Application of intestinal FXR protein and its gene as drug targets in the preparation of drugs for treating osteoarthritis

Abstract

This invention relates to the use of farnesoid X receptor (FXR) antagonists in the preparation of medicaments for the prevention and / or treatment of osteoarthritis (OA). Specifically, this invention provides a novel therapeutic target for OA: the intestinal FXR protein or the intestinal gene encoding the FXR protein (NR1H4). By targeting the intestinal FXR protein or NR1H4 gene to inhibit the activity of the intestinal FXR protein, inhibit the expression of the intestinal NR1H4 gene, or interfere with the biological function of the intestinal FXR protein, OA can be treated, providing a new strategy for OA treatment.

Description

Technical Field

[0001] This invention relates to the field of osteoarthritis (OA) treatment. More specifically, this invention relates to the use of the intestinal FXR protein and its gene NR1H4 as drug targets in the preparation of drugs for treating OA. Background Technology

[0002] Osteoarthritis (OA) is the most common type of arthritis, affecting approximately 595 million people worldwide. It is one of the leading causes of disability, economic loss, and social disruption in people over 50, and its incidence continues to rise. OA is influenced by multiple factors, including genetics and environment, and its pathogenesis remains unclear. Currently, there are no effective intervention targets for OA treatment, therefore no marketed drugs worldwide can safely and effectively slow disease progression. Treatment primarily focuses on symptomatic relief such as pain management and joint replacement. Therefore, there is an urgent need to identify new intervention targets and develop drugs based on these targets. Furthermore, due to the low bioavailability of oral medications within the joint cavity, many potential treatment options currently being developed involve intra-articular injection. However, this is an invasive procedure with low patient compliance and risks of local infection and bleeding.

[0003] The farnesoid X receptor (FXR) is a member of the nuclear receptor (NR) superfamily, encoded in humans by nuclear receptor subfamily 1, group H, member 4 (NR1H4). FXR is primarily expressed in the liver and small intestine, influencing various metabolic pathways through its specific target genes. It regulates bile acid synthesis and homeostasis, glucose and lipid metabolism, and plays a crucial role in intestinal bacterial growth and liver regeneration. Previous reports have indicated its regulatory role in diseases such as diabetes, obesity, and atherosclerosis. An article has reported that intraperitoneal injection of the FXR agonist GW4064 can delay the progression of chondrodysplasia in mice by attenuating osteoclast-mediated abnormal subchondral bone loss (see FASEB J 2022,36:e22243. doi:10.1096 / fj.202101717R). Summary of the Invention

[0004] The inventors have unexpectedly discovered that FXR antagonists can be used to prevent and / or treat osteoarthritis (OA). Specifically, the inventors have used methods such as oral administration or gene knockout of FXR antagonists to reduce intestinal FXR levels, delay OA progression, and provide new intervention targets (especially intestinal FXR proteins and the NR1H4 gene) and new potential therapeutic agents (FXR antagonists) for OA treatment. In particular, the target discovered in this invention can be intestinal FXR, and oral intervention with FXR antagonists can directly act on this target.

[0005] Therefore, according to one aspect of the invention, the present invention provides the use of FXR antagonists in the preparation of medicaments for the prevention and / or treatment of osteoarthritis (OA).

[0006] In one embodiment, the antagonist has one or more of the following effects: (1) reducing or inhibiting the activity of FXR protein; (2) reducing or inhibiting the expression of NR1H4 and / or FXR protein; and / or (3) reducing or inhibiting the biological function of FXR protein.

[0007] In another embodiment, the antagonist is selected from the group consisting of: FXR-binding proteins, FXR-binding peptides, monoclonal / polyclonal antibodies against FXR, small molecule compound inhibitors, gapmers against the NR1H4 gene, antisense RNA, siRNA, esiRNA, shRNA, miRNA, RNA aptamers, TALEN, CRISPR, zinc finger nucleases, Cre-loxP systems (conditional gene knockout systems), or combinations thereof.

[0008] In another embodiment, the small molecule compound inhibitor is selected from the group consisting of:

[0009] 1) Glycoursodeoxycholic acid (GUDCA), tauro-β-muricholic acid (TβMCA), Gly-β-MCA (CAS 66225-78-3) or their medicinal salts;

[0010] 2) Compounds selected from the following general formulas or their pharmaceutical salts,

[0011]

[0012] Where R 1 and R 2 Independently selected from hydrogen, alkyl, and C(=O)R 3 ,

[0013] R 4 Selected from hydrogen, alkyl and C(=O)R 3 ,

[0014] X is selected from C=O and CH2.

[0015] Y is selected from CH2, NR 5 , O, S, SO, SO2 and Se,

[0016] Or X and Y together form C=C,

[0017] Z is selected from COOR 6 SO3R 7 P(=O)(OR) 8 )2 and NR 9 R 10 ,

[0018] R 3 R 5 R 6 R 7 R 8 R 9 and R 10 Independently selected from hydrogen, alkyl, and aryl groups,

[0019] m is an integer from 1 to 6, and

[0020] n is an integer from 1 to 6.

[0021] Or its medicinal salt,

[0022] The conditions are that when the compound is a compound of formula (I), m and n are independently 2, X is C=O, Y is NH, and R 1 and R 2 Both are hydrogen, and R 4 If it is hydrogen, then Z is not SO3H, and

[0023] When the compound is a compound of formula (I), m is 2, n is 1, X is C=O, Y is NH, and R is... 1 and R 2 Both are hydrogen, and R 4 If it is hydrogen, then Z is not COOH.

[0024] 3) Selected from the following aminophenylacetamide derivatives:

[0025] .

[0026] In another implementation, the FXR is an intestinal FXR.

[0027] In another embodiment, the drug is a gastrointestinal absorbent, preferably an intestinal absorbent, and more preferably an oral absorbent.

[0028] In another embodiment, the drug also comprises additional drugs for treating OA.

[0029] According to another aspect of the invention, a pharmaceutical composition for the prevention and / or treatment of OA is provided, comprising: an FXR antagonist according to the invention; and a pharmaceutical carrier.

[0030] In one embodiment, the pharmaceutical composition further comprises additional medicaments for treating OA.

[0031] According to another aspect of the present invention, a method for screening drugs for the prevention or treatment of OA is provided, wherein the method comprises: selecting from the compounds to be screened compounds a compound having one or more of the following effects as a candidate drug for the prevention or treatment of OA: (1) inhibiting the activity of FXR protein; (2) inhibiting the expression of NR1H4 gene and / or FXR protein; and (3) interfering with the biological function of FXR protein, preferably the FXR protein and NR1H4 gene being intestinal FXR protein and intestinal NR1H4 gene. Attached Figure Description

[0032] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 The study found a significant negative correlation between glycoursodeoxycholic acid (GUDCA) and OA prevalence (A) and OA disease severity (B) in the cohort. Specifically: GUDCA, glycoursodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; GHDCA, glycoursodeoxycholic acid; HCA, cholic acid; 7-KLCA, 7-ketolithocholic acid; UDCA, ursodeoxycholic acid; LCA, lithocholic acid; TDCA, tauroursodeoxycholic acid; 12-DHCA, 12-dehydrocholic acid; GHCA, glycoursodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GCA, glycocholic acid; HDC A, porcine deoxycholic acid; GLCA, glycolithocholic acid; 7-KDCA, 7-ketodeoxycholic acid; TCA, taurocholic acid; 23-NDCA, 23-deoxycholic acid; DCA, deoxycholic acid; UCA, ursolic acid; 12-KLCA, 12-ketolithocholic acid; CDCA, chenodeoxycholic acid; GDCA, glycolithocholic acid; CA, cholic acid; TCDCA, taurochenodeoxycholic acid; ILCA, isolithocholic acid; ACA, allocholic acid; BA, bile acid; PBA, primary bile acid; SBA, secondary bile acid; KL, Kellgren-Lawrence;

[0034] Figure 2The validation cohort showed a significant negative correlation between GUDCA and the presence or absence of OA (A) and the severity of OA (B); where: GUDCA, glycoursodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; GHDCA, glycoursodeoxycholic acid; HCA, cholic acid; 7-KLCA, 7-ketolithocholic acid; UDCA, ursodeoxycholic acid; LCA, lithocholic acid; TDCA, tauroursodeoxycholic acid; 12-DHCA, 12-dehydrocholic acid; GHCA, glycoursodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GCA, glycocholic acid; HDCA, cholic acid; GLCA, glycolithocholic acid; 7-KDCA, 7-ketodeoxycholic acid; TCA, taurourcholic acid. DCA, deoxycholic acid; UCA, ursolic acid; 12-KLCA, 12-ketolithocholic acid; CDCA, chenodeoxycholic acid; GDCA, glycodeoxycholic acid; CA, cholic acid; TCDCA, taurine chenodeoxycholic acid; ILCA, isolithocholic acid; ACA, allocholic acid; BA, bile acid; PBA, primary bile acid; SBA, secondary bile acid; KL, Kellgren-Lawrence. *, P<0.05;

[0035] Figure 3 OARSI pathological scores of mouse joint sections stained with Safranin and Fast Green showed that, compared with the littermate control group, the FXR intestinal conditional knockout mouse group had significantly reduced OA progression (***, P<0.001).

[0036] Figure 4 Immunohistochemical staining of mouse joint sections showed that, compared with littermate control groups, the percentage of proteoglycan-positive cells was increased and the percentage of platelet-reactive protein integrin metallopeptidase 5-positive cells was decreased in FXR intestinal conditional knockout mice (**, P<0.01, ***, P<0.001).

[0037] Figure 5 Pain behavior testing in mice showed that, compared with the littermate control group, the FXR intestinal conditional knockout mouse group had significantly reduced OA pain symptoms (****, P<0.0001);

[0038] Figure 6 The results showed that GUDCA intervention significantly reduced the activity of the FXR signaling pathway in the mouse gut, with FGF15 being a direct downstream indicator of FXR protein (*, P<0.05).

[0039] Figure 7 The results showed that compared with the solvent control group, the progression of OA was significantly reduced in the GUDCA gavage group (****, P<0.0001);

[0040] Figure 8The results showed that compared with the solvent control group, the GUDCA gavage group had significantly reduced OA pain symptoms (****, P<0.0001). Detailed Implementation

[0041] Unless otherwise indicated, the terms used herein have their general technical meanings as understood by those skilled in the art.

[0042] Osteoarthritis (OA).

[0043] Farnesoid X receptor (FXR).

[0044] Glycoursodeoxycholic acid (GUDCA):

[0045] .

[0046] The term "Farnesoid X Receptor (FXR)" refers to a member of a nuclear receptor (NR) superfamily, belonging to subfamily 1, group H, member 4, and therefore may be referred to as NR1H4, encoded by the nuclear receptor gene NR1H4 in humans. In a preferred embodiment, the farnesoid X receptor is a mammalian farnesoid X receptor. Preferably, the mammals include, but are not limited to, rodents such as mice, and lagomorphs such as rabbits. Preferably, the mammals are from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammals are from the order Artiodactyla, including bovids (cattle) and suidae (pigs) or perissodactyls, including equines (horses). Most preferably, the mammals are primates, ceboids, or simioids (monkeys) or apes (humans and apes). Particularly preferred mammals are humans. The FXR protein used in the embodiments of this application is the following mouse or human FXR protein.

[0047] Human FXR protein (SEQ ID NO: 1): MVMQFQGLENPIQISPHCSCTPSGFFMEMMSMKPAKGVLTEQVAGPLGQNLEVEPYSQYSNVQFPQVQPQISSSSYYSNLGFYPQQPEEWYSPGIYELRRMPAETLYQGETEVAEMPVTKKPRMGASAGRIKGDELCVVCGDRASGYHYNALTCEGCKGFFRRSITKNAVYKCKNGGNCVMDMYMRRKCQECRLRKCKEMGMLAECMYTGLLTEIQCKSKRLRKNVKQHADQTVNEDSEGRDLRQVTSTTKSCREKTELTPDQQTLLHFIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATNHVQVLVEFTKKLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPSGHSDLLEERIRNSGISDEYITPMFSFYKSIGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIHQPENPQHFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQ

[0048] Human FXR GenBank accession no: NG_029843

[0049] Mouse FXR protein (SEQ ID NO: 2): MVMQFQGLENPIQISLHHSHRLSGFVPEGMSVKPAKGMLTEHAAGPLGQNLDLESYSPYNNVPFPQVQPQISSSSYYSNLGFYPQQPEDWYSPGIYELRRMPAETGYQGETEVSEMPVT KKPRMAAASAGRIKGDELCVVCGDRASGYHYNALTCEGCKGFFRRSITKNAVYKCKNGGNCVMDMYMRRKCQECRLRKCKEMGMLAECMYTGLLTEIQCKSKRLRKNVKQHADQTANEDDSEG RDLRQVTSTTKFCREKTELTADQQTLLDYIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATSHVQILVEFTKKLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPAGHADLLE ERIRKSGISDEYITPMFSFYKSVGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKMYQPENPQHFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQ

[0050] Mouse FXR GenBank accession no: NC_000076

[0051] The expression of the FXR gene (NR1H4) refers to its expression at two levels: one is expression at the DNA level; the other is expression at the RNA level.

[0052] The techniques and reagents used to detect gene expression levels are well known to those skilled in the art. In this invention, the reagent is preferably selected from specific probes (preferably nucleic acid probes with detection markers, typically complementary to the target gene) of the FXR gene (NR1H4 gene), gene chips, or PCR primers for PCR-specific amplification reactions.

[0053] The term "reducing or suppressing the expression of the FXR gene (NR1H4 gene) or FXR protein" refers to reducing the expression level of the FXR gene (NR1H4 gene) or FXR protein to less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or 10% of the original expression level, for example, less than 5%, less than 2%, less than 1%, or even 0%. In one embodiment, the expression of the FXR gene (NR1H4 gene) or FXR protein can be reduced or suppressed by gene knockout or knockdown.

[0054] The term "knockout" refers to a genetic engineering technique in which an exogenous mutated gene is used to replace an endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene and causing it to exhibit mutant traits.

[0055] The term "knockdown" refers to the process of inhibiting gene expression by degrading mRNA containing homologous target genes. It utilizes double-stranded small RNAs to efficiently and specifically degrade intracellular homologous mRNAs, thereby blocking the expression of target genes and causing cells to exhibit a target gene deletion phenotype. Unlike gene knockout, which permanently silences the expression of the target gene, it achieves this effect by degrading mRNA containing homologous target genes.

[0056] Gene knockout or knockdown techniques are well-known in the art, including but not limited to retroviral gene transfer that induces mutations such as point mutations, insertions, deletions, frameshifts, or missense mutations. Another way to knock out genes is using zinc finger nucleases. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA-cutting domain. The zinc finger domain can be modified to target specific DNA sequences, allowing zinc finger nucleases to target unique sequences within complex genomes. Other genome-customized technologies that can be used to knock out genes include TAL effector nucleases (TALENs). Another technology is the CRISPR / Cas genome editing system, which can be used to achieve RNA-guided genome modification.

[0057] Techniques to reduce or suppress the expression of the FXR gene (NR1H4 gene) or FXR protein may also include the use of gapmers, antisense RNA, siRNA, esiRNA, shRNA, miRNA, or RNA aptamers.

[0058] "Antisense RNA" refers to an RNA molecule that is complementary to mRNA, and also includes RNA molecules that are complementary to other RNAs. Since ribosomes cannot translate double-stranded RNA, the specific complementary binding of antisense RNA to mRNA inhibits its translation. Antisense constructs can be delivered, for example, as expression plasmids, wherein when expressed in cells, the plasmid produces RNA complementary to at least one unique portion of the cellular FXR gene (NR1H4 gene).

[0059] Another specific form of the antisense RNA strategy is the gapmer. A gapmer is a chimeric antisense oligonucleotide containing a central block of a deoxynucleotide monomer long enough to induce RNase H cleavage. The design and synthesis of gapmers are well known to those skilled in the art and can be performed by commercial companies.

[0060] Small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, is a class of double-stranded RNA molecules approximately 20-25 base pairs in length that function through the RNA interference (RNAi) pathway. It interferes with the post-transcriptional degradation of mRNA in specific genes expressing complementary nucleotide sequences, thereby preventing translation. The siRNA of this invention can target any segment of approximately 19 to 25 consecutive nucleotides in the target sequence of the FXR gene (NR1H4 gene), examples of which are provided in this application. Techniques for selecting target sequences for siRNA are well known in the art.

[0061] Short hairpin RNA (shRNA) is an RNA sequence consisting of two short inverted repeat sequences that can silence gene expression via RNA interference (RNAi).

[0062] The full English name of "esiRNA" is Endoribonuclease-prepared siRNAs. It is a mixture of siRNAs produced by cutting long double-stranded RNA (dsRNA) with RNase III (a ribonuclease) in E. coli. The length is between 18-25 bp and it can be used to efficiently knock out the expression level of target genes.

[0063] The technique of conditional gene knockout based on the Cre-Loxp system is well known to those skilled in the art. This technique is typically used in conjunction with CRISPR / Cas9 technology to construct animal models of conditional knockout of target genes. The constructed animal models can then be identified by PCR using identification primers.

[0064] In one embodiment of the present invention, the FXR antagonist may be a small molecule compound inhibitor.

[0065] Small molecule inhibitors of FXR may include glycoursodeoxycholic acid (GUDCA), taurine-β-mouse cholic acid (TβMCA), Gly-β-MCA (CAS 66225-78-3), or their pharmaceutical salts, which are well known in the art.

[0066] In addition, CN110437297A and CN105593237A disclose small molecule inhibitors of FXR. Specifically, compounds of formula (I) or (II) or pharmaceutical salts thereof are disclosed:

[0067]

[0068] Where R 1 and R 2 Independently selected from hydrogen, alkyl, and C(=O)R 3 ,

[0069] R 4 Selected from hydrogen, alkyl and C(=O)R 3 ,

[0070] X is selected from C=O and CH2.

[0071] Y is selected from CH2, NR 5 , O, S, SO, SO2 and Se,

[0072] Or X and Y together form C=C,

[0073] Z is selected from COOR 6 SO3R 7 P(=O)(OR) 8 )2 and NR 9 R 10 ,

[0074] R 3 R 5 R 6 R 7 R 8 R 9 and R 10 Independently selected from hydrogen, alkyl, and aryl groups,

[0075] m is an integer from 1 to 6, and

[0076] n is an integer from 1 to 6.

[0077] Or its medicinal salt,

[0078] The conditions are that when the compound is a compound of formula (I), m and n are 2, X is C=O, Y is NH, and R is 2. 1 and R 2 Both are hydrogen, and R4 If it is hydrogen, then Z is not SO3H.

[0079] When the compound is a compound of formula (I), m is 2, n is 1, X is C=O, Y is NH, and R is... 1 and R 2 Both are hydrogen, and R 4 If it is hydrogen, then Z is not COOH.

[0080] According to certain implementations, the compound is a compound of formula (I).

[0081] According to any of the above implementation schemes, R 4 It is hydrogen.

[0082] According to certain implementation schemes, R 1 and R 2 It is hydrogen.

[0083] According to some implementation schemes, X is C=O.

[0084] According to some implementation schemes, m is 2.

[0085] According to some implementation schemes, Y is NH.

[0086] In some implementations, n is an integer from 1 to 6.

[0087] In a preferred embodiment, the general formula compound is

[0088] .

[0089] The term "alkyl" means a straight-chain or branched alkyl substituent containing, for example, 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, etc.

[0090] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic substituent as commonly understood in the art, and the term "C6-C" refers to an unsubstituted or substituted aromatic carbocyclic substituent. 10 "Aryl" includes phenyl and naphthyl. To understand, according to Hückel's rule, the term aryl applies to planar cyclic substituents and contains 4n+2 π electrons.

[0091] In any of the above embodiments, the C-20 carbon atom of the compound or salt of formula (I) or (II) may have an R configuration, an S configuration, or may be a mixture of R and S isomers.

[0092] In any of the above embodiments, when the stereochemistry at the chiral carbon atom is not specified, the chiral carbon atom may have an R configuration, an S configuration, or may be a mixture of R and S isomers.

[0093] CN 117285504 A also discloses other small molecule inhibitors of FXR. Specifically, it discloses aminophenylacetamide derivatives 1-14 with the following structural formulas:

[0094] .

[0095] The term "medicinal salt" is intended to include nontoxic salts synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or a mixture of both. Non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977).

[0096] Pharmaceutical salts may include acetates, adipates, benzenesulfonates, bromides, camphorsulfonates, chlorides, citrates, ethanedisulfonates, propionate, dodecyl sulfate, fumarates, gluconate, gluconate, glucuronide, hippurate, hexanoate, hydrobromide, hydrochloride, iodides, hydroxyethylsulfonates, lactates, lacturonates, maleates, methanesulfonates, methyl bromide, methyl sulfate, naphthalenesulfonates, nitrates, oleates, palmitates, phosphates, polygalacturonates, stearates, succinates, sulfates, sulfosalicylate, tannates, tartrates, terephthalates, toluenesulfonates, and triethyl iodide.

[0097] It should also be understood that the above-mentioned compounds and salts can form solvates or exist in substantially unreintegrated forms, such as anhydrous forms. As used herein, the term "solvent" refers to a molecular complex in which solvent molecules, such as crystalline solvents, are bound in a crystal lattice. When the solvent bound in a solvate is water, the molecular complex is called a hydrate. Pharmaceutical solvates include hydrates, alcohols such as methanol and ethanol compounds, acetonitrile compounds, etc. These compounds can also exist in polymorphic forms.

[0098] This invention also relates to pharmaceutical compositions comprising a pharmaceutical carrier and at least one FXR antagonist (such as a small molecule compound inhibitor) described herein.

[0099] Preferably, the pharmaceutical carrier is a carrier that is chemically inert to the active ingredient and does not have harmful side effects or toxicity under the conditions of use.

[0100] The choice of carrier will be determined in part by the specific FXR antagonist of the present invention selected, and by the specific method of administration of the composition. Therefore, there are a variety of suitable formulations of the pharmaceutical compositions of the present invention. In some embodiments, the formulations are suitable for administration to the digestive tract, and particularly to the small intestine.

[0101] Formulations suitable for oral administration may consist of (a) a liquid solution, such as a therapeutically effective amount of the FXR antagonist of the present invention dissolved in a diluent, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and tablets, each containing a predetermined amount of the active ingredient as a solid or granule; (c) a powder; (d) a suspension in a suitable liquid; and (e) a suitable emulsion. Liquid formulations may include diluents such as water and alcohols, such as ethanol, benzyl alcohol, and polyethylene glycol, with or without pharmaceutical surfactants, suspending agents, or emulsifiers. Capsule forms may be of the common hard or soft-shell gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms may include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginate, microcrystalline cellulose, gum arabic, gelatin, guar gum, colloidal silica, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffers, disintegrants, humectants, preservatives, flavorings, and pharmacologically compatible excipients. Lozenge forms may contain flavorings, typically active ingredients in sucrose and gum arabic or astragalus gum, and tablets containing inert bases such as gelatin and glycerin, or active ingredients in sucrose and arabinose, emulsions, gels, etc., containing the excipients in addition to the active ingredients, are known in the art.

[0102] In some embodiments, the formulation may be adapted to prolong the amount of time the FXR antagonist of the present invention is in contact with the mammalian digestive tract, and particularly with the mammalian small intestine. In this regard, various formulations such as extended-release formulations and formulations designed to prolong the amount of time the FXR antagonist remains in the stomach before being released into the small intestine can be used. Many suitable formulations are provided in Remington: The Science and Practice of Pharmacy, Gennaro, AR, ed., pp. 858-929, Lippincott Williams & Wilkins (2000).

[0103] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. For example, a time-release formulation intended for oral administration to humans may contain 20 to 2000 µmol (approximately 10 to 1000 mg) of the active substance mixed with an appropriate and convenient amount of carrier material, which may vary from about 5% to about 95% of the total composition. Preferably, a pharmaceutical composition can be prepared to provide an easily measurable dosage.

[0104] In one embodiment, the pharmaceutical composition of the present invention further comprises an additional medicament for treating OA. This additional medicament for treating OA is known in the art and includes, for example: anti-inflammatory analgesics such as acetaminophen; nonsteroidal anti-inflammatory drugs such as aspirin, salicylic acid, phenylbutazone, indomethacin, naproxen, diclofenac, meloxicam, nabumetone, etodoxacin, sulindac, and acemetacin; and glucosamine.

[0105] The present invention also relates to a method for preventing or treating OA, the method comprising administering the FXR antagonist or pharmaceutical composition of the present invention to a subject in need.

[0106] This invention is the first to discover the important regulatory role of intestinal FXR in the progression of osteoarthritis (OA). Through various experiments, including investigating the biological function of intestinal FXR proteins and regulating NR1H4 gene expression, it was verified that inhibiting the activity of intestinal FXR proteins and inhibiting the expression of the intestinal NR1H4 gene can alleviate cartilage degeneration and delay the progression of OA. Intestinal FXR proteins or the NR1H4 gene can serve as therapeutic targets for OA, and this invention provides new ideas and strategies for OA treatment and drug development or screening.

[0107] The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. All chemicals used in the following reactions are commercially available products unless otherwise specified.

[0108] Logistic regression and unpaired Student's t-test were used for statistical analysis in this invention. Statistical calculations were performed using SAS and Microsoft Excel. A p-value was considered significant when p < 0.05.

[0109] 1) High-throughput target bile acid metabolomics detection

[0110] (1) Detection platform: UHPLC-PRM-MS (Vanquish, Thermo Fisher Scientific)

[0111] (2) Sources of biological samples: Blood samples from the population included in the discovery and validation cohorts

[0112] (3) Sample pretreatment:

[0113] a. Take 50 μL of sample into an EP tube, add 200 μL of extraction buffer (methanol:acetonitrile = 1:1, containing 0.1% formic acid), and vortex for 30 seconds to mix.

[0114] b. Ultrasound for 10 minutes (ice water bath), -40°C o C. Let stand for 1 hour;

[0115] c. Centrifuge the sample at 4°C, 12000 rpm for 15 minutes;

[0116] d. Transfer the supernatant to a sample vial for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS);

[0117] e. Accurately weigh the corresponding amount of standard into a 10 mL volumetric flask and prepare 10 mmol / L standard stock solutions. Take the corresponding amount of standard stock solution into a 10 mL volumetric flask and prepare a mixed standard solution. Dilute this standard solution sequentially to obtain a series of calibration solutions (containing an isotope-labeled internal standard mixture with the same concentration as in the sample);

[0118] f. Randomly sequenced testing.

[0119] (4) Analytical Separation: A Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph was used, employing a Waters ACQUITY UPLC BEH C18 (150 * 2.1 mm, 1.7 μm, Waters) column for chromatographic separation of the target compounds. Phase A of the liquid chromatography consisted of 1 mmol / L ammonium acetate and 0.1% acetic acid aqueous solution, while phase B consisted of acetonitrile. The column oven temperature was 50℃, the sample tray was set to 4℃, and the injection volume was 1 μL. A Q Exactive HFX high-resolution mass spectrometer was used for mass spectrometry analysis in Parallel Reaction Monitoring (PRM) mode. The ion source parameters were as follows: spray voltage = +3500 V / -3100 V, shield gas (N2) flow rate = 40, auxiliary gas (N2) flow rate = 15, scanning gas (N2) flow rate = 0, auxiliary gas (N2) temperature = 350℃, and capillary temperature = 320℃.

[0120] (5) Data Preprocessing: The raw data were deconvolved, peak aligned, and standardized. TIC chromatograms were used to preliminarily assess the instrument's retention time reproducibility, the quantity of substances measured, and whether there were significant differences between different groups of chromatograms. Data quality control was performed using a relative standard deviation of <30% for control samples and a feature yield >80%. Peak lookup and peak alignment were performed using XCMS software (version 1.41.0).

[0121] 2) DMM surgical modeling

[0122] The surgical modeling process for medial meniscus destabilization (DMM) is as follows:

[0123] (1) Skin preparation: After 12 weeks of age, the hair on the right knee joint of the mice was shaved off, and the surgical area was disinfected with iodine.

[0124] (2) Expose the joint cavity: cut open the skin of the mouse knee joint, cut along the medial edge of the patellar ligament to open the joint capsule and expose the joint cavity;

[0125] (4) Ligament transection: Under a surgical microscope, the ligament connecting the meniscus and the tibia on the medial side is transcribed;

[0126] (5) Sew layer by layer.

[0127] 3) FXR intestinal conditional knockout

[0128] Using Cre-loxP technology, conditional knockout mice that specifically knock out FXR in intestinal cells were obtained by crossing mice carrying Cre recombinase controlled by the chorionic promoter (FXR intestinal conditional knockout mice) (J Biol Chem 2002 Sep 6;277(36):33275-83. doi: 10.1074 / jbc.M204935200) with homozygous Fxr-floxed mice containing the loxp site (litter control group) (Cell 2000 Sep15;102(6):731-44. doi: 10.1016 / s0092-8674(00)00062-3). All of the mice mentioned above have a C57BL / 6N genetic background. The FXR intestinal conditional knockout mice and the littermate control mice and mice were backcrossed with C57BL / 6N mice for more than 10 generations (J Lipid Res 2007 Dec;48(12):2664-72. DOI: 10.1194 / jlr.M700330-JLR200).

[0129] Construction and genotyping of gut-specific Fxr-deficient mice:

[0130] (1) Hybridize homozygous Fxr-floxed mice (livestock control group) with mice carrying Cre recombinase controlled by the chorionic protein promoter;

[0131] (2) Mouse tails were cut off and DNA was extracted. 50 ng of mouse tail DNA was amplified in 10 ml of a system containing 1.5 mM MgCl2, 0.25 mM deoxyribonucleotide triphosphate, 2.5% Me2SO4, 0.25 Utaq polymerase, 0.2 mM FXR-geno-F (5'-atagacaaccccagtgaccc-3'(SEQ ID NO: 3)) and 0.2 mM FXR-geno-R (5'-tctaaaggatagccgaatct-3'(SEQ ID NO: 4)). The cycling conditions were 94℃ for 3 min, followed by 30 cycles of 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, and then extension at 72℃ for 10 min. These primers produced 300 bp and 380 bp Fxr wild-type and littermate control allele products, respectively.

[0132] (3) Genotyping of the Cre transgene was performed as previously described, using microsomal epoxide hydrolase as an internal control. The disruption of Fxr was quantified by real-time quantitative PCR (RT-qPCR) using primers designed for the deleted exons: forward 5'-cacagcgatcgtcatcctctct-3' (SEQ ID NO: 5) and reverse 5'-tctcaggctggtacatcttgca-3' (SEQ ID NO: 6).

[0133] 4) Use safranin-fast green staining and OARSI score to assess the progression of OA.

[0134] Safranin-Fixed Green Staining

[0135] (1) Dewaxing of slides: The slide containing the knee joint is baked in an oven at 65°C and dewaxed using an environmentally friendly dewaxing solution;

[0136] (2) Safranin O / Solid Green preparation: 2.5 g of Safranin O solid was dissolved in 100 ml of 95% ethanol solution. 0.5 g of Solid Green was dissolved in 100 ml of 95% ethanol solution;

[0137] (3) Rehydration of slides: Rehydrate the dewaxed slides by passing them through a gradient of alcohols;

[0138] (4) Fast Green staining: Stain the whole slide in Fast Green staining solution and check the Fast Green staining under a microscope. If the color is light, the staining time can be increased.

[0139] (5) Safranin O staining: The entire slide is stained with Safranin O. After completion, the excess dye is quickly washed away in a water tank. The staining of Safranin O is checked under a microscope. If the color is too light, the slide can be stained with acetic acid again and the staining time of Safranin O can be increased.

[0140] (6) After microscopic examination, it enters the gradient dehydration stage and is then cleared in xylene.

[0141] (7) After the residual xylene on the glass slide evaporates, add neutral resin to cover the glass slide to complete the sealing.

[0142] OASRI rating

[0143] The scoring rules are as follows:

[0144]

[0145] 5) Use behavioral tests such as Von Frey and bipedal weight-bearing difference to assess OA pain levels.

[0146] The specific steps for Von Frey pain threshold measurement are as follows:

[0147] Von Frey fibers are blunt plastic filaments, with different models exhibiting varying degrees of bending resistance. When a fiber of a specific length and diameter is pressed perpendicularly onto the skin, pressure is continuously applied until the fiber bends; the magnitude of the applied pressure can then be calculated (once the fiber bends, continued pressure will cause it to bend further, but will not generate additional pressure). The Von Frey test can detect changes in the mechanical pain threshold at different sites in patients or laboratory animals, primarily used to assess hyperalgesia and hypoalgesia (i.e., a decrease or increase in the tenderness threshold caused by peripheral neuropathy, tissue damage, etc.). This test measures the Von Frey pain threshold weekly.

[0148] (1) Install the detection device, place the mouse in the acrylic box, cover the acrylic box to make the animal to be tested in a dark and quiet environment, and avoid the animal to be tested being too agitated. Allow it to adapt to the environment for 15-30 minutes (depending on the actual situation).

[0149] (2) Assembly and use of Von Frey instrument: Assemble the handheld automatic electronic Von Frey pain monitor, and match the plug and socket (e.g., plug DC corresponds to socket DC); when assembling the Von Frey needle, avoid pressing the sensor head too much and rotate the assembled needle gently.

[0150] (3) Stimulate the middle of the sole of the hind limb of the mouse modeling side with Von Frey fiber needles. Do not change the fiber type during the test. Then stimulate the sole of the foot with the fixed Von Frey fiber and record the mechanical stimulation value when the foot retraction or licking response occurs.

[0151] (4) If the same mouse is measured three times and the results are basically consistent, the result is adopted. The interval between adjacent stimuli is 5-10 min.

[0152] The specific details of the weight bearing bipedal load test are as follows:

[0153] Weight bearing was measured using an incapacitance analgesia meter (IITC. Woodland Hills, CA, USA) to assess hind limb pain in mice and rats. The incapacitance analgesia meter is equipped with two precise force sensors, on which the animal's two hind limbs are positioned, and a restraint device is used to stabilize the animal. After the animal is placed in a suitable position and assumes a specific standing posture, the incapacitance analgesia meter begins measuring the weight-bearing capacity of both hind limbs. Three to five measurements can be taken in one measurement cycle (in a standard standing posture), and the results are averaged. The time interval between test cycles is set by the experimenter, ranging from 2 to 8 seconds (the instrument in this laboratory is set to 5 seconds). At the end of the measurement, the instrument display shows the weight-bearing values ​​for the left and right hind limbs respectively. When the animal is unrestrained, a weight-bearing difference will appear between the modeling hind limb and the control hind limb; the larger the difference, the more pain the modeling hind limb experiences.

[0154] (1) Choose a relatively quiet place and connect the instrument to the power supply;

[0155] (2) One experimenter takes the mice / mice, removes the labels that clearly identify the grouping, and then hands the mice to another experimenter for testing. The first experimenter records the results. After the test is completed, the mice are returned to the first experimenter for re-marking and put back.

[0156] (3) Before the formal test begins, the experimental animals are placed in a restraint device to allow them to adapt to the environment and become quiet. This allows the experimental animals to adapt to the weight-bearing posture of their hind limbs, which can increase the stability of the measurement results.

[0157] (4) Once the experimental animal is positioned in the standard standing posture (body in the center of the restraint device, both hind feet on the sensors, both forelimbs on the inclined plate of the restraint device, maintaining a relatively upright posture), the test will begin.

[0158] (5) As the time of weight-bearing on both hind feet of the experimental animals increased, the symptoms on the painful hind limb became more obvious, resulting in reduced weight-bearing and greater differences in weight-bearing data between the two sides. Therefore, each experimental animal was measured 3-5 times consecutively while maintaining a standard standing posture;

[0159] (6) After the experiment, the instrument should be wiped with gauze and alcohol to remove surface dirt, and the waste at the bottom of the sensor should be cleaned with cotton swabs.

[0160] Example 1: The level of the intestinal FXR-specific antagonist GUDCA was significantly negatively correlated with the prevalence of OA.

[0161] High-throughput targeted metabolomics is a technique that simultaneously detects the levels of multiple target metabolites in a sample. It features high throughput and high sensitivity, playing a crucial role in exploring risk factors and pathogenesis of diseases. Bile acids are important metabolites in the human body and are major ligands for FXR (extracorporeal membrane oxygenation). Therefore, based on two independent cohorts (included from the Xiangya Osteoarthritis Study and the Xiangya Walking Study), we performed bile acid metabolomics analysis on OA patients and controls. We found that glycoursodeoxycholic acid (GUDCA), a previously reported FXR-specific antagonist, was significantly negatively correlated with the presence and severity of OA in the cohort (n = 1714; OA patients: 421; controls: 1293). Figure 1 Furthermore, consistent results with the discovery cohort were also found in the validation cohort (n = 154; number of OA patients: 77, number of controls: 77). Figure 2 The results suggest that decreased GUDCA levels may be a risk factor for OA. Furthermore, since GUDCA is primarily found in the gut, this also suggests that specific antagonism of intestinal FXR may have a protective effect against OA.

[0162] Example 2: Intestinal FXR protein deficiency effectively delays OA progression in a DMM model

[0163] Based on the correlation between GUDCA and OA found in Example 1, and considering that animal experiments can more directly study the relationship between intestinal FXR and OA, we further used FXR intestinal conditional knockout mice and their littermate wild-type controls to determine the effect of intestinal FXR protein deficiency on OA progression in mice. In this example, DMM surgery was performed on 12-week-old FXR intestinal conditional knockout mice and their littermate control mice. OARSI pathological scores of mouse joint sections stained with Safranin-Fix-Green showed that OA progression was significantly reduced in the FXR intestinal conditional knockout mouse group compared to the littermate control group. Figure 3 Immunohistochemical staining of mouse joint sections showed that, compared with the littermate control group, the percentage of proteoglycan-positive cells and the percentage of platelet-reactive protein-integrin-metallopeptidase 5-positive cells were increased in the FXR intestinal strip knockout group. Figure 4 Pain behavior testing in mice showed that, compared with the littermate control group, the FXR intestinal conditional knockout mouse group had significantly reduced OA pain symptoms. Figure 5 The above results indicate that the absence of intestinal FXR protein can significantly delay the progression of OA in mice.

[0164] Example 3: Gavage administration of the FXR-specific antagonist GUDCA safely and effectively delayed the progression of OA in a DMM model.

[0165] GUDCA is an FXR-specific antagonist, and previous reports have shown that gavage can effectively reduce the activity of the intestinal FXR signaling pathway. In this study, 12-week-old C57BL / 6J mice (3 groups, 8 mice per group) underwent DMM surgery to establish a mouse model, and were intervened by gavage administration of GUDCA (50 mg / kg / day). FGF15 is a direct downstream indicator of FXR protein. Western blot results (FGF15 mouse monoclonal antibody, Santa Cruz, Cat. sc-514647) confirmed that the activity of the mouse intestinal FXR signaling pathway was significantly reduced (n = 3 / group). Figure 6 Safranin-Fix-Green staining and OARSI pathological scoring of mouse joint sections showed that, compared with the solvent control group, the GUDCA gavage group had a significantly reduced OA progression. Figure 7 Mouse pain behavior testing showed that, compared with the solvent control group, the GUDCA gavage group had significantly reduced OA pain symptoms. Figure 8 The above results indicate that GUDCA can significantly delay the progression of OA in C57BL / 6J mice with DMM surgery by reducing the activity of the FXR signaling pathway.

[0166] The combined results of the population cohort study in Example 1, the gene knockout mouse experiment in Example 2, and the drug intervention experiment in Example 3 fully demonstrate that intestinal FXR plays a key regulatory role in the progression of OA disease, and that antagonists targeting intestinal FXR (such as GUDCA) have potential value in treating OA, providing a new direction for the treatment and drug development of OA.

[0167] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

Claims

1. Use of farnesol X receptor antagonists in the preparation of medicaments for the prevention and / or treatment of osteoarthritis.

2. The application according to claim 1, wherein the antagonist has one or more of the following effects: (1) Reduce or inhibit the activity of FXR protein; (2) Reduce or suppress the expression of the NR1H4 gene and / or FXR protein; and / or (3) Reduce or inhibit the biological function of FXR protein.

3. The application according to claim 1 or 2, wherein the antagonist is selected from the group consisting of: FXR-binding proteins, FXR-binding peptides, monoclonal / polyclonal antibodies against FXR, small molecule compound inhibitors, gapmers against the NR1H4 gene, antisense RNA, siRNA, esiRNA, shRNA, miRNA, RNA aptamers, TALEN, CRISPR, zinc finger nucleases, Cre-loxP systems, or any combination thereof.

4. The application according to any one of claims 1 to 3, wherein the small molecule compound inhibitor is selected from the group consisting of: 1) Glyursodeoxycholic acid (GUDCA), taurine-β-mouse cholic acid (TβMCA), Gly-β-MCA (CAS 66225-78-3) or their medicinal salts; 2) Compounds selected from the following general formulas or their pharmaceutical salts, Where R 1 and R 2 Independently selected from hydrogen, alkyl, and C(=O)R 3 , R 4 Selected from hydrogen, alkyl and C(=O)R 3 , X is selected from C=O and CH2. Y is selected from CH2, NR 5 , O, S, SO, SO2 and Se, Or X and Y together form C=C, Z is selected from COOR 6 SO3R 7 P(=O)(OR) 8 )2 and NR 9 R 10 , R 3 R 5 R 6 R 7 R 8 R 9 and R 10 Independently selected from hydrogen, alkyl, and aryl groups, m is an integer from 1 to 6, and n is an integer from 1 to 6. Or its medicinal salt, The conditions are that when the compound is a compound of formula (I), m and n are 2, X is C=O, Y is NH, and R is 2. 1 and R 2 Both are hydrogen, and R 4 If it is hydrogen, then Z is not SO3H. When the compound is a compound of formula (I), m is 2, n is 1, X is C=O, Y is NH, and R is... 1 and R 2 Both are hydrogen, and R 4 If it is hydrogen, then Z is not COOH; and 3) Selected from the following aminophenylacetamide derivatives: 。 5. The application according to any one of claims 1 to 4, wherein the FXR is an intestinal FXR.

6. The application according to any one of claims 1 to 5, wherein the drug is a gastrointestinal absorbent, preferably an intestinal absorbent, and more preferably an oral absorbent.

7. The application according to any one of claims 1 to 6, wherein the medicament further comprises another medicament for treating osteoarthritis.

8. A pharmaceutical composition for the prevention and / or treatment of osteoarthritis, comprising: an FXR antagonist as described in any one of claims 1 to 7 and a pharmaceutical carrier.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises an additional medicament for treating OA.

10. A method for screening drugs for the prevention or treatment of osteoarthritis, wherein the method comprises: From the compounds to be screened, select compounds having one or more of the following effects as candidate drugs for the prevention or treatment of osteoarthritis: (1) Inhibit the activity of FXR protein; (2) Inhibit the expression of the NR1H4 gene and / or FXR protein; and (3) Interferes with the biological function of FXR protein. Preferably, the FXR protein and NR1H4 gene are intestinal FXR protein and intestinal NR1H4 gene.

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