Synthesis and application of polycholic acid-short chain fatty acid with novel structure

Abstract

The invention belongs to the technical field of medicines, and discloses synthesis and application of polycholic acid-short chain fatty acid with a novel structure. The polymer is formed by connecting polycholic acid (as a main chain) with one or more short-chain fatty acids through covalent bonds. The polycholic acid is introduced as a skeleton structure of the polymer and the short-chain fatty acid is introduced as a side chain group, so that the functional polymer with biological activity is constructed. The invention also provides a nano-drug prepared by taking the polymer as a carrier. The nano-drug has good biocompatibility and drug loading capacity, effectively improves the effects of losing weight, reducing fat and relieving liver inflammation, effectively relieves liver fibrosis and aging, can enhance the fatty liver treatment effect and further reduce insulin resistance, and has wide clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the synthesis and application of a novel polycholic acid-short-chain fatty acid. Background Technology

[0002] Diabetes mellitus is one of the leading causes of disability and death worldwide due to its high incidence, disability rate, and mortality rate, and is currently the eighth leading cause of combined disability and death. Type 2 diabetes (T2D) is the most common type, accounting for over 96% of all diabetes cases.

[0003] Type 2 diabetes (T2D) is a complex disease involving multiple factors, characterized by its multi-gene and multi-mechanism pathogenesis. Its occurrence is closely related to various risk factors, with obesity considered one of the most significant contributing factors. Obesity is not only a key pathophysiological basis for T2D development but also often accompanies a series of cardiovascular and metabolic abnormalities, such as hypertension, hyperlipidemia, non-alcoholic fatty liver disease, and a chronic low-grade inflammatory state, ultimately leading to serious complications such as cardiovascular disease and chronic kidney disease. Furthermore, diabetes itself can further exacerbate obesity, creating a vicious cycle.

[0004] In recent years, a growing body of research has demonstrated a complex interplay between aging, obesity, and diabetes. Aging can induce chronic inflammation, metabolic disorders, and abnormal accumulation of adipose tissue. These pathological states, in turn, accelerate the aging process, further propelling disease progression and creating a vicious cycle that continues to worsen. Adipose tissue not only secretes various pro-inflammatory factors, exacerbating systemic inflammation, but also promotes cellular senescence, leading to mitochondrial dysfunction and impaired muscle mass. Furthermore, fat redistribution, particularly the abnormal migration of subcutaneous fat into the intermuscular space, can result in muscle degeneration, lipotoxicity, insulin resistance, and systemic chronic inflammation.

[0005] Therefore, implementing comprehensive interventions targeting multiple interrelated pathological states such as aging, inflammation, obesity, and metabolic abnormalities to break the vicious cycle they form is key to improving the quality of life and health of the elderly population.

[0006] The relevant reference CN119661825A discloses polyzwitterionic cholic acid, its synthesis, and its application in drug delivery. The polymer includes polycholic acid and zwitterionic polymers covalently linked to polycholic acid. It can prevent and treat obesity and non-alcoholic fatty liver disease, but the effect is not very good. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a novel polycholic acid-short-chain fatty acid structure that enhances weight loss and fat reduction, alleviates liver inflammation, effectively alleviates liver fibrosis and aging, strengthens the treatment of fatty liver, and further reduces insulin resistance. Furthermore, it can serve as a drug carrier, particularly suitable for the oral delivery of macromolecular biological drugs.

[0008] To address the above technical problems, the present invention provides a polymer comprising polycholic acid and short-chain fatty acids covalently linked to polycholic acid; The structure of the short-chain fatty acid is as follows: Wherein, R is selected from: C 1-6 alkyl; The monomer of polycholic acid is shown in the following formula. Among them, R 1 and R 2 Each is independently selected from: -H, -OH, C 1-6 alkyl.

[0009] Preferably, the polymer structure is as follows: Wherein, B represents the unit structure of polycholic acid; Each X is independently a non-cholic acid monomer or a short-chain fatty acid moiety, and at least one X is a short-chain fatty acid moiety; n is an integer between 1 and 50.

[0010] In another preferred embodiment, n is an integer from 2 to 100; more preferably, n is an integer from 2 to 50; most preferably, n is an integer from 2 to 30; and most preferably, an integer from 2 to 10.

[0011] In another preferred embodiment, n is an integer from 2 to 10; more preferably, it is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0012] In another preferred embodiment, the derivatization from the short-chain fatty acid refers to the process in which the active groups (such as -COOH) in the short-chain fatty acid react with the active groups (such as -OH) in the polycholic acid to form polycholic acid-butyric acid.

[0013] Preferably, the polymer is selected from the following structures: Where n is an integer from 1 to 50.

[0014] In another preferred embodiment, in polycholic acid, some or all of the terminal hydroxyl groups and the hydroxyl groups in the unit structure form covalent bonds with the carboxyl groups of short-chain fatty acids or cholic acids.

[0015] Preferably, the short-chain fatty acid is selected from the following structures:

[0016] A method for synthesizing a polymer, comprising the following steps: (A) Prepare polycholic acid and short-chain fatty acids; (B) Polycholic acid-short-chain fatty acid polymers were synthesized by a one-pot method.

[0017] Preferably, the one-pot reaction steps are as follows: cholic acid monomer, short-chain fatty acid, p-toluenesulfonic acid, and dimethylaminopyridine are dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine, with a mixing ratio of 3–7:1; the mixture is stirred at a reaction temperature of 30–50°C under nitrogen protection; then diisopropylcarbodiimide is added to the resulting reaction mixture, and the reaction is carried out for 1–3 hours; after the reaction is completed, the resulting product is precipitated in cold anhydrous methanol, the precipitate is collected by centrifugation and dried to obtain polycholic acid-short-chain fatty acid polymer.

[0018] A nanomedicine formulation comprising a polymer-formed nanoparticle or a pharmaceutically acceptable carrier.

[0019] Nanomedicine formulations may also include drugs optionally encapsulated in the nanoparticles.

[0020] In another preferred embodiment, the drug is as defined in the third aspect.

[0021] In another preferred embodiment, the drug is a biological macromolecular drug.

[0022] In another preferred embodiment, the drug is an antibody drug.

[0023] In another preferred embodiment, the nanomedicine formulation is an oral formulation or an injectable formulation; preferably, the nanomedicine formulation is an oral formulation.

[0024] In another preferred embodiment, the nanomedicine formulation is a formulation selected from the group consisting of capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, tinctures, or combinations thereof.

[0025] The use of a polymer in the preparation of a medicament for treating or preventing a disease selected from the group consisting of obesity, aging, diabetes, non-alcoholic fatty liver disease, or combinations thereof.

[0026] In a first aspect of the invention, a polymer is provided, the polymer comprising polycholic acid and a short-chain fatty acid covalently linked to the polycholic acid.

[0027] In another preferred embodiment, the polycholic acid is the backbone (backbone) of the polymer.

[0028] In another preferred embodiment, the polycholic acid has a degree of polymerization of an integer from 1 to 100.

[0029] In another preferred embodiment, the degree of polymerization of the polycholic acid is an integer from 2 to 100; more preferably, an integer from 2 to 50; even more preferably, an integer from 2 to 30; and most preferably, an integer from 2 to 20.

[0030] In another preferred embodiment, the degree of polymerization of the polycholic acid is an integer from 2 to 15; more preferably, it is an integer from 2 to 10, and even more preferably, it is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0031] In another preferred embodiment, the molecular weight of the polycholic acid is 600-20000, more preferably 600-10000; and even more preferably 600-5000.

[0032] In another preferred embodiment, the molecular weight of the polycholic acid is 1000-5000, more preferably 1000-3000.

[0033] In another preferred embodiment, the short-chain fatty acid is covalently linked to the polycholic acid backbone.

[0034] In another preferred embodiment, the short-chain fatty acid is covalently linked to the ends and / or repeating unit structures of the polycholic acid.

[0035] In another preferred embodiment, each unit structure of the polycholic acid is independently linked to 0, 1, or 2 short-chain fatty acids.

[0036] In another preferred embodiment, the polycholic acid is terminally linked to a short-chain fatty acid, and each unit structure is independently linked to 0, 1, or 2 butyric acids.

[0037] In another preferred embodiment, the monomer of the polycholic acid is a free bile acid.

[0038] In another preferred embodiment, the monomer of the polycholic acid is selected from one or more of the following structures:

[0039] In another preferred embodiment, the polymer is as shown in Formula II; in, X 1 X 2 and X 3 Each is independently selected from the following groups: -H, -OH, C 1-6Alkyl groups, bile acids, short-chain fatty acids; and X 1 X 2 and X 3 At least one of them is a short-chain fatty acid; n is an integer between 1 and 50.

[0040] In another preferred example, X 1 ,X 2 and X 3 Each can be independently -H, -OH, or a short-chain fatty acid.

[0041] In another preferred example, X 1 ,X 2 and X 3 All of them are short-chain fatty acids.

[0042] In another preferred example, X 1 Selected from the following groups: -H, -OH, C 1-6 Alkyl groups (e.g., -CH3) (preferably X) 1 =-H and X 2 (These are short-chain fatty acids).

[0043] In another preferred example, X 1 It is a short-chain fatty acid and X 2 Selected from the following groups: -H, -OH, C 1-6 Alkyl groups (e.g., -CH3) (preferably X) 2 (For H).

[0044] In another preferred example, X 3 It is a short-chain fatty acid.

[0045] In another preferred embodiment, the short-chain fatty acid refers to one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, or hexanoic acid. Preferably, acetic acid, propionic acid, or butyric acid; most preferably, butyric acid.

[0046] In a second aspect of the invention, a method for preparing a polymer as described in the first aspect is provided, comprising the steps of: (A) Prepare polycholic acid and short-chain fatty acids; (B) Polycholic acid-short-chain fatty acid polymers were synthesized by a one-pot method.

[0047] A method for synthesizing a polymer, comprising the following one-pot reaction steps: cholic acid monomer, short-chain fatty acid, p-toluenesulfonic acid, and dimethylaminopyridine are dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine at a ratio of 3–7:1; the mixture is stirred at a reaction temperature of 30–50°C under nitrogen protection; then diisopropylcarbodiimide is added to the resulting reaction mixture, and the reaction is carried out for 1–3 hours; after the reaction is complete, the resulting product is precipitated in cold anhydrous methanol, the precipitate is collected by centrifugation and dried to obtain a polycholic acid-short-chain fatty acid polymer. This reaction step is simple and yields a high rate of reaction.

[0048] In another preferred embodiment, the polycholic acid is prepared by a cholic acid polymerization step, which includes: providing cholic acid monomers and causing the cholic acid monomers to undergo a polymerization reaction to obtain polycholic acid.

[0049] In another preferred embodiment, the polymerization of cholic acid monomers is carried out in a mixed solvent of dichloromethane and pyridine.

[0050] In another preferred embodiment, in the polymerization reaction of cholic acid monomers, the volume ratio of dichloromethane to pyridine in the mixed solvent is 1 to 10:1; more preferably, it is 3 to 7:1; more preferably, it is 4 to 6:1; and most preferably, it is 5:1.

[0051] In another preferred embodiment, in the polymerization reaction of the cholic acid monomer, the dichloromethane is anhydrous dichloromethane, and / or the pyridine is anhydrous pyridine.

[0052] In another preferred embodiment, the polymerization reaction of the cholic acid monomer takes place for 0.5 to 5 hours, more preferably 1 to 3 hours, and even more preferably 2 hours.

[0053] In another preferred embodiment, the polymerization reaction of the cholic acid monomer is carried out at a reaction temperature of 30–100°C, more preferably 30–50°C, and even more preferably 35–45°C.

[0054] In another preferred embodiment, the method for preparing polycholic acid includes the steps of: dissolving cholic acid monomer, p-toluenesulfonic acid, and dimethylaminopyridine in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (1–10:1; preferably, 3–7:1; more preferably, 4–6:1; most preferably, 5:1); stirring under nitrogen protection at 30–100°C (optimally 40±5°C); then adding diisopropylcarbodiimide to the resulting reaction mixture and reacting for 0.5–5 hours (preferably 1–3 hours, more preferably 2 hours); after the reaction is complete, precipitating the resulting product in cold anhydrous methanol, centrifuging to collect the precipitate, and drying it to obtain polycholic acid.

[0055] In another preferred embodiment, the polycholic acid-short-chain fatty acid is prepared by a one-pot method, wherein the cholic acid-butyric acid polymerization step includes: providing cholic acid monomers and butyric acid, causing the cholic acid monomers to undergo a polymerization reaction, while the butyric acid is covalently linked to the cholic acid to obtain polycholic acid-butyric acid.

[0056] In another preferred embodiment, the one-pot synthesis of polycholic acid-short-chain fatty acids is carried out in a mixed solvent of dichloromethane and pyridine.

[0057] In another preferred embodiment, in the one-pot synthesis of polycholic acid-short-chain fatty acids, the volume ratio of dichloromethane to pyridine in the mixed solvent is 1 to 10:1; more preferably, it is 3 to 7:1; more preferably, it is 4 to 6:1; and most preferably, it is 5:1.

[0058] In another preferred embodiment, in the one-pot synthesis of polycholic acid-short-chain fatty acids, the dichloromethane is anhydrous dichloromethane, and / or the pyridine is anhydrous pyridine.

[0059] In another preferred embodiment, in the one-pot synthesis of polycholic acid-short-chain fatty acids, the polymerization reaction takes 0.5 to 5 hours, more preferably 1 to 3 hours, and even more preferably 2 hours.

[0060] In another preferred embodiment, in the one-pot synthesis of polycholic acid-short-chain fatty acids, the reaction temperature of the polymerization reaction is 30–100°C, more preferably 30–50°C, and even more preferably 35–45°C.

[0061] In another preferred embodiment, the one-pot synthesis of polycholic acid-short-chain fatty acids comprises the following steps: dissolving cholic acid monomers, short-chain fatty acids, p-toluenesulfonic acid, and dimethylaminopyridine in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (1–10:1; preferably, 3–7:1; more preferably, 4–6:1; most preferably, 5:1); stirring under nitrogen protection at 30–100°C (ideally 40±5°C); then adding diisopropylcarbodiimide to the resulting reaction mixture and reacting for 0.5–5 hours (preferably 1–3 hours, more preferably 2 hours); after the reaction is complete, precipitating the resulting product in cold anhydrous methanol, centrifuging to collect the precipitate, and drying it to obtain the polycholic acid-short-chain fatty acid polymer.

[0062] In a third aspect of the invention, a drug delivery carrier is provided, the drug delivery carrier comprising nanoparticles formed from a polymer as described in the first aspect.

[0063] In another preferred embodiment, the drug delivery carrier is capable of delivering drugs.

[0064] In another preferred embodiment, the drug is encapsulated in the nanoparticles.

[0065] In another preferred embodiment, the drug is a biological macromolecular drug.

[0066] In another preferred embodiment, the biopharmaceutical includes: protein and polypeptide drugs, nucleic acid drugs, poorly soluble drugs, antibody drugs, antibody-drug conjugates (ADCs), vaccine drugs, or combinations thereof.

[0067] In another preferred embodiment, the protein-peptide drugs include: insulin, glargine insulin, lispro insulin, aspart insulin, detemir insulin, protamine human insulin, degludec insulin, exenatide, liraglutide, semaglutide, dulaglutide, liximab, benaglutide, loxenatide, semaglutide, abiglutide, growth hormone, glatiramer acetate, lupron (leuprolide acetate), texipatide, motixafortide (BL-8040, BKT140), pemoxatide (EPO-018B), parathyroid hormone (PTH), trofinetide, interferon, TNF-α, C5, coagulation factor IX, and interleukin (IL-2). Recombinant human coagulation factor VIII, recombinant human erythropoietin, recombinant human epidermal growth factor, recombinant human interferon α1b, recombinant human granulocyte colony-stimulating factor, recombinant insulin, recombinant interferon, recombinant coagulation factor, recombinant erythropoietin, recombinant granulocyte colony-stimulating factor, enzyme-replaced recombinant protein drugs, recombinant growth hormone, recombinant human prourokinase, recombinant hirudin, recombinant human bone morphogenetic protein, recombinant human granulocyte-macrophage colony-stimulating factor, fibroblast growth factor 1 (icvFGF1), fibroblast growth factor 4 (icvFGF4), fibroblast growth factor, or combinations thereof.

[0068] In another preferred embodiment, nucleic acid drugs include: DNA drugs, mRNA drugs, RNAi interference drugs, gene editing drugs, siRNA drugs, circular RNA drugs, or combinations thereof.

[0069] In another preferred embodiment, the poorly soluble drug includes: PROTAC, molecular gel, or a combination thereof.

[0070] In another preferred embodiment, the poorly soluble drug includes: a Bcl-xl protein degrader (e.g., DT2216), an androgen receptor degrader (e.g., ARV-110, ARV-766, AR-LDD), an estrogen receptor degrader (e.g., ARV-471), an IRAK4 degrader (e.g., KT-474, KT-413), a STAT3 degrader (e.g., KT-333), a BTK degrader (e.g., NX-2127, NX-5948), a TRK degrader (e.g., CG001419), a BRD9 degrader (e.g., CFT-8634, FHD-609), thalidomide, lenalidomide, pomalidomide, or combinations thereof.

[0071] In another preferred embodiment, the antibody drug includes: PD-1 monoclonal antibody.

[0072] In another preferred embodiment, the antibody drugs include: adalimumab, corticotropin, bevacizumab, pembrolizumab, nivolumab, sintilimab, cimiplimab, toripalimab, tislelizumab, camrelizumab, prolgolimab, penpulimab, dostarlimab, zimberelimab, serplulimab, atezolizumab, and durvalumab. (Durvalumab), Avelumab, Sugemalimab, Envafolimab, Cadonilimab, Bamlanivimab, Tebentafusp, Rituximab, Catuximab, Emicizumab, Amivantamab, Faricimab, Mosunetuzumab, Blinatumomab, Cadonilimab, Moxetumomab pasudotox), iodine 131derlotuximab biotin, iodine 131tositumomab, iodine 131Metuximab, Fc fusion protein, scFv antibody and TCR fusion protein (Tebentafusp), and antibody and enzyme fusion protein (Pabinafusp alfa), caplacizumab, envafolimab, adalimumab, chimeric antibody I131-Metuximab, loncastuximabtesirine), catutoxab, moxetumomabpasudotox, obinutuzumab, mogamulizumab, edrecolomab, nebacumab, benralizumab, inebilizumab, or combinations thereof.

[0073] In another preferred embodiment, the vaccine drug includes: polio vaccine, cholera vaccine, typhoid live vaccine, SARS-CoV-2 vaccine, HPV vaccine, pneumonia vaccine, Asian swine fever vaccine, or combinations thereof.

[0074] In a fourth aspect of the present invention, a method for preparing a drug delivery carrier is provided, comprising the steps of: (1) Provide the polymer as described in the first aspect; (2) Nanoparticles are formed by water-in-oil-in-water (W / O / W) or water-in-oil-in-solid (S / O / W) technology.

[0075] In another preferred embodiment, the polymer and optionally a drug are mixed to form drug-loaded nanoparticles using a water-in-oil-in-water (W / O / W) or water-in-oil-in-solid (S / O / W) technique.

[0076] A nanoparticle-loaded drug delivery composition comprising the polymer as described in the first aspect.

[0077] In another preferred embodiment, the nano-drug-loaded composition may also include an optional drug.

[0078] In another preferred embodiment, the polymer exists either in the form of nanoparticles or in a form where no nanoparticles are formed.

[0079] In another preferred embodiment, the drug is as defined in the third aspect.

[0080] In another preferred embodiment, the drug is a biological macromolecular drug.

[0081] In another preferred embodiment, the drug is an antibody drug.

[0082] In another preferred embodiment, the nano-drug-carrying composition is used to prepare a drug delivery carrier.

[0083] In another preferred embodiment, the nano-drug-loaded composition is used to prepare nanomedicine formulations.

[0084] In a seventh aspect of the invention, a use of the polymer as described in the first aspect is provided, wherein the use is one or more uses selected from the group consisting of: a. Used for delivering medicine; b. Used in the preparation of nanodelivery carriers; c. Used in the preparation of nanomedicines.

[0085] In another preferred embodiment, the nanomedicine may be either unencapsulated from any other drug (such as a biopharmaceutical) or encapsulated with a biopharmaceutical.

[0086] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0087] 1. The polycholic acid-short-chain fatty acid in this invention has excellent biocompatibility and stability, effectively improving the effects of weight loss and fat reduction and alleviating liver inflammation, and effectively alleviating liver fibrosis and aging, and can enhance the treatment effect of fatty liver and further reduce insulin resistance.

[0088] 2. The polycholic acid-short-chain fatty acid polymer of the present invention can promote the absorption and circulation of short-chain fatty acids in the gastrointestinal tract and in vivo, and can play a good anti-inflammatory role.

[0089] 3. This invention uses polycholic acid-short-chain fatty acids, which are non-immunogenic and easy to load with drugs, as the basic unit. The prepared polycholic acid-short-chain fatty acid nanocarrier raw materials are inexpensive, readily available, and have good biocompatibility. The synthesis conditions are mild, making it an excellent oral nanocarrier.

[0090] 4. The synthesis method of the polymer of the present invention involves dissolving cholic acid monomer, short-chain fatty acid, p-toluenesulfonic acid and dimethylaminopyridine in a mixed solution of anhydrous dichloromethane and anhydrous pyridine, with a mixing ratio of 3 to 7:1; stirring at a reaction temperature of 30-50°C under nitrogen protection; then adding diisopropylcarbodiimide to the resulting reaction mixture and reacting for 1-3 hours; after the reaction is completed, precipitating the obtained product in cold anhydrous methanol, centrifuging to collect the precipitate and drying it in a one-pot method to obtain polycholic acid-short-chain fatty acid polymer. This one-pot method has simple reaction steps and high yield. Attached Figure Description

[0091] Figure 1 The 1H NMR spectra of polycholic acid-butyric acid shown in Examples 1, 2 and 3 are displayed. Figure 2 Size exclusion chromatograms (GPCs) of polycholic acid-butyric acid shown in Examples 1, 2 and 3 are displayed. Figure 3 The matrix-assisted laser desorption / ionization mass spectra of polycholic acid-butyric acid shown in Examples 1, 2 and 3 are displayed. Figure 4 The effect of polycholic acid-butyric acid on gene expression in differentiated 3T3-L1 cells was shown in Example 5 (n=4). Figure 5 The effect of polycholic acid-butyric acid on gene expression in HepG2 cells was shown in Example 5 (n=4). Figure 6 The liver, fat, and body weight of obese mice treated in Example 6 are shown (n=5). Figure 7 The results of the glucose tolerance test (n=5) and insulin sensitivity test (n=5) of obese mice treated in Example 6 are shown. Figure 8 Oil Red staining of liver sections from obese mice treated in Example 7 is shown. Figure 9 The image shows H&E staining of various organs in obese mice after treatment in Example 7. Detailed Implementation

[0092] Through long-term and in-depth research, the inventors have, for the first time, combined short-chain fatty acids with bile acids to prepare a polycholic acid-short-chain fatty acid compound, and used this polymer as a basis to prepare nanoparticles. Experimental verification has shown that these nanoparticles can be used alone as drugs to delay aging and prevent and treat cardiovascular diseases, diabetes, obesity, and non-alcoholic fatty liver disease. Furthermore, these nanoparticles possess excellent drug loading and delivery capabilities, making them suitable as delivery carriers for biopharmaceutical drugs. Based on these findings, the inventors have completed this invention.

[0093] the term

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0095] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0096] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0097] As used herein, the term “room temperature” or “normal temperature” refers to a temperature of 4–40°C, preferably 25 ± 5°C.

[0098] butyric acid

[0099] Butyrate is a short-chain fatty acid (SCFA) primarily produced by gut microbiota through glycolysis and fermentation in the colon. Existing research indicates that butyrate mediates its biological functions through multiple mechanisms, mainly including the following two pathways: (a) signal transduction via G-protein coupled receptors (GPCRs), particularly activation of free fatty acid receptors 2 and 3 (FFAR2 / FFAR3); and (b) regulation of gene expression as an inhibitor of histone deacetylase (HDAC).

[0100] In the intestinal tract, butyrate has a variety of beneficial effects, including preventing and inhibiting the development of colon cancer, improving inflammatory response, relieving oxidative stress, enhancing epithelial defense barrier function, and regulating visceral sensitivity and intestinal motility.

[0101] The potential applications of butyrate in the parenteral system are also attracting increasing attention. Studies have shown that it can be used to intervene in a variety of diseases, especially metabolic diseases, including hypercholesterolemia, obesity, insulin resistance, and ischemic stroke, demonstrating promising clinical translation prospects.

[0102] bile acids

[0103] Bile acids (BAs) are one of the main components of bile, synthesized from cholesterol in the liver and stored in the gallbladder. Bile acids perform multiple physiological functions in the body, participating in the absorption, distribution, metabolism, and excretion of nutrients. Besides acting as surfactants to promote lipid absorption, bile acids are also considered important nutrient sensing factors and metabolic regulators. They can activate signaling pathways such as the Farnesoid X Receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5), thereby regulating glucose metabolism, energy balance, and immune responses, and maintaining metabolic homeostasis.

[0104] Furthermore, as natural products derived from cholesterol metabolism, bile acids exhibit good biocompatibility. On one hand, their hydrophobic structure can participate in the formation of micelles for drug delivery; on the other hand, they also demonstrate certain biological activities in energy metabolism regulation and anti-inflammation, showing good potential for pharmacological development. Bile acids can be divided into two main categories according to their structure: one is free bile acids, including cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid; the other is the product of free bile acids combined with glycine or taurine, called conjugated bile acids, mainly including glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, and taurochenodeoxycholic acid.

[0105] Polycholic acid-short-chain fatty acids and their preparation methods

[0106] One object of this invention is to provide a polycholic acid-short-chain fatty acid and its synthetic route. Firstly, the compound (or polymer) is synthesized from cholic acid and short-chain fatty acid. Secondly, the compound can be used to prepare drug-loaded nanoparticles for drug delivery.

[0107] In this document, the terms "polymer of the present invention", "cholic acid-short chain fatty acid polymer" and "polycholic acid-short chain fatty acid" are used interchangeably and refer to polymers as defined in the first aspect of the present invention.

[0108] In one specific embodiment, a novel structure of polycholic acid-butyric acid (also known as the polymer of the present invention) is provided.

[0109] In another embodiment, the polymer is as defined in the first aspect.

[0110] In another embodiment, the polymer is capable of forming a nanodelivery carrier (specifically, nanomicelles).

[0111] In some embodiments, the polymer has the following structural formula: in, Each X is independently either a cholic acid monomer or a short-chain fatty acid moiety; and at least one X is a short-chain fatty acid moiety. n is an integer between 1 and 50.

[0112] In other implementations, B is bile acid.

[0113] In other embodiments, the bile acid monomers may be those selected from Table A1 below: Table A1

[0114] In other embodiments, the polycholic acid backbone may be selected from those in Table A1.1 below: Table A1.1

[0115] Where m is an integer from 1 to 100.

[0116] In other embodiments, when all hydroxyl groups in polycholic acid are linked to butyric acid, the polymer is as described in Table C: Table C

[0117] It should be understood that in the polymer, the hydroxyl groups of polycholic acid can be ungrafted, butyric acid-linked, or cholic acid-linked, and the cholic acid monomers are selected from Table A1.

[0118] In another embodiment, the polymer is used to prepare a nanoparticle-loaded drug composition.

[0119] In another embodiment, the present invention also provides the use of the polymer in treating diseases, preferably including, but not limited to, cardiovascular diseases, obesity, diabetes, non-alcoholic fatty liver disease, and aging.

[0120] In another specific embodiment, the present invention also provides a method for preparing the polymer, comprising the steps of: (A) Provides polycholic acid; (B) Polycholic acid is reacted with short-chain fatty acids to obtain the polymer.

[0121] In some specific embodiments, the method for preparing the polycholic acid-butyric acid provided by the present invention includes the following steps: (1) Dissolve bile acids, p-toluenesulfonic acid, and dimethylaminopyridine in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (5:1). Stir under nitrogen protection at 30℃-100℃ (optimal 40±5℃). Then add diisopropylcarbodiimide to the reaction mixture and react for 2 hours. After the reaction is complete, precipitate the product in cold anhydrous methanol, collect the precipitate by centrifugation, and dry to obtain a white powder. (2) Dissolve the cholic acid polymer, butyric acid, EDCI, and dimethylaminopyridine in dichloromethane. Stir overnight under nitrogen protection. After the reaction is complete, precipitate the crude product in cold methanol, collect the precipitate by centrifugation, and dry it to obtain a white powder.

[0122] In another specific embodiment, the present invention also provides a method for preparing the polymer, comprising the step of directly synthesizing the cholic acid-butyric acid polymer by a one-pot method.

[0123] Bile acids, butyric acid, p-toluenesulfonic acid, and dimethylaminopyridine were dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (5:1). The mixture was stirred at 30°C–100°C (optimal 40±5°C) under nitrogen protection. Then, diisopropylcarbodiimide was added to the reaction mixture, and the reaction was allowed to proceed for 2 hours. After the reaction was complete, the product was precipitated in cold anhydrous methanol, centrifuged to collect the precipitate, and dried to obtain a white powder.

[0124] In another embodiment, the preparation method is as described in the second aspect.

[0125] Nanodelivery carriers, nanomedicines and their preparation methods

[0126] Another objective of this invention is to provide a nanocarrier (or nanodelivery carrier) and its preparation method. Firstly, this nanocarrier exhibits good biocompatibility and drug loading capacity. Secondly, this carrier can treat and prevent obesity, fatty liver, and cardiovascular diseases. Furthermore, the preparation method provided by this invention is simple, the materials are readily available, and the reaction conditions are mild, thus possessing promising industrialization prospects.

[0127] In one specific embodiment, a method for preparing the nanodelivery carrier is provided, comprising the steps of: The mixture was prepared using a water-in-oil-in-water technique. Polycholic acid-butyric acid (100 mg) was dissolved in 2 mL of chloroform. Phosphate-buffered saline (PBS, 100 μL) with or without the drug was added dropwise to the solution, and the mixture was homogenized using a high-pressure homogenizer for 10 min. The dispersant was then added dropwise to a continuous phase of 5% PVA and homogenized. The mixture was added dropwise to 200 mL of 0.2% PVA and stirred overnight to evaporate the solvent. The nanoparticles were collected by centrifugation at 12,000 rpm for 20 min at 4 °C and then washed three times with deionized water. Optionally, the encapsulation efficiency in the above-mentioned drug-loaded nanomicelles ranged from 100% to 95%, and the drug loading ranged from 0% to 15%.

[0128] Pharmaceutical compositions, nanomedicine formulations and methods of administration

[0129] Because the polymers of the present invention and the micelles formed therefrom have excellent preventive and therapeutic effects on diseases such as diabetes, obesity and non-alcoholic fatty liver disease, and have excellent drug loading and delivery capabilities, they can be used as drug delivery carriers. Therefore, micelles or blank micelles encapsulated with other drugs formed by the polymers of the present invention, as well as pharmaceutical compositions or nanomedicine formulations containing the polymers of the present invention as the main active ingredient, can be used to treat, prevent and alleviate a variety of diseases or symptoms.

[0130] The pharmaceutical compositions or nanomedicine formulations of the present invention comprise micelles of the present invention and pharmaceutically acceptable carriers within a safe and effective range. "Safe and effective range" refers to a amount of micelles or other drugs contained therein sufficient to significantly improve the condition without causing serious side effects.

[0131] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds.

[0132] The administration method of the micelles or pharmaceutical compositions of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and local administration. Considering the excellent delivery properties of the micelles of the present invention, the micelles and pharmaceutical compositions of the present invention are particularly suitable for oral administration. Therefore, the nanomedicine formulations of the present invention are preferably oral formulations.

[0133] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures.

[0134] The main advantages of this invention include:

[0135] 1. The polycholic acid-short-chain fatty acid of the present invention can improve the weight reduction effect in type II diabetic mice and further improve the insulin sensitivity of mice.

[0136] 2. The polycholic acid-short-chain fatty acid of the present invention can promote the absorption and circulation of short-chain fatty acids in the gastrointestinal tract and in vivo, and can play a good anti-inflammatory role.

[0137] 3. This invention uses non-immunogenic, easily drug-loadable cholic acid-short-chain fatty acids as the basic unit, and the prepared polycholic acid-short-chain fatty acid nanocarrier raw material is inexpensive and readily available. The synthesis conditions are mild, making it an excellent oral nanocarrier.

[0138] In summary, this invention synthesizes polycholic acid-short-chain fatty acids and uses them to prepare nanoparticles. The polycholic acid-short-chain fatty acids in this invention exhibit excellent biocompatibility and stability, improving the effects of weight loss and fat reduction, alleviating liver inflammation, effectively alleviating liver fibrosis and aging, enhancing the treatment effect of fatty liver, and further reducing insulin resistance. Furthermore, this carrier can effectively load various macromolecular drugs, including insulin and genes, as well as poorly soluble small molecule drugs.

[0139] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0140] General preparation methods for polymers

[0141] The polymers in the examples can be synthesized by the following methods: (1) Synthesis of polycholic acid: Ursodeoxycholic acid, p-toluenesulfonic acid, and dimethylaminopyridine were dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (5:1). The mixture was stirred at 40°C under nitrogen protection. Then, diisopropylcarbodiimide was added to the reaction mixture, and the reaction was allowed to proceed for 2 hours. After the reaction was completed, the product pBA was precipitated in cold anhydrous methanol, the precipitate was collected by centrifugation and dried to obtain a white powder of pBA. (2) Synthesis of polycholic acid-butyric acid: Polycholic acid, butyric acid, PCB, EDCI, and dimethylaminopyridine were dissolved in dichloromethane. The mixture was stirred overnight under nitrogen protection. After the reaction was completed, the crude product was precipitated in cold methanol, the precipitate was collected by centrifugation and dried to obtain a white final product. (3) One-pot preparation of polycholic acid-butyric acid: Ursodeoxycholic acid, butyric acid, p-toluenesulfonic acid, and dimethylaminopyridine were dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine (5:1). The mixture was stirred at 40°C under nitrogen protection. Then, diisopropylcarbodiimide was added to the reaction mixture, and the reaction was allowed to proceed for 2 hours. After the reaction was completed, the precipitate was transferred to cold anhydrous methanol, centrifuged to collect the precipitate, and dried to obtain a white powdery final product.

[0142] Example 1: Synthesis of polycholic acid

[0143] Ursodeoxycholic acid (UDCA) (5.4 mmol), p-toluenesulfonic acid (0.652 mmol), and dimethylaminopyridine (0.652 mmol) were dissolved in 60 mL of a mixture of anhydrous dichloromethane and anhydrous pyridine (5:1). The mixture was stirred at 40 °C under nitrogen protection. Then, diisopropylcarbodiimide (DIC, 6.92 mmol) was added to the reaction mixture, and the reaction was allowed to proceed for 2 hours. After the reaction was complete, the product pBA was precipitated in 400 mL of cold anhydrous methanol. The precipitate was collected by centrifugation and dried to obtain a white powder. Its 1H NMR spectrum is shown below. Figure 1 As shown. Based on GPC and NMR characterization results, the weight-average molecular weight (MW) of the product was determined to be 2880, and the degree of polymerization was approximately 4. Figure 2 and Figure 3 ).

[0144] Example 2: Synthesis of polycholic acid-butyric acid

[0145] Polycholic acid, butyric acid, EDCI, and dimethylaminopyridine were dissolved in dichloromethane. The mixture was stirred overnight under nitrogen protection. After the reaction was complete, the crude product was precipitated in cold methanol, the precipitate was collected by centrifugation and dried to obtain a white powder.

[0146] Example 3: One-pot preparation method of polycholic acid-butyric acid

[0147] Polycholic acid, butyric acid, EDCI, and dimethylaminopyridine were dissolved in dichloromethane. The mixture was stirred overnight under nitrogen protection. After the reaction was complete, the crude product was precipitated in cold methanol, the precipitate was collected by centrifugation and dried to obtain a white powder.

[0148] Example 4: Preparation of a polycholic acid-butyric acid nanocarrier

[0149] The nanoparticles were prepared using a water-in-oil-in-water technique. Polycholic acid-butyric acid (100 mg) was dissolved in 2 mL of chloroform. Phosphate-buffered saline (PBS, 100 μL) was added dropwise to the solution, and the mixture was homogenized using a high-pressure homogenizer for 10 min. The dispersant was then added dropwise to a continuous phase of 5% PVA and homogenized. The mixture was added dropwise to 200 mL of 0.2% PVA and stirred overnight to evaporate the solvent. The nanoparticles were collected by centrifugation at 12,000 rpm for 20 min at 4 °C and then washed three times with deionized water.

[0150] Example 5: In vitro evaluation of the therapeutic effect of polycholic acid-butyric acid

[0151] 3T3-L1 preadipocytes were cultured and induced to differentiate. Preadipocytes were cultured in complete medium containing high-glucose DMEM supplemented with 10% FBS and 1% penicillin and streptomycin in a 37°C, 5% CO2 incubator. For adipocyte differentiation, cells were treated for 2–4 days with differentiation medium supplemented with 10 μg / mL insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). Finally, the differentiation medium was replaced every two days with complete medium containing 10 μg / mL insulin.

[0152] HepG2 cells and differentiated 3T3-L1 cells were co-incubated with polycholic acid-butyrate for 3 days. After incubation, the cells were washed 2-3 times with PBS. The PBS was discarded, and 0.5 mL of Trizol was added and allowed to stand for 3-5 minutes. RNA was then extracted from the cells using a kit, and the expression levels of various RNAs in the cells were detected by RT-PCR. The experimental results demonstrated that polycholic acid-butyrate could inhibit the expression of inflammatory factors in hepatocytes and differentiated adipocytes, and also had a significant inhibitory effect on aging-related genes. Figure 4 and Figure 5 ).

[0153] Example 6: Application of polycholic acid-butyric acid in the treatment and prevention of obesity and type II diabetes.

[0154] An obese mouse model was established by feeding C57 mice a high-fat diet. After 8 weeks of high-fat diet feeding, the mice's body weight increased significantly. Subsequently, the obese mice were randomly divided into four groups: a PBS group, a butyrate group, an ursodeoxycholic acid group, and a polycholic acid-butyrate group, respectively. Administered the medication every three days for six weeks. After the administration period, the mice's body weight was measured, and glucose tolerance and insulin tolerance tests were performed.

[0155] Oral glucose tolerance test (OGTT): Mice were fasted overnight (20:00-8:00), but water was allowed. Note that the bedding should be changed before fasting. Fasting blood glucose levels were measured before administration, and then each mouse was orally administered a 20% glucose solution (1g / kg). Blood glucose levels in the tail vein were measured at 15 min, 30 min, 60 min, 90 min, and 120 min after administration.

[0156] In the insulin tolerance test (ITT), mice were fasted for 4 hours but allowed free access to water. The bedding was changed before fasting. Blood glucose levels were measured after fasting, followed by subcutaneous injection of insulin (1 IU / kg). Blood glucose levels were measured at 15, 30, 60, 90, and 120 minutes post-injection.

[0157] Experimental results show that ( Figure 6 Polycholic acid-butyric acid (PCA-B) can significantly reduce the body weight of obese mice. Furthermore, based on changes in blood glucose levels and the area under the curve, PCA-B can improve glucose tolerance and insulin tolerance in mice, and also improve insulin sensitivity to some extent.

[0158] Example 7: Application of polycholic acid-butyric acid in the treatment of fatty liver

[0159] Obese mice were administered polycholic acid-butyric acid (50 mg / kg) for six consecutive weeks, while ursodeoxycholic acid and butyric acid were administered orally in the PBS group as controls. After the administration was completed, liver sections of the mice were collected and stained with H&E and Oil Red O.

[0160] Experimental results show that ( Figure 6-9 Polycholic acid-butyric acid can alleviate symptoms such as obesity and diabetes caused by a high-fat diet, and at the same time significantly reduce vacuolation and fat content in the liver of mice, demonstrating a good preventive and therapeutic effect on fatty liver.

[0161] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A polymer, characterized in that, Including polycholic acid and short-chain fatty acids covalently linked to polycholic acid; The structure of the short-chain fatty acid is as follows: Wherein, R is selected from: C 1-6 alkyl; The monomer of polycholic acid is shown in the following formula. Among them, R 1 and R 2 Each is independently selected from: -H, -OH, C 1-6 alkyl.

2. A polymer according to claim 1, characterized in that: The monomer of the polycholic acid is selected from one or more of the following structures:

3. A polymer according to claim 1, characterized in that: The polymer has the following structural formula. Wherein, B represents the unit structure of polycholic acid; Each X is independently a non-cholic acid monomer or a short-chain fatty acid moiety, and at least one X is a short-chain fatty acid moiety; n is an integer between 1 and 50.

4. A polymer according to claim 1, characterized in that: The polymer is selected from the following structures: Where n is an integer from 1 to 50.

5. A polymer according to claim 1, characterized in that, The short-chain fatty acid is selected from the following structures:

6. A method for synthesizing a polymer, characterized in that, Includes the following steps: (A) Prepare polycholic acid and short-chain fatty acids; (B) Polycholic acid-short-chain fatty acid polymers were synthesized by a one-pot method.

7. A method for synthesizing a polymer according to claim 6, characterized in that, The one-pot reaction steps are as follows: cholic acid monomer, short-chain fatty acid, p-toluenesulfonic acid, and dimethylaminopyridine are dissolved in a mixed solution of anhydrous dichloromethane and anhydrous pyridine at a ratio of 3–7:1; the mixture is stirred at a reaction temperature of 30–50°C under nitrogen protection; then diisopropylcarbodiimide is added to the resulting reaction mixture, and the reaction is carried out for 1–3 hours; after the reaction is completed, the resulting product is precipitated in cold anhydrous methanol, the precipitate is collected by centrifugation and dried to obtain polycholic acid-short-chain fatty acid polymer.

8. A nanomedicine formulation, characterized in that: This includes nanoparticles formed from a polymer as described in any one of claims 1-6, or pharmaceutically acceptable carriers.

9. The use of a polymer according to any one of claims 1-6 in the preparation of a medicament for treating or preventing a disease, wherein the disease is selected from the group consisting of: obesity, aging, diabetes, tumors, cardiovascular diseases, nervous system diseases, metabolic diseases, non-alcoholic fatty liver disease, or combinations thereof.

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