the N-terminus of the conjugated polypeptide described in the first aspect of the present invention. Cell-penetrating peptides, also known as targeting peptides, protein transduction domains, Trojan peptides, etc., are a large class of short amino acid sequences (5-30 residues) capable of crossing biological membranes and delivering various compounds into cells, including small molecules, proteins, viruses, nucleic acids, imaging agents, and drugs. The cell-penetrating peptides described in the present invention include any cell-penetrating peptides known in the art.

[0024] In some embodiments, the conjugate may be a detection reagent, which may be any substance having detectable physical or chemical properties. Exemplarily, detection reagents include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.) and radiolabels (e.g., 125 I, 35 S, 14 C 3 H or 32 P, etc.), in particular, radioactive labels (e.g., 55 Mn, 162 Dy、 157 Gd, 52 Cr or 56 Fe, etc.), metal ions (e.g., 111 In、 68 Ga、 72 As、 97 Ru、 67 Ga、 89 Zr or 201 Tl, etc.), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), electron transfer agents (e.g., metal-binding proteins, etc.), luminescent and chemiluminescent labels (e.g., fluorescein, 2,3-dihydrophthalazine, or luminol, etc.), magnetic beads, and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene or polypropylene, etc.).

[0025] Furthermore, the present invention also provides pharmaceutically acceptable salts corresponding to the aforementioned conjugated polypeptides, wherein the pharmaceutically acceptable salts corresponding to the conjugated polypeptides refer to acidic salts formed by the conjugated polypeptides with inorganic and / or organic acids and basic salts formed by the conjugated polypeptides with inorganic and / or organic bases. Preferably, pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred, but other salts are also useful. The pharmaceutically acceptable salts of the conjugated polypeptides can be formed, for example, by reacting the conjugated polypeptides with a certain amount of acid or base in a medium, such as a medium in which salts precipitate or an aqueous medium (lyophilized after the reaction).

[0026] Specific pharmaceutically acceptable salts include those that, within the bounds of reliable medical judgment, are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit or risk. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. The pharmaceutically acceptable salts of the conjugated polypeptides described in this invention include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0027] In some embodiments, suitable acids for preparing pharmaceutically acceptable salts of the coupled polypeptide include, but are not limited to: maleic acid, acylated amino acids, acetic acid, 2,2-dichloroacetic acid, lauric acid, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, benzoic acid, 4-acetaminobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, hexanoic acid, cinnamic acid, citric acid, cyclohexylsulfamic acid, and dodecyl sulfate. 1-Hydroxy-2-naphthoic acid, oxalic acid, palmitic acid, decanoic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, 2-hydroxyethylsulfonic acid, formic acid, L-glutamic acid, α-ketoglutarate, glycolic acid, fumaric acid, galactopyric acid, gentianic acid, glucoheponic acid, D-gluconic acid, D-glucuronic acid, hippuric acid, hydrobromic acid, hydrochloric acid, sebacic acid, stearic acid, succinic acid, hydroiodic acid, (+)-L-lactic acid, (-)-L-malic acid, phosphoric acid, L-pyroglutamic acid, tannic acid, L-tartaric acid, gluconic acid, salicylic acid, 4-aminosalicylic acid, sulfuric acid, thiocyanate, p-toluenesulfonic acid, undecenoic acid and / or valeric acid.

[0028] In some embodiments, suitable bases for preparing pharmaceutically acceptable salts of the coupled polypeptide include, but are not limited to: inorganic bases such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, and / or sodium hydroxide; organic bases such as primary, secondary, tert-, butyric, aliphatic, and aromatic amines, including: L-arginine, choline, dimethylaminoethanol, diethanolamine, phenethylbenzylamine, dimethylamine, propylamine, dibenzylethylenediamine, diethylamine, ethanolamine, ethylamine, ethylenediamine, diisopropylamine, diethylaminoethanol, isopropylamine, meglumine, hebamin, 1H-imidazole, L-lysine, pyridine, quinine ring, quinoline, isoquinoline, secondary amine, morphine, 4-(2-hydroxyethyl)morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)pyrrolidine, triethanolamine, triethylamine, N-methyl-D-glucosamine, trimethylamine, tris(hydroxymethyl)aminomethane, and / or tromethamine.

[0029] A second aspect of the present invention provides a pharmaceutical composition.

[0030] Furthermore, the pharmaceutical composition comprises the conjugated polypeptide described in the first aspect of the present invention.

[0031] In some embodiments, the pharmaceutical composition comprises a therapeutically and / or preventively effective amount of the conjugated polypeptide described in the first aspect of the invention.

[0032] In some embodiments, the pharmaceutical composition may also contain a second therapeutic agent.

[0033] In some embodiments, the second therapeutic agent is another drug that can be used to treat and / or prevent glomerular basement membrane disease.

[0034] In some embodiments, the other drugs that can be used to treat and / or prevent glomerular basement membrane disease include, but are not limited to: hydrocortisone acetate, prednisolone acetate, dexamethasone, betamethasone, cyclophosphamide, tacrolimus, cyclosporine, mycophenolate mofetil, methylprednisolone, dermatan sulfate, glucosamine sulfate, glycosaminoglycans, nifedipine, and amlodipine.

[0035] In this invention, the second therapeutic agent is not particularly limited; any drug that can be used to treat and / or prevent glomerular basement membrane disease, or as an adjunct treatment and / or prevention of glomerular basement membrane disease, falls within the scope of the second therapeutic agent. Since this invention has experimentally demonstrated that the coupled polypeptide has therapeutic and / or preventive effects against glomerular basement membrane disease, those skilled in the art can combine the coupled polypeptide with other drugs for the treatment and / or prevention of glomerular basement membrane disease as needed. Therefore, using the coupled polypeptide described in the first aspect of this invention as the sole active ingredient, or combining the coupled polypeptide described in the first aspect of this invention with any one or more of the aforementioned second therapeutic agents as the active ingredient for the treatment and / or prevention of glomerular basement membrane disease, both fall within the protection scope of this invention.

[0036] A third aspect of the present invention provides a pharmaceutical preparation.

[0037] Furthermore, the pharmaceutical preparation comprises the conjugated polypeptide described in the first aspect of the present invention.

[0038] Furthermore, the pharmaceutical formulation comprises a pharmaceutically acceptable carrier and / or excipient;

[0039] Optionally, the dosage form of the pharmaceutical preparation includes injections, lyophilized powder for injection, microspheres, powders, powder aerosols, capsules, tablets, pills, nasal sprays, aerosols, enteric coatings, microemulsions, or complex emulsions.

[0040] In some embodiments, the pharmaceutical formulation may be prepared according to any preparation method known in the art. For this purpose, if desired, the conjugated polypeptide may be combined with one or more solid or liquid excipients to form a suitable administration or dosage form for human use.

[0041] In some embodiments, the dosage form of the pharmaceutical preparation includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0042] In some embodiments, the gastrointestinal dosage forms include solutions, granules, tablets, capsules, suspensions, powders, sustained-release formulations, effervescent tablets, emulsions, syrups, drops, and / or chewable tablets.

[0043] In some embodiments, the non-gastrointestinal dosage form includes injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and / or skin dosage forms.

[0044] In this invention, the dosage form of the pharmaceutical preparation is not particularly limited. In some embodiments, the pharmaceutical compositions of this invention as described above can be prepared into injectable or oral formulations, including but not limited to: oral liquid preparations, granules, injections, tablets, capsules, pills, powders, sustained-release preparations, suppositories, aerosols, nano-preparations, tinctures, syrups, tinctures, and elixirs. Those skilled in the art can select suitable dosage forms known in the art according to actual needs (e.g., improving drug bioavailability, improving patient medication adherence, etc.).

[0045] In some embodiments, the conjugated polypeptides, pharmaceutical compositions comprising the conjugated polypeptides, or pharmaceutical formulations comprising the conjugated polypeptides provided by the present invention can be administered in unit dose form. Routes of administration include, but are not limited to, enteric or extraintestinal routes, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum, or rectal administration. Dosage forms include, but are not limited to, tablets, pills, powders, granules, solutions, capsules, drops, aerosols, suspensions, emulsions, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems.

[0046] In some implementations, a wide variety of excipients known in the art can be used to formulate unit dosage forms containing coupled peptides into tablets. Examples of excipients include, but are not limited to: diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, calcium carbonate, microcrystalline cellulose, etc.; humectants and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, polyvinylpyrrolidone, etc.; disintegrants, such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, sodium dodecyl sulfate, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, etc.; absorption enhancers, such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; and lubricants, such as talc, silica, corn starch, stearates, etc. Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0047] In some implementations, a wide variety of excipients known in the art can be used to formulate unit dosage forms containing coupled polypeptides into pills. Examples of excipients include, but are not limited to: diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.

[0048] In some embodiments, to encapsulate a unit dosage form containing a conjugated polypeptide, the active ingredient (conjugated polypeptide) is mixed with the various excipients described above, and the resulting mixture is placed in a hard capsule or soft capsule. Alternatively, the active ingredient (conjugated polypeptide) can be formulated as a microcapsule, suspended in an aqueous medium to form a suspension, or filled into a hard capsule or formulated as an injectable preparation.

[0049] In some embodiments, to formulate a unit dosage form containing a coupled polypeptide into an injectable preparation, such as a solution, emulsion, lyophilized powder for injection, or suspension, all diluents commonly used in the art can be used, including but not limited to: water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Additionally, to prepare an isotonic injection, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable preparation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added.

[0050] In some implementations, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparation, if necessary.

[0051] In this invention, the effective therapeutic and / or preventive dose of the drug, pharmaceutical composition, or pharmaceutical preparation can be prescribed in various ways depending on factors such as the actual formulation method, administration route, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and responsiveness. Skilled physicians can usually easily determine the prescription and the effective dosage for the desired treatment and / or prevention. Any dosage that produces the expected therapeutic and / or preventive effect on the disease or related symptoms falls within the scope of protection of this invention.

[0052] The fourth aspect of the present invention provides a method for preparing the conjugated polypeptide described in the first aspect of the present invention.

[0053] Furthermore, the method includes preparing the conjugated polypeptide described in the first aspect of the present invention by modifying the polypeptide sequence TDIPPCPHGWSSLWKGFSFIMF with butyric acid or 2-aminobutyric acid.

[0054] Furthermore, the present invention also provides a method for treating and / or preventing glomerular basement membrane disease, the method comprising the steps of administering to a subject in need a therapeutic and / or preventative effective amount of the conjugated polypeptide of the first aspect of the present invention, the pharmaceutical composition of the second aspect of the present invention, and / or the pharmaceutical preparation of the third aspect of the present invention.

[0055] In some embodiments, the subject includes both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of the invention, the subject is a human. The term subject includes confirmed patients.

[0056] In some implementation methods, the routes of administration include, but are not limited to: oral, local, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, intranasal, intrapulmonary, and rectal administration. In specific implementation methods, the clinician may determine the beneficial dose for the subject based on factors such as the subject's type, age, weight, general disease condition, and method of administration.

[0057] In some embodiments, the effective amount refers to the amount of drug, pharmaceutical composition, or pharmaceutical preparation that effectively produces the desired preventive, alleviating, or therapeutic effect. The effective amount of the drug, pharmaceutical composition, or pharmaceutical preparation described in this invention may vary depending on factors such as the active ingredient (in this invention, particularly the conjugated polypeptide or its pharmaceutically acceptable salt, pharmaceutical composition, or pharmaceutical preparation as described above), the symptoms and their severity, and the age of the mammal being treated. However, the specific dosage can be conventionally determined by those skilled in the art based on their knowledge in the field and the disclosure of this invention. Doses capable of producing the aforementioned effects are all within the protection scope of this invention.

[0058] The fifth aspect of the invention provides for any of the following applications:

[0059] (1) The use of the conjugated polypeptide according to the first aspect of the present invention in the preparation of a medicament for the treatment and / or prevention of glomerular basement membrane disease;

[0060] (2) The use of the conjugated polypeptide according to the first aspect of the present invention in the preparation of a pharmaceutical composition for the treatment and / or prevention of glomerular basement membrane disease;

[0061] (3) The use of the conjugated polypeptide described in the first aspect of the present invention in the preparation of pharmaceutical formulations for the treatment and / or prevention of glomerular basement membrane disease.

[0062] In this invention, treatment and / or prevention refer to delaying disease progression, preventing disease progression, and / or reducing the severity of symptoms that will or are expected to develop. Therefore, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing underlying metabolic causes of symptoms, inhibiting disorders or diseases, for example, preventing the development of disorders or diseases, alleviating disorders or diseases, regressing disorders or diseases, reducing symptoms caused by diseases or disorders, or stopping the symptoms of diseases or disorders. In one specific embodiment of the invention, the disease is anti-glomerular basement membrane disease.

[0063] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0064] This invention discloses for the first time a butyric acid-conjugated polypeptide drug (m-P14-2A (2-aminobutyric acid), m-P14-BA (butyric acid)) for the treatment of glomerular basement membrane disease. Comparative experiments have demonstrated that not all short-chain fatty acids and small-molecule polypeptides conjugated with m-P14 have therapeutic effects on glomerular basement membrane disease. The butyric acid-conjugated polypeptide drug (m-P14-2A, m-P14-BA) provided by this invention can significantly reduce kidney damage in an anti-glomerular basement membrane disease model, and its therapeutic effect is significantly superior to that of m-P14. In other words, the butyric acid-conjugated polypeptide drug (m-P14-2A, m-P14-BA) provided by this invention has achieved unexpected technical effects in the treatment of glomerular basement membrane disease, providing a novel treatment strategy in this technical field and laying the foundation for the final transformation of therapeutic polypeptides into clinical trials, with broad clinical application prospects. Attached Figure Description

[0065] Figure 1 Flowchart of m-P14-BA treatment in an experimental anti-GBM disease rat model;

[0066] Figure 2Clinical and immunological indicators of m-P14-BA treatment in anti-GBM rats: Figure A: From the day of model establishment, rats in each group were treated with m-P14-BA. The urinary protein level of rats in each group gradually increased, and the increase in urinary protein was significantly reduced in the early and late m-P14-BA treatment groups; Figure B: At week 6, the urinary protein level in the early and late m-P14-BA treatment groups was significantly lower than that in the disease control group; Figure C: At week 6, the blood urea nitrogen level in the early and late m-P14-BA treatment groups was significantly lower than that in the disease control group; Figure D: At week 6, the anti-α3 levels of rats in the early and late m-P14-BA treatment groups... Antibody production was significantly reduced in the 127-148 group; Figure E: Antibody production of anti-α3(IV)NC1 whole protein in rats treated with m-P14-BA was significantly reduced in the 6th week. Among them, ****: P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05, ns: no statistical difference.

[0067] Figure 3 Effects of m-P14-BA treatment on anti-GBM glomerulonephritis in WKY rats (pathological characteristics), where Figures A-D: representative immunofluorescence images of glomerular IgG deposition in each group of rats; Figures E-H: representative images of glomerular crescent formation in each group of rats; Figure I: statistical graph of the proportion of renal crescent formation in each group of rats; Figure J: statistical graph of immunofluorescence intensity of glomerular IgG deposition in each group of rats. ****: P < 0.0001, ***: P < 0.001, **: P < 0.01;

[0068] Figure 4 The therapeutic effects of stearic acid, albumin, and disulfide bond-coupled peptides were compared. Among them, there was no statistically significant difference in urinary protein (left) in rats 24 hours after treatment with albumin, inter-chain disulfide bonds, stearic acid-coupled peptides, and m-P14 peptide antibody compared with the disease control group; there was also no statistically significant difference in truncated peptides and intra-chain disulfide bond-modified peptides (right) compared with the disease control group.

[0069] Figure 5 The therapeutic effect of fatty acid-coupled m-P14 peptides: After treatment with butyrate-coupled peptides m-P14-BA and m-P14-2A, the urinary protein (left), serum creatinine (middle), and blood urea nitrogen (right) in rats were significantly reduced.

[0070] Figure 6 Butyrate-coupled peptide (m-P14-BA) in rats treated with late treatment (LT) significantly reduced 24-hour urinary protein compared to the disease control group, suggesting that late treatment was effective.

[0071] Figure 7Butyrate-modified peptides (m-P14-BA and m-P14-2A) can significantly reduce the level of autoantibodies in the peripheral circulation (left), and also reduce the number of CD4+ T cells (middle) and B cells (right) in the spleen. Detailed Implementation

[0072] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0073] Example: Peptide drugs obtained from short-chain fatty acid-coupled peptides (m-P14) and their application in the treatment of glomerular basement membrane disease.

[0074] 1. Determine the m-P14 backbone modification scheme for small molecule peptide drugs.

[0075] The m-P14 (TDIPPCPHGWSSLWKGFSFIMF) small molecule peptide is a hydrophobic peptide that is rapidly metabolized in vivo. The inventors of this application, through extensive and in-depth research, have determined modification schemes for the m-P14 backbone peptides (including: m-P14(short), m-P14(inter-chain SS), m-P14(intra-chain SS), m-P14-2A, m-P14-BA, m-P14-stearic acid, and m-P14-albumin). The design of coupling short-chain fatty acids is innovative. The peptide drugs derived from 2-aminobutyric acid (2A) and butyric acid (BA) conjugated peptides were named m-P14-2A and m-P14-BA, respectively. The peptide drugs derived from inter-chain SS and intra-chain SS disulfide bond conjugated peptides were named m-P14(inter-chain SS) and m-P14(intra-chain SS), respectively. The peptide drugs derived from stearic acid and albumin conjugated peptides were named m-P14-stearic acid and m-P14-albumin, respectively. The names and sequences of the designed and synthesized peptide backbone-modified peptides are detailed in Table 1 below.

[0076] Table 1. Peptide names and sequences related to polypeptide backbone modification.

[0077]

[0078] The peptides used in the experiment were synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd. using the F-moc method on an automated peptide synthesizer. The peptides were purified by high-performance liquid chromatography (HPLC) and then screened for final purity ≥98% by mass spectrometry. They were stored at -80℃ for the construction of experimental animal models.

[0079] Peptide α3127-148 sequence: TDIPPCPHGWISLWGFSFIMF (SEQ ID NO:3);

[0080] m-P14-BA sequence: Butyric acid-KKKKK-Ahx-TDIPPCPHGWSSLWKGFSFIMF.

[0081] 2. Experimental Materials and Methods

[0082] This invention utilizes the pathogenic T-cell epitope α3127-148 (α-P14) against GBM nephritis to actively immunize WistarKyoto (WKY) rats, thus establishing an animal model of glomerular basement membrane (GBM) nephritis. The experimental groups were divided into an early treatment group (administered from day 0) and a treatment group (administered after the appearance of hematuria or proteinuria). m-P14-BA was injected as the therapeutic drug into the α-P14-immunized rats, and a negative control group (physiological saline) was administered the same way. Renal function was evaluated by detecting urinary protein and blood urea nitrogen levels; circulating antibody levels and epitope expansion were detected by detecting anti-α3127-148 peptide antibodies or anti-α3(IV)NC protein antibodies; and the degree of renal pathological damage was evaluated by renal tissue immunofluorescence and PAS staining. Specific experimental materials and methods are as follows:

[0083] 2.1 Laboratory Animals

[0084] The experimental animals used in this study were 5-6 week old, 60-80g female Wistar Kyoto (WKY) rats, specific pathogen-free (SPF) grade. The animals were ordered with the approval of the Laboratory Animal Ethics Committee (ethics number: J2023109). The rats used in the experiment were purchased from Beijing Vital River Company and housed in a barrier environment (SPF grade) at the Laboratory Animal Center of Peking University First Hospital.

[0085] 2.2 Antigen Preparation

[0086] Remove the α3127-148 peptide from the -80℃ freezer. Centrifuge the peptide powder at 3000 rpm for 5 minutes to allow the powder to settle at the bottom of the tube. Dissolve the peptide in 10 μL of DMSO, then add an appropriate amount of sterile PBS and mix thoroughly to ensure complete dissolution. Add an equal volume of complete Freund's adjuvant to a 5 mL centrifuge tube and mix thoroughly with the peptide PBS solution at a 1:1 ratio. Vortex for at least 40 minutes to fully emulsify.

[0087] 2.3 Animal Immunization and Treatment Grouping

[0088] An experimental anti-GBM disease model was established by subcutaneous injection of a completely Freund's adjuvant-emulsified α3127-148 peptide solution into the bilateral hind paw pads at a dose of 400 μg / kg. Rats were randomly divided into four groups of six each. From the day of immunization, the disease control group received no treatment; the early treatment group received intraperitoneal injection of m-P14-BA until the day of sacrifice; the late treatment group received intraperitoneal injection of m-P14-BA upon the appearance of GBM disease symptoms (detection of hematuria or proteinuria); the control group received an equal volume of physiological saline intraperitoneally as a control, continuing until day 42 after model establishment. For detailed experimental procedures, please refer to [link to experimental protocol]. Figure 1 .

[0089] 2.4 Large Mouse Notebook Data Collection

[0090] Plasma specimens: After anesthetizing rats, blood was collected from the inner canthus vein of the rats using a capillary tube and placed in an EDTA anticoagulant tube. The blood was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant plasma was collected and stored at -40°C.

[0091] Urine specimens: At a fixed time each week, rats were placed in metabolic cages with ample water and food, and 24-hour urine was collected. The total 24-hour urine volume was recorded. The urine was centrifuged at 3000 rpm for 30 minutes at 4°C, and 1 mL of the supernatant was collected and stored at -40°C.

[0092] Kidney specimens: Six weeks after immunization, rats were anesthetized and samples were collected. 5% sodium pentobarbital was injected intraperitoneally at a dose of 0.1 mL / 100 g. After disinfection, the rat peritoneum was surgically opened, and the left kidney was removed. The kidney was repeatedly rinsed in physiological saline, and two pieces of cortex were cut. One piece was fixed in 4% paraformaldehyde solution for paraffin section preparation, and the other piece was placed in a cryovial and frozen in liquid nitrogen for frozen section preparation.

[0093] 2.5 Biochemical Detection of Rat Blood and Urine

[0094] After collecting blood and urine samples from rats, urinary protein was detected using the biuret method, and blood urea nitrogen concentration was detected using urease. This part of the testing was entrusted to technicians using an automated biochemical analyzer.

[0095] 2.6 IgG Immunofluorescence Staining of Kidney Tissue

[0096] 1) Frozen kidney cortex was embedded with OCT embedding agent to prepare frozen sections with a thickness of 8 μm.

[0097] 2) First, pre-cool the acetone at -20℃, then fix the sections with acetone at -20℃ for 10 minutes.

[0098] 3) Wash the tissue three times with PBS and circle the tissue with a histochemical pen.

[0099] 4) Blocking: Add 3% BSA blocking solution to the tissue and block for 1 hour at 37°C.

[0100] 5) Incubation with primary antibody: Dilute goat anti-rat IgG (Jackson Pharmaceuticals, USA, FITC labeled) with PBS at a ratio of 1:50, cover the tissue with the antibody dilution solution, and incubate at 37°C for 1 hour.

[0101] 6) Wash 3 times with PBS.

[0102] 7) Mounting: Add 1 drop of DAPI-containing mounting medium and mount the slide from one corner using a coverslip.

[0103] 8) Store in the dark at 4°C and photograph it in a dark room using a fluorescence microscope within one week.

[0104] 2.7 Rat kidney pathology: periodic acid-schiff stain (PAS)

[0105] 1) The renal cortex was thoroughly fixed with 4% formalin fixative. The tissue was dehydrated using alcohol of varying concentrations and then soaked in xylene solution to achieve transparency. The transparent tissue was then embedded in molten paraffin. After the paraffin solidified, 3μm thin sections were cut using a microtome, flattened, and dried in an incubator to prepare paraffin tissue sections. This part of the procedure was performed by Qu Lei, a pathologist in the Nephrology Department of Peking University Hospital.

[0106] 2) Before staining paraffin sections, remove the paraffin from the sections with xylene, soak them in two separate containers for 15 minutes each, and then place them in new xylene containers in sequence to ensure thorough dewaxing.

[0107] 3) Hydrate the sections in a gradient of alcohols, using 100% alcohol, 75% alcohol, and 50% alcohol respectively. Finally, wash the sections three times with PBS.

[0108] 4) Add periodic acid solution and incubate at room temperature in the dark for 20 minutes, then wash 3 times with PBS.

[0109] 5) Add Scheff's reagent, let it sit at room temperature for 30 minutes, then soak it in water at 40-60℃ for 40 minutes. After it cools naturally, rinse it with tap water for 10 minutes.

[0110] 6) Hematoxylin staining of the nucleus: Add hematoxylin staining solution to the tissue and let it stand at room temperature for 8 minutes to stain. Rinse with running water for 5 minutes to return to blue.

[0111] 7) After staining, dehydrate and clear the sections with gradient alcohol and xylene, drop 1 drop of neutral resin onto the section, and carefully cover it with a coverslip from one corner to mount the section.

[0112] 8) After the sections are placed in a fume hood to air dry naturally, they are photographed under an optical microscope in bright field.

[0113] 2.8 Detection of circulating antibodies in rats using enzyme-linked immunosorbent assay (ELISA)

[0114] 1) Antigen: Dissolve the α3127-148 peptide or α3(IV)NC protein in DMSO, and then dilute the peptide to 2 mg / mL with 0.05 mmol / L carbonate buffer.

[0115] 2) Plate coating: Add 100 μL of 2 μg / mL α3127-148 peptide solution to each well of the 96-well microplate and coat the plate with 0.05 mmol / L carbonate buffer as non-antigen coating wells, 100 μL per well. Place the microplate at 4°C and let it stand overnight.

[0116] 3) Remove the microplate, pour out the liquid inside, add 300 μL of 0.1% PBST solution to each well and wash 5 times, then pat dry on absorbent paper.

[0117] 4) Remove the rat plasma to be tested from the -40℃ freezer. After the plasma has thawed and vortexed, dilute the plasma sample with 1% BSA-0.1% PBST at a ratio of 1:100. Add 100 μL to each well. Set up positive control wells and blank control wells for each plate. Incubate at 37℃ for 30 minutes.

[0118] 5) Wash the plate 5 times with PBST.

[0119] 6) Dilute alkaline phosphatase-labeled goat anti-rat IgG with 0.1% PBST at a dilution ratio of 1:5000, add 100 μL to each well, and incubate at 37°C for 30 minutes.

[0120] 7) Wash the plate 5 times with PBST.

[0121] 8) Prepare alkaline phosphatase colorimetric solution. Prepare fresh and use immediately. Add 100 μL to each well and develop color at room temperature.

[0122] 9) Use an ELISA reader to read the OD value at a wavelength of 405nm. Read the OD value every 5 minutes and record the OD value.

[0123] 3. Experimental Results

[0124] 3.1m-P14-BA treatment can effectively reduce kidney damage in a rat model of anti-GBM nephropathy.

[0125] In rats, proteinuria appeared 3-4 weeks after α3127-148 peptide modeling, and the level of urinary protein gradually increased with the progression of the disease. Figure 2 A. Both the early and late treatment groups of m-P14-BA showed a decrease in urinary protein levels. The most significant decrease was observed in the early treatment group: at week 6, the m-P14-BA early treatment group vs. the disease control group: 70.14±65.50 vs. 202.6±48.22 mg / 24h, P=0.0026. The 24-hour urinary protein level in the late treatment group was also significantly lower than that in the disease group: late treatment group vs. the disease control group: 105.9±50.72 vs. 202.6±48.22 mg / 24h, P=0.007. (See [link to relevant documentation]). Figure 2 B.

[0126] At week 6, the blood urea nitrogen (BUN) levels in the disease control group and the healthy control group were significantly higher after modeling. After treatment, the BUN levels in both the early and late m-P14-BA treatment groups were significantly lower: early treatment group vs. disease control group: 6.153±0.624 vs. 7.772±1.01 mmol / L, P = 0.0075; late treatment group vs. disease control group: 6.215±0.620 vs. 7.772±1.01 mmol / L, P = 0.0092. (See [link to relevant documentation]). Figure 2 C.

[0127] In summary, m-P14-BA treatment significantly reduced clinical indicators of kidney damage in rats in all groups, and m-P14-BA treatment can significantly improve experimental anti-GBM rat nephritis.

[0128] 3.2m-P14-BA therapy inhibits α3-P14-induced intramolecular epitope expansion.

[0129] This experiment used ELISA to detect circulating antibody levels and antigenic epitopes in rats after m-P14-BA treatment. At week 6, both the disease control and treatment groups produced circulating autoantibodies against the immunogen α3127-148 peptide. Compared to the disease control group, the levels of autoantibodies were significantly lower in both the early and late m-P14-BA treatment groups (1.277±0.269 vs. 1.63±0.118, P = 0.015; 1.182±0.371 vs. 1.63±0.118, P = 0.018). [See attached image]. Figure 2 D.

[0130] Epitope expansion was observed in all rats of the disease control group after immunization, i.e., antibodies against the intact α3(IV)NC1 protein appeared in circulation. Compared with the disease control group, at week 6, the circulating antibody level against the intact α3(IV)NC1 protein was significantly lower in the m-P14-BA early treatment group (0.138±0.14 vs. 0.44±0.158, P=0.0057); the circulating antibody level against the intact α3(IV)NC1 protein was not significantly lower in the m-P14-BA late treatment group (0.235±0.186 vs. 0.44±0.158, P=0.0669). Figure 2 Early treatment with E. m-P14-BA can inhibit the expansion of intramolecular antigenic epitopes caused by the immunogen α3127-148 peptide.

[0131] 3.3m-P14-BA treatment alleviates renal pathological damage in a rat model of anti-GBM disease.

[0132] Rats were sacrificed after 6 weeks of treatment, and the kidney cortex was collected for staining and observation of renal pathological damage. Compared with healthy control rats, the disease control group rats exhibited typical crescentic glomerulonephritis. Figure 3 In AD, immunofluorescence examination of frozen sections of the kidneys reveals IgG deposition along GBM lines. Figure 3 EH). In rats treated with m-P14-BA, the pathological damage to the kidneys decreased to varying degrees.

[0133] Compared with the disease control group, rats in the early treatment group of m-P14-BA showed only a small number of crescent formations in their kidneys, and the proportion of crescent formations in the late treatment group was also significantly reduced (early treatment group vs. disease control group: 12.17±5.90 vs. 61.5.0±21.38%, P=0.0003; late treatment group vs. disease control group: 20.83±13.91 vs. 61.5.0±21.38%, P=0.0029), see [link to relevant data]. Figure 3 I; Immunofluorescence staining of IgG in frozen kidney sections was significantly reduced in the early treatment group of m-P14-BA (early treatment group vs. disease control group: 0.47±0.4 vs. 1.8±0.29, P<0.0001), and glomerular IgG deposition in rats was also reduced to some extent compared with the late treatment group of m-P14-BA (1.2±0.30 vs. 1.8±0.29, P=0.0065). See Figure 3 J.

[0134] In summary, m-P14-BA treatment resulted in significant reductions in urinary protein, blood urea nitrogen levels, circulating antibody production, and pathological changes in the proportion of glomerular crescents and IgG deposition. These results indicate that m-P14-BA intervention can significantly alleviate experimental anti-GBM glomerulonephritis in both the early and late treatment groups.

[0135] Comparative Study on the Therapeutic Efficacy of Peptide Drugs Obtained from Short-Chain Fatty Acid Coupled with Peptide (m-P14) in the Treatment of Glomerular Basement Membrane Disease

[0136] We used the above-mentioned modified peptide and the control therapeutic peptide m-P14 to treat an anti-GBM nephritis rat model. The specific experimental and detection methods were the same as those described in the above examples.

[0137] The results showed that stearic acid-coupled peptide (mP14-SA), albumin-coupled peptide (mP14-ALB), inter-chain disulfide bond modified peptide (mP14-SS), intra-chain disulfide bond modified peptide (intra-chain SS), m-P14 peptide antibody (mP14-Ab), and truncated peptide (short) all had no significant therapeutic effect. Figure 4 However, peptides (m-P14-2A and m-P14-BA) conjugated with butyric acid (2-aminobutyric acid (2A) and butyric acid (BA)) both showed significant therapeutic effects. Figure 5 Furthermore, after discontinuation of administration, the recurrence rate of disease in rats treated with butyrate-conjugated peptides was lower than that in the m-P14 treatment group, indicating that its effect was superior to m-P14, i.e., the modification was successful. We then used butyrate-conjugated peptides for late-stage treatment of a rat anti-GBM disease model, i.e., administration after the onset of the disease. The butyrate-conjugated peptides showed good therapeutic effects. Figure 6 ).

[0138] The above results indicate that stearic acid-modified peptides (m-P14-stearic acid), albumin-modified peptides (m-P14-albumin), and disulfide bond-modified peptides (inter-chain SS, intra-chain SS) have no therapeutic effect on the rat model of GBM nephritis. However, butyric acid-modified peptides (m-P14-2A, m-P14-BA) have significant therapeutic effects. It is evident that although stearic acid modification, albumin modification, and disulfide bond modification are all well-known and common modification strategies for prolonging the half-life of small molecule peptides, their use in modifying the small molecule peptide m-P14, which has a clear therapeutic effect against GBM, has resulted in no therapeutic effect. In other words, before experimental verification, those skilled in the art could not have predicted whether butyric acid-modified peptides (m-P14-2A (2-aminobutyric acid), m-P14-BA (butyric acid)) would have a therapeutic effect against GBM. Thus, the butyric acid-coupled peptide claimed in this application has achieved unexpected technical effects.

[0139] The above results all suggest that butyrate-coupled peptides have a better therapeutic effect, and their effect in delaying the onset of the disease is stronger than that of m-P14. We further investigated their therapeutic mechanism, and the results showed that m-P14-BA and m-P14-2A can significantly inhibit the production of autoantibodies, and their effect is stronger than that of m-P14. Figure 7 ).

[0140] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A butyric acid-based conjugated polypeptide, characterized in that, The conjugated polypeptide is obtained by modifying the polypeptide sequence with butyric acid or 2-aminobutyric acid. The conjugated polypeptide obtained by modifying the polypeptide sequence with butyric acid is: C4H8O2-(K)n-linker-TDIPPCPHGWSSLWKGFSFIMF; The conjugated polypeptide obtained by modifying the polypeptide sequence with 2-aminobutyric acid is: C4H9NO2-(K)n-linker-TDIPPCPHGWSSLWKGFSFIMF; The n=5.

2. The conjugated polypeptide according to claim 1, characterized in that, The linker includes 6-aminohexanoic acid and / or β-alanine.

3. The conjugated polypeptide according to claim 2, characterized in that, The linker is 6-aminohexanoic acid.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the conjugated polypeptides according to claims 1-3.

5. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises any one of the conjugated polypeptides according to claims 1-3.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical formulation contains a pharmaceutically acceptable carrier and / or excipient.

7. The pharmaceutical preparation according to claim 6, characterized in that, The dosage forms of the pharmaceutical preparations include injections, powders, powder inhalers, capsules, tablets, pills, aerosols, microemulsions, or compound emulsions.

8. The pharmaceutical preparation according to claim 6, characterized in that, The dosage forms of the pharmaceutical preparations include lyophilized powder for injection, microspheres, enteric-coated or nasal sprays.

9. The method for preparing the coupled polypeptide according to any one of claims 1-3, characterized in that, The method includes preparing the conjugated polypeptide according to any one of claims 1-3 by modifying the polypeptide sequence TDIPPCPHGWSSLWKGFSFIMF with butyric acid or 2-aminobutyric acid.

10. Use of the conjugated polypeptide according to any one of claims 1-3 in the preparation of a medicament for the treatment and / or prevention of glomerular basement membrane disease.

11. Use of the conjugated polypeptide according to any one of claims 1-3 in the preparation of a pharmaceutical composition for the treatment and / or prevention of glomerular basement membrane disease.

12. Use of the conjugated polypeptide according to any one of claims 1-3 in the preparation of a pharmaceutical formulation for the treatment and / or prevention of glomerular basement membrane disease.

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