Use of farnesol x receptor agonist in preventing and / or treating fibrosis

Abstract

The present invention relates to the field of biotechnology, and particularly relates to use of a farnesol X receptor agonist in preventing and / or treating fibrosis. The present invention discloses for the first time that farnesol X receptor (FXR) agonists can inhibit the transcriptional activity of fibrosis-related genes and the protein expression of the fibrosis-related genes, and thus inhibit the transformation of fibroblasts into myofibroblasts, thereby significantly alleviating fibrotic conditions and achieving the prevention and / or treatment of fibrotic diseases.

Description

Application of Farnesol X Receptor Agonists in the Prevention and Treatment of Fibrosis Technical Field

[0001] This invention relates to the field of chemical medicine, and in particular to the application of farnesol X receptor agonists in the prevention and treatment of fibrosis. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic disease characterized by progressive extracellular matrix deposition, ultimately leading to respiratory failure and death. The pathogenesis of IPF involves several risk factors, including environmental exposure, smoking, chronic viral infections, and certain comorbidities. The pathological features of interstitial pulmonary fibrosis are honeycomb-like, traction bronchiectasis, and fibroblastic foci, representing abnormal remodeling of lung structure. Once the underlying pathology progresses to clinical and radiological abnormalities, the patient's prognosis is poor. Currently, treatment options for IPF patients remain limited, with lung transplantation being the only treatment that can significantly improve patient survival. After more than a decade of clinical trials, two drugs for IPF treatment—pirfenidone and nintedanib—have received FDA approval. While these drugs can slow the decline in lung function in IPF patients, they do not improve survival or quality of life and have serious toxic side effects. Therefore, there is an urgent need to find new targets and drugs for treating pulmonary fibrosis. Summary of the Invention

[0003] To address at least one of the above problems, this invention discloses an in vivo and in vitro evaluation of the antifibrotic activity of multiple farnesoid X receptor (FXR) agonists, thereby screening out FXR agonists with good preventive and therapeutic effects on fibrosis, especially pulmonary fibrosis.

[0004] A first aspect of the invention provides the use of a farnesol X receptor agonist in any one of a1)-a3):

[0005] a1) Prepare medicines for the prevention and / or treatment of fibrotic diseases;

[0006] a2) In vitro inhibition of fibroblast transformation into myofibroblasts;

[0007] a3) Prepare reagents for in vitro inhibition of fibroblast transformation into myofibroblasts.

[0008] In some embodiments, the farnesoid X receptor agonist includes any one or more combinations of TERN101, Cilofexor, PX20606, Nidufexor, Tropifexor, EDP305, Omesdafexor, or their precursors or pharmaceutically acceptable salts or derivatives thereof.

[0009] The use of farnesol X receptor agonists, their precursors, and / or pharmaceutically acceptable salts thereof in any of a1)-a3):

[0010] a1) Prepare medicines for the prevention and / or treatment of fibrotic diseases;

[0011] a2) In vitro inhibition of fibroblast transformation into myofibroblasts;

[0012] a3) Prepare reagents for in vitro inhibition of fibroblast transformation into myofibroblasts.

[0013] In some embodiments, the farnesoid X receptor agonist comprises A, wherein A is selected from any one of B, wherein B comprises: TERN101, Cilofexor, PX20606, Nidufexor, Omesdafexor (MET642), EDP305, Tropifexor.

[0014] In some implementations, B includes: TERN101, Cilfexor, PX20606, Nidufexor, Omesdafexor (MET642), and Tropifexor.

[0015] In some embodiments, the farnesoid X receptor agonist further comprises C, wherein C comprises at least one of B, and the component in C does not contain or is not a component of A.

[0016] In some embodiments, the pharmaceutically acceptable salt comprises at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0017] In some embodiments, the metal salt comprises at least one of alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and aluminum salts.

[0018] In some embodiments, the salt formed with an organic base comprises a salt formed with one or more of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0019] In some embodiments, the salt formed with the inorganic acid comprises a salt formed with one or more of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0020] In some embodiments, the salt formed with the organic acid comprises a salt formed with one or more of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0021] In some embodiments, the salt formed with the basic amino acid comprises a salt formed with one or more of the following basic amino acids: arginine, lysine, ornithine.

[0022] In some embodiments, the salt formed with the acidic amino acid comprises a salt formed with one or more of the following acidic amino acids: aspartic acid, glutamic acid.

[0023] In some embodiments, the drug described in a1) prevents and / or treats fibrotic diseases by at least one of b1)-b6);

[0024] b1) Inhibits the transformation of fibroblasts into myofibroblasts;

[0025] b2) Inhibit the expression of tissue fibrosis-related proteins;

[0026] b3) Inhibit signaling pathways related to tissue fibrosis;

[0027] b4) Improves symptoms of tissue fibrosis;

[0028] b5) Inhibits or improves inflammation caused by tissue fibrosis;

[0029] b6) Improve the survival rate of patients with tissue fibrosis.

[0030] In some embodiments, in a2) and a3), at least one of c1)-c2) inhibits the transformation of fibroblasts into myofibroblasts:

[0031] c1) Inhibits the expression of tissue fibrosis-related proteins;

[0032] c2) Inhibit signaling pathways related to tissue fibrosis.

[0033] In some embodiments, the drug described in a1) also contains other active ingredients for the prevention and / or treatment of fibrotic diseases.

[0034] In some embodiments, the reagent described in a3) further comprises other active ingredients that inhibit the in vitro conversion of fibroblasts into myofibroblasts.

[0035] In some embodiments, the tissue fibrosis-related proteins (i.e., b2, c1) include one or more of the following: fibronectin (Fn1), type I collagen α1 (COL1A1), α-smooth muscle actin (α-SMA), and phosphorylated SMAD3.

[0036] In some embodiments, the inhibition of the expression of tissue fibrosis-related proteins (i.e., b2, c1) includes inhibiting the expression of tissue fibrosis-related proteins (preferably fibronectin (Fn1), type I collagen α1 (COL1A1), and / or α-smooth muscle actin (α-SMA)) at the transcriptional level and / or inhibiting the expression of tissue fibrosis-related proteins (preferably fibronectin (Fn1), type I collagen α1 (COL1A1), and / or phosphorylated SMAD3) at the translational level.

[0037] In some embodiments, the tissue fibrosis-related signaling pathway (i.e., b3, c2) is the TGF-β / SMAD signaling pathway.

[0038] In some embodiments, the fibrosis described in a1) includes one or more of the following: renal fibrosis, pulmonary fibrosis, liver fibrosis, myocardial fibrosis, bone marrow fibrosis, adipose tissue fibrosis, pancreatic fibrosis, retroperitoneal fibrosis, mesenteric fibrosis, breast fibrosis, cystic fibrosis, gastrointestinal tract fibrosis, or skin fibrosis.

[0039] In some embodiments, the fibrosis described in a1) includes pulmonary fibrosis.

[0040] In some embodiments, the pulmonary fibrosis includes one or more of the following: idiopathic pulmonary fibrosis, sarcoidosis, cystic fibrosis, radiation-induced fibrosis, familial pulmonary fibrosis, silicosis, asbestos deposition, coal miner's pneumoconiosis, carbon monoxide poisoning, allergic pneumonia, interstitial lung disease, pulmonary hypertension or chronic obstructive pulmonary disease, nonspecific interstitial pneumonia, common interstitial pneumonia, and airway fibrosis.

[0041] In some embodiments, the symptoms of pulmonary fibrosis include one or more of the following: collagen deposition in the lungs, pulmonary scarring (fibrosis), organ parenchyma, and thickening of the alveolar walls.

[0042] In some implementations, the drug described in a1) further comprises pharmaceutically acceptable excipients.

[0043] In some embodiments, the pharmaceutically acceptable excipients include at least one of diluents, excipients, binders, humectants, surfactants, lubricants, and disintegrants.

[0044] In some implementations, the dosage form of the drug described in a1) is a dosage form suitable for children or for adults.

[0045] In some embodiments, the dosage form is selected from gastrointestinal dosage forms or non-gastrointestinal dosage forms.

[0046] In some embodiments, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0047] In some embodiments, the non-gastrointestinal dosage form includes at least one of the following: injectable dosage forms (e.g., injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (e.g., sprays, aerosols, powder inhalers, etc.); skin dosage forms (e.g., topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, films, etc.); and cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0048] In some implementations, the drug described in a1) is administered to animals.

[0049] In some implementations, the animal is a mammal; further selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and further still, humans, including minors, adults, and the elderly.

[0050] In some implementations, the reagent described in a3) further comprises pharmaceutically acceptable excipients.

[0051] In some implementations, the application described in a2) does not involve the treatment of the disease.

[0052] In some embodiments, the application described in a2) includes the step of treating the fibroblasts with the farnesoid X receptor agonist, its precursor, and / or a pharmaceutically acceptable salt thereof, and optionally, other active ingredients that inhibit the in vitro conversion of fibroblasts into myofibroblasts.

[0053] In some embodiments, the fibroblasts described in a2) and a3) are TGF-β-treated fibroblasts.

[0054] In some implementations, the fibroblasts described in a2) and a3) are derived from animals.

[0055] In some embodiments, the animal is a mammal; further selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and even further selected from mice (preferably mice).

[0056] In some implementations, the fibroblasts described in a2) and a3) are derived from the kidney, lung, liver, myocardium, bone marrow, adipose tissue, pancreas, peritoneum, mesentery, mammary gland, cystic tissue, digestive tract, or skin; further, they are derived from the lung.

[0057] In some embodiments, the final concentration of the farnesol X receptor agonist, its precursor, and / or its pharmaceutically acceptable salt when treating fibroblasts is 1-100 μM; further, 5-15 μM; and even further, 8-12 μM.

[0058] In some implementations, the processing time is 24-72 hours; further, 36-60 hours; and even further, 46-50 hours.

[0059] A second aspect of the present invention provides a method for preventing and / or treating fibrotic diseases by administering a drug to a subject, said drug being the drug described in the first aspect of the present invention.

[0060] In some embodiments, the fibrosis includes one or more of the following: renal fibrosis, pulmonary fibrosis, hepatic fibrosis, myocardial fibrosis, bone marrow fibrosis, adipose tissue fibrosis, pancreatic fibrosis, retroperitoneal fibrosis, mesenteric fibrosis, mammary fibrosis, cystic fibrosis, gastrointestinal tract fibrosis, or skin fibrosis.

[0061] In some embodiments, the fibrosis includes pulmonary fibrosis.

[0062] In some embodiments, the pulmonary fibrosis includes one or more of the following: idiopathic pulmonary fibrosis, sarcoidosis, cystic fibrosis, radiation-induced fibrosis, familial pulmonary fibrosis, silicosis, asbestos deposition, coal miner's pneumoconiosis, carbon monoxide poisoning, allergic pneumonia, interstitial lung disease, pulmonary hypertension or chronic obstructive pulmonary disease, nonspecific interstitial pneumonia, common interstitial pneumonia, and airway fibrosis.

[0063] In some embodiments, the symptoms of pulmonary fibrosis include one or more of the following: collagen deposition in the lungs, pulmonary scarring (fibrosis), organ parenchyma, and thickening of the alveolar walls.

[0064] In some embodiments, the subject is an animal; further, a mammal; even further, selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and even further, humans, including minors, adults, and the elderly.

[0065] In some embodiments, the dosage of the drug is 1-30 mg / kg per dose for mice; the dose per unit body weight for different subjects can be calculated using equivalent dose conversion relationships. For example, the effective dose for humans can be derived from the dose for experimental animals based on the equivalent dose conversion relationships known to those skilled in the art (usually referring to the guidance of drug regulatory agencies such as the FDA and SFDA, or "Huang Jihan et al. Equivalent dose conversion between animals and between animals and humans in pharmacological experiments. Chinese Journal of Clinical Pharmacology and Therapeutics, 2004 Sep; 9(9): 1069-1072").

[0066] According to one aspect of the present invention, the use of a farnesoid X receptor agonist in the preparation of a medicament or formulation is provided for: (A1) preventing or treating fibrotic diseases; and / or, (A2) inhibiting the conversion of fibroblasts into myofibroblasts in vitro.

[0067] In some embodiments, the farnesoid X receptor agonist includes any one or more combinations of TERN101, Cilofexor, PX20606, Nidufexor, Omesdafexor (MET642), EDP305, Tropifexor, or their precursors or pharmaceutically acceptable salts or derivatives thereof.

[0068] In some embodiments, the drug or preparation is used for one or more of the following: (B1) inhibiting the transformation of fibroblasts into myofibroblasts in vitro; (B2) inhibiting the expression of tissue fibrosis-related proteins; (B3) inhibiting tissue fibrosis-related signaling pathways; (B4) improving symptoms of tissue fibrosis; (B5) inhibiting or improving inflammation caused by tissue fibrosis; (B6) improving the survival rate of patients with tissue fibrosis; (B7) preventing or treating tissue fibrosis.

[0069] In some embodiments, the tissue fibrosis-related proteins include one or more of the following: fibronectin (Fn1), type I collagen α1 (COL1A1), and α-smooth muscle actin (α-SMA).

[0070] In some embodiments, the inhibition of tissue fibrosis-related protein expression includes inhibiting the expression of tissue fibrosis-related proteins at both the transcriptional and translational levels.

[0071] In some embodiments, the tissue fibrosis-related signaling pathway is the TGF-β / SMAD signaling pathway.

[0072] In some embodiments, the farnesol X receptor agonist includes tablets, capsules, solutions, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, or patches.

[0073] In some embodiments, the farnesoid X receptor agonist is administered via intramuscular injection, intranasal, intratracheal, gastric, rectal, mucosal presentation, intravenous delivery, or intradermal or subcutaneous administration.

[0074] In some embodiments, the fibrosis includes one or more of the following: renal fibrosis, pulmonary fibrosis, hepatic fibrosis, myocardial fibrosis, bone marrow fibrosis, adipose tissue fibrosis, pancreatic fibrosis, retroperitoneal fibrosis, mesenteric fibrosis, mammary fibrosis, cystic fibrosis, gastrointestinal tract fibrosis, or skin fibrosis.

[0075] In some embodiments, the fibrosis includes pulmonary fibrosis.

[0076] In some embodiments, the pulmonary fibrosis includes one or more of the following: idiopathic pulmonary fibrosis, sarcoidosis, cystic fibrosis, radiation-induced fibrosis, familial pulmonary fibrosis, silicosis, asbestos deposition, coal miner's pneumoconiosis, carbon monoxide poisoning, allergic pneumonia, interstitial lung disease, pulmonary hypertension or chronic obstructive pulmonary disease, nonspecific interstitial pneumonia, common interstitial pneumonia, and airway fibrosis.

[0077] In some embodiments, the symptoms of pulmonary fibrosis include one or more of the following: collagen deposition in the lungs, pulmonary scarring (fibrosis), organ parenchyma, and thickening of the alveolar walls.

[0078] According to one aspect of the present invention, an in vitro non-diagnostic, non-therapeutic method for inhibiting the transdifferentiation of lung fibroblasts into myofibroblasts is provided, comprising the step of contacting the lung fibroblasts with a therapeutically effective amount of the farnesol X receptor agonist. Beneficial effects:

[0079] This invention discloses for the first time that farnesoid X receptor (FXR) agonists can inhibit the transcriptional activity of fibrosis-related genes and the protein expression of fibrosis-related genes, inhibit the transformation of fibroblasts into myofibroblasts, thereby significantly improving fibrosis symptoms and achieving the prevention and / or treatment of fibrosis.

[0080] The farnesoid X receptor (FXR) agonist disclosed herein can inhibit the transcriptional activity of fibrosis-related genes and their protein expression to varying degrees, and can significantly improve fibrosis symptoms. The farnesoid X receptor agonist disclosed herein can be used to prepare drugs for the prevention or treatment of pulmonary fibrosis. Attached Figure Description

[0081] Figure 1 shows the TR-FRET experimental validation of the in vitro activity of the FXR clinical compound.

[0082] Figure 2 shows the effect of RT-qPCR on the transcriptional activity of fibrosis-related genes by FXR clinical compounds.

[0083] Figure 3 shows the effect of Western blot analysis of the clinical compound FXR on the expression of fibrosis-related gene proteins.

[0084] Figure 4 illustrates the effect of the FXR clinical compound on the progression of pulmonary fibrosis in mice. Figure 4A shows the experimental procedure; Figure 4B shows the effect of the FXR clinical compound on the progression of pulmonary fibrosis in mice. Detailed Implementation

[0085] This invention primarily targets the farnesoid X receptor and investigates the pharmacological effects of a clinical-stage farnesoid X receptor compound on pulmonary fibrosis. It is the first time that the anti-pulmonary fibrosis activity of a clinical-stage farnesoid X receptor (FXR) compound has been demonstrated in vitro and in vivo.

[0086] This invention uses time-resolved fluorescence resonance energy transfer (TR-FRET) and luciferase reporter gene assays to demonstrate the activation effect of the compounds on the farnesoid X receptor (FXR). RT-qPCR was used to detect that a series of farnesoid X receptor (FXR) compounds could inhibit the mRNA levels of pulmonary fibrosis characterizing genes (FN1, COL1A1, α-SMA). Western blot analysis showed that the farnesoid X receptor (FXR) compounds could inhibit the protein levels of pulmonary fibrosis characterizing genes (FN1, COL1A1, α-SMA). This invention also uses a bleomycin (BLM)-induced pulmonary fibrosis mouse model to verify that the series of compounds provided in this invention that can activate the farnesoid X receptor (FXR) can delay the progression of pulmonary fibrosis in vivo and improve the symptoms of pulmonary fibrosis.

[0087] definition

[0088] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0089] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0090] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0091] The term "fibronectin (Fn1)" used in this article refers to a multifunctional extracellular matrix protein and a high-molecular-weight glycoprotein. It can bind to various components on the cell surface and in the extracellular matrix, including collagen, fibrin, heparin, DNA, and actin, and is widely distributed on the cell surface, extracellular fluid, and basement membranes associated with connective tissue. Fn1 possesses a wide range of biological activities, including promoting cell adhesion, cell-fiber and matrix connections, and phagocytic function of macrophages; it is also closely related to tissue healing, inflammation, fibrosis, and sclerosis. Therefore, changes in Fn1 levels are closely related to the severity and prognosis of various clinical diseases.

[0092] The term "type I collagen α1 (COL1A1)" used in this article refers to a member of the collagen family that is involved in the epithelial-mesenchymal transition (EMT), which is closely related to the development of malignant tumors. Fibrotic fibroblasts are mainly enriched in fibrotic lesions and can be labeled with COL1A1.

[0093] The term "α-smooth muscle actin (α-SMA)" used in this article is encoded by the ACTA2 gene and is a subtype of vascular smooth muscle actin. It is mainly expressed in vascular smooth muscle cells and participates in vascular movement and contraction. In addition to being expressed in smooth muscle cells around the bronchi and large blood vessels, α-SMA is also expressed in fibroblasts, and its expression increases with the progression of pulmonary fibrosis.

[0094] The term "farnesoid X receptor (FXR)" used in this article belongs to the NR1H4 nuclear receptor family and plays an important role in regulating bile acid, glucose, lipid metabolism, and inflammatory responses. Bile acids are natural ligands of FXR, therefore FXR can also be referred to as a bile acid receptor.

[0095] The term "TR-FRET" used in this article refers to time-resolved fluorescence resonance energy transfer, a technique that combines fluorescence resonance energy transfer (FRET) and time-resolved (TR) methods. TR-FRET utilizes energy transfer from two fluorophores: a donor (a lanthanide element such as europium or terbium) and an acceptor (a fluorophore such as FITC or 5-FAM). The donor's emission spectrum overlaps with the acceptor's excitation spectrum. The donor is excited by an external energy source (e.g., a laser), and if it is within a sufficiently close distance to the acceptor, it can resonantly transfer energy to the acceptor. The acceptor is then excited, emitting light at a specific wavelength. Coupled to two biomolecules capable of specific binding, the binding of these biomolecules brings the donor and acceptor closer, resulting in energy transfer. TR technology leverages the unique properties of lanthanides in the rare earth elements, whose fluorescence lasts longer than ordinary fluorescence. The half-life of ordinary fluorescence is in the nanosecond range, while that of lanthanides is in the millisecond range, a difference of six orders of magnitude. Therefore, during detection, there is a time delay of approximately 100 microseconds in the TR (transient fluorescence) signal, making the ordinary background fluorescence signal in the reaction system almost zero. Thus, the background of TR is very low, accurately reflecting the actual situation of the sample.

[0096] As used herein, the terms "therapeutic effective amount (or dose)" or "effective amount (or dose)" refer to an amount of compound sufficient to cause a statistically significant improvement in one or more symptoms of the disease being treated. The precise amount depends on numerous factors, such as the activity of the composition, the method of delivery used, the immunostimulatory capacity of the composition, the intended patient and patient considerations, etc., and can be readily determined by those skilled in the art. Therapeutic effects may include, directly or indirectly, the reduction of one or more symptoms of the disease.

[0097] As used herein, the term "pharmaceuticalally acceptable" means that it can be administered to humans and / or other animals as subjects without producing excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to auxiliary materials that coexist with the active ingredient in a pharmaceutical preparation without producing excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH adjusters, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceuticalally acceptable excipient" refers to a highly safe excipient suitable for a specific pharmaceutical preparation and routinely used in pharmaceutical practice. This includes, but is not limited to, liposomes, liposomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

[0098] The term "pharmaceutically acceptable salt" as used herein refers to, for example, metal salts and control amine salts, where the cation does not significantly contribute to the toxicity or biological activity of the salt. However, other salts may be used in the separation or purification steps employed during preparation and are therefore covered within the scope of this invention.

[0099] The terms “pulmonary fibrosis,” “interstitial lung disease (ILD),” or “interstitial pulmonary fibrosis” as used in this article encompass more than 130 types of chronic lung diseases, named for their hardening of the lungs by damaging lung tissue, causing inflammation in the alveolar walls, and scarring or fibrosis of the pulmonary interstitium (the tissue between alveoli). Apnea is the first symptom of these diseases, and a dry cough may also occur. Symptoms and X-rays are often insufficient to accurately distinguish between different types of pulmonary fibrosis. In some patients with pulmonary fibrosis, the cause is known, while in others it is unknown or idiopathic. The course of this disease is generally unpredictable. Its progression includes thickening and hardening of lung tissue, inflammation, and dyspnea. Some patients require supplemental oxygen as part of their treatment. Pulmonary fibrosis typically includes any of the following features: (a) collagen deposition in the lungs, (b) pulmonary scarring (fibrosis) (including in the alveoli and interstitial spaces), and / or (c) the presence of areas of particularly thickened alveolar walls, one or more of which may contribute to chronic stiffening of the lungs and / or a decreased ability of the lung tissue to transport oxygen.

[0100] As used in this article, the term "subject" can refer to any organism capable of generating a cellular immune response, such as humans, pets, livestock, show animals, zoo specimens, or other animals. For example, a subject can be a human, a non-human primate, a dog, a cat, a rabbit, a rat, a mouse, a guinea pig, a horse, a cow, a sheep, a goat, a pig, etc.

[0101] As used herein, the term "treatment" refers to a method for obtaining a beneficial or desired outcome (including clinical outcomes) through the use of compounds or compositions of the present invention. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: reducing the severity and / or frequency of one or more symptoms caused by a disease, disorder, or condition; reducing the degree of a disease, disorder, or condition or causing its remission; stabilizing a disease, disorder, or condition (e.g., preventing or delaying the worsening of a disease, disorder, or condition); delaying or slowing the progression of a disease, disorder, or condition; improving the state of a disease, disorder, or condition; reducing the dosage of one or more other medicines required to treat a disease, disorder, or condition; and / or improving quality of life.

[0102] As used in this article, the term "effective dose" refers to the effective dose and time period required to achieve the desired therapeutic or preventative effect.

[0103] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. The actual scope of protection of this invention is set forth in the claims. In the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications. Unless otherwise specified, the equipment, instruments, reagents, and / or kits used in the following embodiments are commercially available or obtained through conventional methods known to those skilled in the art.

[0104] Example

[0105] The compounds used in the examples and their structures are shown in Table 1.

[0106] Table 1: Structural Formulas of Compounds

[0107] Example 1. Evaluation of the activity of clinical drugs targeting the farnesol X receptor using the TR-FRET assay.

[0108] Experimental Procedure: Step 1: Add phosphate buffer containing 0.005% Tween 20 (20mM Phosphate Buffer (KH2PO4, K2HPO4) + 50mM KCl + 5mM TECP + 0.5mM EDTA) to the EP tube, then add the following in sequence to a final concentration of 40nM. His-FXR-LBD protein (amino acid sequence: MAHHHHHHVDDDDKMLEVLFQGPELTPDQQTLLHFIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATNHVQVLVEFTKKLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPSGHSDLLEERIRNSGISDEYITPMFSFYKSIGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIHQPENPQHFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQ, SEQ ID NO: 1), with a final concentration of 4 nM Anti-His-Tb (ThermoFisher, catalog number PV5863), and SRC2-2 accessory peptide (Nanjing Peptide Valley, sequence: FITC-Ahx-LKEKHKILHRLLQDSSSPV, SEQ ID NO: 1), with a final concentration of 400 nM. NO: 2), after slowly inverting and mixing, add 79.2 μL of the resulting mixed solution to each well of a 96-well plate. Second step: Prepare compounds with different concentration gradients. Dilute seven compounds (TERN101, Cilofexor, PX20606, Nidufexor, Tropifexor, Omesdafexor (MET642), and EDP305) at an initial concentration of 2 mM to the 12th well in a 1:3 gradient. Add 0.8 μL of each to the mixed solution obtained in the first step and mix well. Third step: Thoroughly mix the mixed solution from the first step and the compounds added in the second step in the 96-well plate, transfer 22.5 μL to each well of a 384-well black plate, with 3 replicates per group. Incubate the 384-well black plate at room temperature in the dark for 15 min and read the values ​​using a microplate reader. The excitation and emission wavelengths are 495 nm and 520 nm, respectively. Plot the graph using Graphpad Prism 8.0 software to obtain the 520 / 495 ratio curve shown in Figure 1. As can be seen from the data results in Figure 1, all seven FXR ligands exhibit good activation activity.

[0109] Example 2: Pharmacological activity of farnesol X receptor clinical drugs against pulmonary fibrosis

[0110] Experimental Procedure: Primary lung fibroblasts were extracted from the lung tissue of 6-8 week old C57BL / 6 mice (Vitollife Laboratory Animal Technology Co., Ltd.) and cultured in DMEMF12 medium (Gibco) containing 10% FBS for later use. Experimental groups were set up as follows: control group (DMSO), TGF-β group, and groups treated with TGF-β in combination with seven FXR agonists (TERN101, Cilofexor, PX20606, Nidufexor, Tropifexor, Omesdafexor (MET642), and EDP305) (compound groups) (cells were treated with TGF-β and the compounds simultaneously for 48 hours). The treatment method for the compound group was as follows: primary lung fibroblasts were seeded into six-well plates. When the cells adhered and reached a density of 70%, TGF-β (Proteintech, Cat) was added to a final concentration of 10 ng / mL. TGF-β (No. HZ-1011) stimulates fibroblasts to transform into myofibroblasts, at which point fibrosis-related genes are significantly upregulated, mimicking the occurrence of fibrosis at the cellular level. Simultaneously, an FXR agonist at a final concentration of 10 μM is added. The treatment methods for the control group and the compound group differ as follows: TGF-β is replaced with an equal volume of DMEMF12 medium containing 10% FBS, and the FXR agonist is replaced with an equal volume of DMSO. The treatment methods for the TGF-β group differ as follows: the FXR agonist is replaced with an equal volume of DMSO. After culturing in a cell culture incubator for 48 h, the cells are washed three times with sterile PBS, with the last wash removing any residual PBS. Cells are collected with 1 mL of trizol (Takara), and 200 μL of chloroform is added to an EP tube. The tube is gently inverted 10-20 times to mix, and incubated at room temperature for 5 min. At this point, the solution separates into three layers (upper layer: RNA, middle layer: DNA, lower layer: organic solvent). The cells are then centrifuged at 12000 rpm for 15 min at 4 °C. Transfer 400 μL of the supernatant to a new EP tube, add an equal volume of isopropanol, invert 10-20 times to mix, and incubate on ice for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C. Discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, and centrifuge at 12000 rpm for 5 min at 4°C. Discard the supernatant, and dry the precipitate at room temperature until it becomes clear. Dissolve the precipitate in an appropriate amount of RNase-free water, mix well, and then measure the RNA concentration.

[0111] 1 μg of total RNA was reverse transcribed into cDNA. The reaction solution was prepared according to the experimental procedures of the SYBR Green Pro Taq HS premixed qPCR kit from Acrylic Acid. Three replicates were set for each sample. Ct values ​​were detected using the qPCR assay. The calculation method was: Expression ratio = 2 - [(Target gene Ct in experimental group - Internal reference gene Ct in experimental group) - (Target gene Ct in control group - Internal reference gene Ct in control group)]. The primer sequences used in the experiment are shown in Table 2. The experimental results are shown in Figure 2. In this study, a series of compounds of the farnesol X receptor showed significant inhibitory effects on the transcriptional activity of fibrosis-related genes (FN1, Col1a1, α-SMA), with slight variations depending on the compound.

[0112] Table 2. Primer Sequences

[0113] Example 3: Pharmacological activity of farnesol X receptor clinical drugs against pulmonary fibrosis

[0114] Experimental Procedure: Primary lung fibroblasts were extracted from the lung tissue of 6-8 week old C57BL / 6 mice (Vitollife Laboratory Animal Technology Co., Ltd.) and cultured in DMEMF12 medium (Gibco) containing 10% FBS for later use. Experimental groups were set up as follows: control group (CTL), TGF-β group, and groups treated with TGF-β in combination with seven FXR agonists (TERN101, Cilofexor, PX20606, Nidufexor, Tropifexor, Omesdafexor (MET642), and EDP305) (compound groups) (cells were treated with TGF-β and the compounds simultaneously for 48 hours). The treatment method for the compound group was as follows: Primary lung fibroblasts were seeded into six-well plates. When the cells adhered and reached a density of 70%, TGF-β (Proteintech, Cat) was added to a final concentration of 10 ng / mL. TGF-β (No. HZ-1011) stimulates fibroblasts to transform into myofibroblasts, at which point fibrosis-related genes are significantly upregulated, thus mimicking the occurrence of fibrosis at the cellular level. Simultaneously, an FXR agonist at a final concentration of 10 μM is added. The treatment methods for the control group and the compound group differ as follows: TGF-β is replaced with an equal volume of DMEMF12 medium containing 10% FBS, and the FXR agonist is replaced with an equal volume of DMSO. The treatment methods for the TGF-β group differ as follows: the FXR agonist is replaced with an equal volume of DMSO. After culturing in a cell culture incubator for 48 hours, the cells were washed with sterile PBS, and RIPA lysis buffer (Proteintech, PR20035) was added, along with protease inhibitors (Selleck Chemicals) and phosphatase inhibitors (Selleck Chemicals). The cells were lysed on ice for 30 min, centrifuged at 12000 rpm for 10 min, and the supernatant was used for BCA quantification (Thermo Fisher). A suitable amount of 5× loading buffer was added, and the cells were boiled at 95°C for 10 min. The resulting samples were then subjected to Western blot experiments. The results, as shown in Figure 3, indicate that a series of FXR compounds inhibited the protein expression of fibrosis-related genes to varying degrees. Specifically, these compounds significantly reduced the protein expression levels of key proteins closely related to the fibrosis process, including phosphorylation of fibronectin-1 (FN1), collagen α1 chain (Col1a1), and / or SMAD3. This downregulation of protein expression suggests that the studied compounds slowed or prevented the development of fibrosis by intervening in fibrosis-related signaling pathways. This discovery provides important molecular mechanism evidence for the development of novel antifibrotic drugs and lays the experimental foundation for future clinical applications.

[0115] Example 4: Evaluation of the pharmacological activity of a clinical compound targeting the farnesoid X receptor in inhibiting pulmonary fibrosis in mice.

[0116] Sufficient male C57 / BL6 mice aged 6-8 weeks were prepared. After respiratory anesthesia, 50 μL of bleomycin BLM (Selleck Chemicals) was administered intratracheally at a dose of 3 mg / kg. Bleomycin is a multi-component antibiotic composed of basic glycopeptides produced by Streptomyces verticillata that can induce pulmonary fibrosis. Starting the day after bleomycin treatment, seven compounds were continuously administered by gavage every morning for 21 days: the seven compounds included six FXR modifiers (TERN101, Cilofexor, PX20606, Nidufexor, Tropifexor, and Omesdafexor) and the yang-ginseng compound nintedanib, all dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na). The daily dosages were TERN101 (10 mg / kg mouse), Cilofexor (30 mg / kg mouse), PX20606 (10 mg / kg mouse), Nidufexor (10 mg / kg mouse), Tropifexor (1 mg / kg mouse), Omesdafexor (10 mg / kg mouse), and Nintedanib (10 mg / kg mouse). After administration, mouse lung tissue was fixed in paraformaldehyde and stained with hematoxylin and eosin (HE) to assess pathological changes in the lung tissue. The experimental flowchart is shown in Figure 4A. The experimental results are shown in Figure 4B. After nebulized bleomycin, the alveolar walls of the mice were significantly thickened, the lungs were severely consolidated, and collagen was deposited in the lungs, showing symptoms of pulmonary fibrosis. After treatment with the seven compounds, the lungs of the mice in the treatment group showed varying degrees of improvement, and the lung morphology approached normal, with treatment effects comparable to those of the Yangshen compound nintedanib.

[0117] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Application of farnesol X receptor agonists in any of a1)-a3): a1) Prepare medicines for the prevention and / or treatment of fibrotic diseases; a2) In vitro inhibition of fibroblast transformation into myofibroblasts; a3) Prepare reagents for in vitro inhibition of fibroblast transformation into myofibroblasts.

2. The application according to claim 1, characterized in that, The farnesoid X receptor agonists include any one or more combinations of TERN101, Cilofexor, PX20606, Nidufexor, Tropifexor, EDP305, Omesdafexor, or their precursors or pharmaceutically acceptable salts or derivatives.

3. The application according to any one of claims 1-2, characterized in that, The medicine described in a1) also contains other active ingredients for the prevention and / or treatment of fibrotic diseases; and / or The reagent described in a3) also contains other active ingredients that inhibit the in vitro transformation of fibroblasts into myofibroblasts.

4. The application according to any one of claims 1-3, characterized in that, The drug described in a1) also contains pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients include at least one of diluents, excipients, binders, humectants, surfactants, lubricants, and disintegrants.

6. The application according to any one of claims 1-5, characterized in that, The drug dosage form described in a1) is selected from gastrointestinal dosage forms or non-gastrointestinal dosage forms; Preferably, the gastrointestinal dosage form comprises at least one of the following: powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; and / or The non-gastrointestinal dosage forms include at least one of the following: injectable dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

7. The application according to any one of claims 1-6, characterized in that, The application described in a2) includes the following steps: treating the fibroblasts with the farnesoid X receptor agonist and, optionally, other active ingredients that inhibit the in vitro conversion of fibroblasts into myofibroblasts; The fibroblasts described in and / or a2) and a3) are derived from animals.

8. The application according to any one of claims 1-7, characterized in that, The drug described in a1) prevents and / or treats fibrotic diseases by at least one of b1)-b6); b1) Inhibits the transformation of fibroblasts into myofibroblasts; b2) Inhibit the expression of tissue fibrosis-related proteins; b3) Inhibit signaling pathways related to tissue fibrosis; b4) Improves symptoms of tissue fibrosis; b5) Inhibits or improves inflammation caused by tissue fibrosis; b6) Improve the survival rate of patients with tissue fibrosis; and / or a2) and a3) inhibit the transformation of fibroblasts into myofibroblasts through at least one of c1)-c2): c1) Inhibits the expression of tissue fibrosis-related proteins; c2) Inhibit signaling pathways related to tissue fibrosis.

9. The application according to claim 8, characterized in that, The tissue fibrosis-related proteins include one or more of the following: fibronectin, type I collagen α1, α-smooth muscle actin, and phosphorylated SMAD3. and / or The inhibition of tissue fibrosis-related protein expression includes inhibiting the expression of tissue fibrosis-related proteins at the transcriptional level and / or at the translational level; and / or The tissue fibrosis-related signaling pathway is the TGF-β / SMAD signaling pathway.

10. The application according to any one of claims 1-9, characterized in that, The fibrosis described in a1) includes one or more of the following: renal fibrosis, pulmonary fibrosis, hepatic fibrosis, myocardial fibrosis, bone marrow fibrosis, adipose tissue fibrosis, pancreatic fibrosis, retroperitoneal fibrosis, mesenteric fibrosis, breast fibrosis, cystic fibrosis, gastrointestinal fibrosis, or skin fibrosis; further including pulmonary fibrosis.

11. The application according to claim 10, characterized in that, The pulmonary fibrosis includes one or more of the following: idiopathic pulmonary fibrosis, sarcoidosis, cystic fibrosis, radiation-induced fibrosis, familial pulmonary fibrosis, silicosis, asbestos deposition, coal miner's pneumoconiosis, carbon monoxide poisoning, allergic pneumonia, interstitial lung disease, pulmonary hypertension or chronic obstructive pulmonary disease, nonspecific interstitial pneumonia, common interstitial pneumonia, and airway fibrosis; and / or The symptoms of pulmonary fibrosis include one or more of the following: collagen deposition in the lungs, pulmonary scarring, organ parenchyma, and thickening of the alveolar walls.

12. A method for preventing and / or treating fibrotic diseases, comprising administering a drug to a subject, said drug being the drug described in any one of claims 1-11.

13. The method according to claim 12, characterized in that, The fibrosis includes one or more of the following: renal fibrosis, pulmonary fibrosis, hepatic fibrosis, myocardial fibrosis, bone marrow fibrosis, adipose tissue fibrosis, pancreatic fibrosis, retroperitoneal fibrosis, mesenteric fibrosis, breast fibrosis, cystic fibrosis, gastrointestinal tract fibrosis, or skin fibrosis; further including pulmonary fibrosis.

14. The method according to claim 13, characterized in that, The pulmonary fibrosis includes one or more of the following: idiopathic pulmonary fibrosis, sarcoidosis, cystic fibrosis, radiation-induced fibrosis, familial pulmonary fibrosis, silicosis, asbestos deposition, coal miner's pneumoconiosis, carbon monoxide poisoning, allergic pneumonia, interstitial lung disease, pulmonary hypertension or chronic obstructive pulmonary disease, nonspecific interstitial pneumonia, common interstitial pneumonia, and airway fibrosis; and / or The symptoms of pulmonary fibrosis include one or more of the following: collagen deposition in the lungs, pulmonary scarring, organ parenchyma, and thickening of the alveolar walls.

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