Application of dual inhibitor YS-121 in the preparation of drugs for preventing and / or treating polycystic kidney disease

Abstract

The present invention discloses the use of a dual inhibitor YS-121 in the preparation of a drug for preventing and / or treating polycystic kidney disease. The dual inhibitor YS-121 has a structure as shown in the following formula: wherein the pharmaceutically acceptable derivative is selected from at least one of a pharmaceutically acceptable salt, a polymorph, a co-crystal, a radiolabeled form and a combination thereof. The present invention reports for the first time the use of YS-121 in the treatment and / or prevention of autosomal dominant polycystic kidney disease. Experiments have shown that the administration of a drug containing YS-121 can significantly reduce the kidney-to-body weight ratio of polycystic kidney individuals by inhibiting the proliferation of renal tubular epithelial cells, significantly reduce the morphological damage of renal tissue in polycystic kidney individuals, and significantly improve the size of cell vesicles in vitro, which has an improvement effect on autosomal dominant polycystic kidney disease, and provides valuable reference significance for the prevention and treatment of polycystic kidney disease in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a dual inhibitor YS-121 in the preparation of a drug for preventing and / or treating polycystic kidney disease. Background Art

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease, mainly caused by mutations in Pkd1 and Pkd2, which encode polycystin-1 (PC1) and polycystin-2 (PC2) 1. It is characterized by the formation of tubular cysts, which continue to grow over time, leading to massive bilateral tubular necrosis and eventually renal failure.

[0003] There are two types of polycystic kidney disease: autosomal recessive (infantile) polycystic kidney disease, which occurs in infancy and is clinically rare; and autosomal dominant (adult) polycystic kidney disease, which is often discovered in young and middle-aged people but can also occur at any age.

[0004] At present, kidney transplantation and dialysis are the main treatments for end-stage renal failure caused by polycystic kidney disease. Tolvaptan, as the only drug for the treatment of ADPKD, has a single mode of action and many adverse reactions. Therefore, finding a specific and effective drug for the treatment of ADPKD is an important issue that needs to be solved in the clinic. Summary of the invention

[0005] The main purpose of the present invention is to provide a dual inhibitor YS-121 for use in the preparation of a drug for preventing and / or treating polycystic kidney disease, so as to overcome the deficiencies of the prior art.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0007] The present invention provides a dual inhibitor YS-121 or a pharmaceutically acceptable derivative thereof for use in the preparation of a drug for preventing and / or treating polycystic kidney disease. The dual inhibitor YS-121 has a structure as shown in formula (I):

[0008]

[0009]

[0010] Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

[0011] The present invention also provides a pharmaceutical composition for preventing and / or treating polycystic kidney disease, which comprises a dual inhibitor YS-121 or a pharmaceutically acceptable derivative thereof; the dual inhibitor YS-121 has a structure as shown in formula (I):

[0012]

[0013] Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

[0014] The embodiment of the present invention also provides the use of the aforementioned pharmaceutical composition in preparing a drug for preventing and / or treating autosomal dominant polycystic kidney disease.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention reports for the first time the use of the dual inhibitor YS-121 or a pharmaceutically acceptable derivative thereof in the treatment and / or prevention of autosomal dominant polycystic kidney disease. Experiments have shown that administration of YS-121 can significantly reduce the kidney-to-body weight ratio of polycystic kidney individuals, significantly reduce the morphological damage of renal tissue of polycystic kidney individuals, and significantly improve the size of cell vesicles in vitro. In addition, the drug can also significantly reduce the expression of proliferation proteins of renal cysts and reduce the replication efficiency of DNA. The above indicates that YS-121 has an improving effect on autosomal dominant polycystic kidney disease, and provides valuable reference significance for the prevention and treatment of polycystic kidney disease in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1a This is a general picture of the kidneys of the control group and the treatment group Ksp-Cre; Pkd1flox / flox mice in a typical embodiment of the present invention;

[0018] Figure 1b is a statistical graph of kidney weight ratio of PKD mice in a control group and a treatment group in a typical embodiment of the present invention;

[0019] Figure 2a This is a diagram showing the results of HE staining of kidneys of PKD mice in a control group and a treatment group in a typical embodiment of the present invention;

[0020] Figure 2bThis is a statistical result diagram of the vesicle index of the HE staining results of the kidneys of PKD mice in the control group and the treatment group in a typical embodiment of the present invention;

[0021] Figure 3 is a graph showing the cytotoxic effect of the compound represented by formula (I) on MDCK cells detected by CCK-8 method in a typical embodiment of the present invention;

[0022] Figure 4 is a graph showing the inhibitory effect of different concentrations of the compound represented by formula (I) on vesicle growth in a typical embodiment of the present invention;

[0023] Figure 5 is a graph showing the inhibitory effect of different concentrations of the compound represented by formula (I) on vesicle growth in a typical embodiment of the present invention;

[0024] Figure 6a-6b This is a graph showing the expression of PCNA, a kidney proliferation index, after treatment with the compound of formula (I) using immunohistochemistry in a typical embodiment of the present invention;

[0025] Figure 7a-Figure 7b This is a graph showing the expression of the kidney proliferation index Ki67 after treatment with the compound of formula (I) using immunohistochemistry in a typical embodiment of the present invention;

[0026] Figure 8a-8b In a typical example of the present invention, the method of using Clik it Edu to detect DNA replication is used to determine the pH of the compound represented by formula (I). + / - and pN - / - Immunofluorescence expression in cells after drug administration. DETAILED DESCRIPTION

[0027] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Specifically, as one aspect of the technical solution of the present invention, it relates to the use of a dual inhibitor YS-121 or a pharmaceutically acceptable derivative thereof in the preparation of a medicament for preventing and / or treating polycystic kidney disease, wherein the dual inhibitor YS-121 has a structure as shown in formula (I):

[0029]

[0030] Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

[0031] The term "pharmaceutically acceptable salt" as used herein refers to salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction or other problems or complications within the scope of sound medical judgment, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, and the contents are incorporated by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic acids and bases and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed of amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or salts formed by using other methods in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, gluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hydroiodide, 2-hydroxyethanesulfonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like.

[0032] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include suitable non-toxic ammonium salts, quaternary ammonium salts and amine cations formed using, for example, halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

[0033] As used herein, "pharmaceutically acceptable salts" refer to forms of the disclosed compounds in which the parent compound is modified by preparing acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, such as those formed from non-toxic inorganic or organic acids. Such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid.

[0034] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing basic or acidic sites by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are used.

[0035] The term "pharmaceutically acceptable derivative" includes any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound that, when administered to a subject, can provide (directly or indirectly) the dual inhibitor YS-121 or a metabolite or residue having antibacterial activity.

[0036] The salts of the dual inhibitor YS-121 are preferably pharmaceutically acceptable, but non-pharmaceutically acceptable salts are also acceptable within the scope of the present invention because they are useful intermediates for preparing pharmaceutically acceptable salts.

[0037] Suitable pharmaceutically acceptable salts include, but are not limited to, pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, aminosulfonic acid and hydrobromic acid, or pharmaceutically acceptable organic acids such as salts of acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxy acid, fumaric acid, malic acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, p-amino, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and valeric acid.

[0038] The corresponding base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, alkylammonium, for example, salts formed by triethylamine, alkoxyamines, such as those formed with ethanolamine, and salts formed from ethylenediamine, choline or amino acids such as arginine, lysine or histidine. Pharmaceutically acceptable salts and their formation and general information are well known to those skilled in the art, as described in general textbooks such as "Handbook of Pharmaceutical Salts" PHStahl, CGWermuth, 1st edition, 2002, Wiley-VCH.

[0039] In the present invention, the chemical name of the compound represented by formula (I) is 2-[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thiooctanoic acid.

[0040] In some preferred embodiments, the dual inhibitor YS-121 is a dual inhibitor of mPGES-1 and 5-lipoxygenase.

[0041] In some preferred embodiments, the polycystic kidney disease is autosomal dominant polycystic kidney disease.

[0042] In some preferred embodiments, the medicament is capable of at least reducing the kidney-to-body weight ratio in individuals with polycystic kidney disease.

[0043] In some preferred embodiments, the medicament is at least capable of inhibiting renal cyst formation and / or growth in individuals with polycystic kidney disease.

[0044] In some preferred embodiments, the medicament is capable of at least reducing the extent of kidney cysts in an individual with polycystic kidney disease.

[0045] In some preferred embodiments, the drug is at least capable of inhibiting the formation and / or growth of renal cysts in an individual and reducing the degree of renal cysts in an individual.

[0046] In some preferred embodiments, the drug is at least capable of inhibiting Forsklin-induced MDCK vesicle size.

[0047] In some preferred embodiments, the medicament is at least capable of improving kidney tissue morphological damage in individuals with polycystic kidney disease.

[0048] In some preferred embodiments, the drug can reduce the pH of normal mouse renal epithelial cells (pH + / - The DNA replication efficiency of cells.

[0049] In some preferred embodiments, the drug can reduce the polycystic kidney disease in mouse renal epithelial cells (pN - / - cells, PKD mouse kidney epithelial cells) DNA replication efficiency.

[0050] Another aspect of the embodiments of the present invention further provides a pharmaceutical composition for preventing and / or treating polycystic kidney disease, comprising a dual inhibitor YS-121 or a pharmaceutically acceptable derivative thereof; the dual inhibitor YS-121 has a structure as shown in formula (I):

[0051]

[0052] Wherein, the pharmaceutically acceptable derivative is selected from at least one of pharmaceutically acceptable salts, polymorphs, co-crystals, radiolabeled forms and combinations thereof.

[0053] In some preferred embodiments, the pharmaceutical composition also includes at least one pharmaceutically acceptable carrier, excipient, adjuvant or diluent. The carrier and / or excipient can be any known pharmaceutically acceptable carrier and excipient for those skilled in the art suitable for such use. The "pharmaceutical carrier" described in this specification has a meaning well known to those skilled in the art, and it can include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, similar substances and combinations thereof.

[0054] Furthermore, the pharmaceutically acceptable carrier includes a liquid carrier.

[0055] In some preferred embodiments, the dosage of the pharmaceutical composition administered to individuals with polycystic kidney disease is 2 mg / kg.

[0056] In some preferred embodiments, the pharmaceutical composition is administered subcutaneously, intramuscularly, via the tail vein, or intraperitoneally.

[0057] In some preferred embodiments, the pharmaceutical composition is capable of at least reducing the kidney-to-body weight ratio in individuals with polycystic kidney disease.

[0058] In some preferred embodiments, the pharmaceutical composition is at least capable of inhibiting renal cyst formation and / or growth in individuals with polycystic kidney disease.

[0059] In some preferred embodiments, the pharmaceutical composition is capable of at least reducing the extent of renal cysts in individuals with polycystic kidney disease.

[0060] In some preferred embodiments, the pharmaceutical composition is at least capable of inhibiting the formation and / or growth of renal cysts in an individual and reducing the degree of renal cysts in the individual.

[0061] In some preferred embodiments, the pharmaceutical composition is at least capable of inhibiting the size of MDCK vesicles induced by Forsklin.

[0062] In some preferred embodiments, the pharmaceutical composition is at least capable of improving kidney tissue morphological damage in individuals with polycystic kidney disease.

[0063] In some preferred embodiments, the pharmaceutical composition can reduce the pH of normal mouse renal epithelial cells (pH + / - The DNA replication efficiency of cells.

[0064] In some preferred embodiments, the pharmaceutical composition can reduce the expression of polycystic kidney disease in mouse renal epithelial cells (pN - / -cells, PKD mouse kidney epithelial cells) DNA replication efficiency.

[0065] Another aspect of the embodiments of the present invention further provides use of the aforementioned pharmaceutical composition in the preparation of a drug for preventing and / or treating autosomal dominant polycystic kidney disease.

[0066] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0067] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0068] Example 1: Vesicle growth inhibition experiment

[0069] MDCK cells can form cell colonies in three-dimensional matrix gel. If stimulated by forsklin, round vesicles can be formed, and the vesicles continue to grow. While stimulating forsklin, 1μmol / l, 10μmol / l, and 100μmol / l of YS-121 were given and the cells were cultured together. Fresh culture medium was replaced every day to maintain drug activity. On the 4th, 6th, 8th, 10th, and 12th day of culture, the vesicles were tracked in situ and photographed, and the vesicle diameter was measured to evaluate the effect of the compound represented by formula (I) in inhibiting vesicle growth. The inhibitory effect of YS-121 on vesicle growth is shown in Figure 2. Figure 4 The first row shows the results of culturing with a culture medium containing only 10 μmol / l forsklin, the second row shows the results of culturing with a culture medium containing 1 μmol / l YS-121 and 10 μmol / l forsklin, the third row shows the results of culturing with a culture medium containing 10 μmol / l YS-121 and 10 μmol / l forsklin, and the fourth row shows the results of culturing with a culture medium containing 100 μmol / l YS-121 and 10 μmol / l forsklin. The inhibitory effect curve of YS-121 shown in formula (I) on vesicle growth is shown in Figure 4 As shown, the above results indicate that the compound YS-121 represented by formula (I) can significantly inhibit the growth of vesicles.

[0070] Example 2: Determination of the toxic effect of compound YS-121 on MDCK

[0071] The CCK-8 method was used to determine whether the compound YS-121 represented by formula (I) had a toxic effect on MDCK.

[0072] First, the MDCK cell suspension in the logarithmic growth phase was inoculated into a 96-well culture plate (8×10 3 Cells / well), 100μl DMEM / F12 (1:1) culture medium was given to each well, and cultured in a 37℃, 5% CO2 incubator for 24 hours. Synchronize with a mixed culture medium containing 0.4% FBS for 12 hours. Discard the old culture medium, and then add fresh culture medium containing 0.1μmol / l; 1μmol / l; 10μmol / l; 100μmol / l; 1000μmol / l YS-121 to the 96-well plate, and continue to culture for 24 hours. 10μl CCK-8 detection solution was given to each well, and incubated at 37℃ in the dark for 60 minutes. The OD value of each well was detected by an enzyme reader (the detection wavelength was 450nm). Set up zero wells (containing culture medium and CCK-8) and control wells (containing cells, dissolution medium of the same concentration of compounds, culture medium and CCK-8), set 5 replicate wells for each group, and repeat the experiment three times. The results of the cytotoxicity determination of the compounds are shown in the following figure. Figure 4 As shown, the control is a control well containing cells, dissolution medium with the same concentration of the compound, culture medium, CCK-8, but not containing YS-121. The results show that YS-121, the compound represented by formula (I) at a concentration of 100 μmol / l and below, has no cytotoxic effect on MDCK cells, indicating that the inhibition of vesicle formation and growth by YS-121, the compound represented by formula (I), is not related to its cytotoxicity.

[0073] Example 3: H&E staining for PKD kidney histopathological observation

[0074] Experimental methods

[0075] Paraffin section preparation

[0076] (1) Fixation of tissue specimens: The renal cortical tissues of mice in each group in step 1 were fixed in 4% paraformaldehyde at room temperature for 24 hours, wrapped with gauze, marked, and rinsed with running water overnight;

[0077] (2) Dehydration and transparency: Dehydrate in 50%, 60%, 70%, 80% and 90% alcohol for 2 hours each, then dehydrate in 95% alcohol and 100% alcohol-I / II / III for 1 hour each, and transparentize in xylene-I / II for 30 minutes each;

[0078] (3) Wax immersion and embedding: Wax immersion was performed in a 58°C thermostat, with paraffin-I immersion for 1.5 hours and paraffin-II immersion for 2 hours. The samples were placed in an embedding box and embedded in paraffin at 60°C. After cooling and solidification, the wax blocks were taken out.

[0079] (4) Sectioning and spreading: Cut slices with a microtome to a thickness of 5 μm, spread the slices in a 50°C water bath, pick up the slices and mount them on a clean glass slide, and bake them in a 60°C oven overnight. After sectioning, mark them and save them for later use.

[0080] H&E staining

[0081] (1) Dewaxing and rehydration: The sections were dewaxed twice in xylene (15 min / time), then immersed in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0082] (2) Hematoxylin staining: The sections were placed in hematoxylin staining solution for 15 minutes, rinsed with tap water for 3 minutes, and separated with hydrochloric acid and alcohol for 10 seconds (99 ml of 70% alcohol + 1 ml of concentrated hydrochloric acid);

[0083] (3) Blueing and dehydration: Rinse with tap water for 10 minutes to turn it blue. Dehydrate the slices in 50%, 70%, 80%, and 90% alcohol for 5 minutes each;

[0084] (4) Eosin counterstaining: stain with 1% eosin solution for 2 minutes, dehydrate in 95% alcohol and 100% alcohol for 3 minutes respectively until the boundary is clear;

[0085] (5) Transparency and sealing: After xylene is transparent for 3 minutes, the slides are sealed with neutral gum;

[0086] (6) After sealing, the sections were placed in a 50°C oven for drying and the changes in the pathological structure of the renal tissue were observed under a light microscope.

[0087] Example 4: Immunohistochemical staining

[0088] (1) Drying: Place the paraffin sections in a 60°C oven and dry for at least 60 minutes;

[0089] (2) Dewaxing: Immerse the baked paraffin sections completely in xylene for dewaxing: xylene I for 20 minutes and xylene II for 20 minutes;

[0090] (3) Hydration: The dewaxed paraffin sections were completely immersed in different concentrations of ethanol for hydration treatment: 100% ethanol for 10 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 85% ethanol for 5 minutes, 70% ethanol for 5 minutes, and tap water or PBS for several times;

[0091] (4) Antigen repair: Add an appropriate amount of sodium citrate antigen repair solution to a pressure cooker, immerse the rinsed paraffin sections in the sodium citrate antigen repair solution (the liquid level does not cover the tissue), put the pressure cooker in a microwave and heat for 10 minutes until the antigen repair solution boils, open the lid and check for bubbles (bubbles indicate that the sodium citrate antigen repair solution has boiled), cover the lid and continue heating for 5 minutes, then open the lid and cool naturally at room temperature, usually about 30 minutes;

[0092] (5) Rinse the repaired paraffin sections with PBS three times, 5 minutes each time;

[0093] (6) Blocking endogenous catalase: Use 3% hydrogen peroxide to completely immerse the paraffin sections, block them in the dark at room temperature for 30 minutes, and then rinse the paraffin sections with PBS three times, each time for 5 minutes;

[0094] (7) Blocking endogenous antigens: Use 5% BSA antigen blocking solution prepared with 0.1% PBST at room temperature for 60 minutes;

[0095] (8) Primary antibody incubation: add 0.1% PBS diluted primary antibody working solution, incubate at 4°C overnight, and rinse with PBS three times, 5 minutes each time;

[0096] (9) Secondary antibody incubation: add an appropriate amount of HRP-labeled secondary antibody working solution of the corresponding species and incubate at room temperature for 60 min;

[0097] (10) DAB color development: Prepare 1× DAB color development solution according to the manufacturer's instructions for use of the reagent, add it dropwise to the dried paraffin tissue, let it react for a while, observe the color development under a microscope, terminate the staining with tap water in time, and rinse the paraffin sections with tap water several times;

[0098] (11) Hematoxylin counterstaining: Immerse the rinsed paraffin sections in hematoxylin stain for 10-20 seconds, then wash off the hematoxylin stain with tap water, and then soak the paraffin sections in PBS with a pH of 7.2-7.4 for 10 minutes;

[0099] (12) Tissue dehydration: Tissue dehydration was performed as follows: 70% ethanol for 5 min, 85% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 10 min, and 100% ethanol for 10 min;

[0100] (13) Paraffin clearing: Tissue clearing was performed as follows: xylene I for 20 min, xylene II for 20 min;

[0101] (14) Sealing: Add appropriate amount of resin to seal the slide, and make sure to remove all bubbles.

[0102] Example 5: Clik it Edu detects cell DNA replication

[0103] EdU with azide binds to DNA and then reacts with fluorescent alkyne to label the replicated DNA.

[0104] (1) Cell plating, add cell slices (sterilized), cell density 3*10 5 / well / 5ml (6cm plates), cultured in normal complete medium for 1 day;

[0105] (2) adding drug treatment on the second day, for example, culturing with complete medium containing 1 μM YS-121, 10 μM YS-121, and 100 μM YS-121 for 24 h;

[0106] (3) positive control: 3 mM HU for 1 h;

[0107] (4) EdU staining:

[0108] Add EdU at a final concentration of 40 μM and incubate for 20 min;

[0109] Discard the old culture medium and wash once with 1× PBS, 2 ml PBS / well;

[0110] Pre-extraction (on ice, pre-cool reagents):

[0111] Wash once with 1×CSK (2 ml per well, leave for a short time, and aspirate thoroughly);

[0112] Add CSK 0.5% Triton for 5 min (2 ml per well, aspirate thoroughly)

[0113] Wash once with 1×CSK (2 ml per well, leave for a short time, and aspirate thoroughly);

[0114] Wash once with 1× PBS (2 ml per well, leave for a short time, and aspirate clean);

[0115] · Fix with 4% PAF paraformaldehyde for 7 min at room temperature;

[0116] Remove paraformaldehyde and wash twice with 1×PBS, 2 min each time;

[0117] Preparation of Click-IT reaction cocktails

[0118] Component A 44μl Component B 1μl Component C 5μl 647 or 488 azide 0.05μl

[0119] Each slide requires 50 μl of mixed solution

[0120] Add the mixed solution and incubate at room temperature away from light for 30 minutes;

[0121] Wash once with PBS;

[0122] (5) PCNA staining:

[0123] Add pre-cooled methanol and place at -20°C for 15 min (to expose the antigen epitope);

[0124] Wash twice with PBS, 2 min each time;

[0125] PBST containing 5% BSA, block for 1 h at room temperature;

[0126] Primary antibody: PCNA (50 μl), diluted 1:200 in PBST containing 5% BSA, incubated at room temperature for 1 h (or overnight at 4°C);

[0127] Wash 3 times with PBST, 2 min each time (add PBST to the well plate first, and place the slides face up);

[0128] Secondary antibody diluted in PBST containing 5% BSA, rabbit antibody (50 μl), diluted 1:200, incubated at room temperature for 0.5 h, washed once with PBST for 2 min;

[0129] DAPI (1:500) staining, diluted in PBS, 5 min;

[0130] Wash three times with PBS, 2 min each time;

[0131] Seal the slides and observe under a microscope.

[0132] Data Analysis:

[0133] SPSS 16.0 software was used to analyze the experimental data. The t-test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. The data were expressed as mean±SEM. When P<0.05, it was considered statistically significant.

[0134] Experimental results

[0135] Figure 1a and Figure 1b The control group and the treatment group Ksp-Cre; Pkd1 flox / flox The kidney size and kidney weight ratio of mice are shown in the statistical chart, where the treatment group mice were subcutaneously injected with methyl acetate dissolved in 0.3 mg / ml of the compound represented by formula (I) YS-121, at a dose of 2 mg / kg, for 7 consecutive days from the 4th day after birth to the 11th day after birth; the control group mice were injected with an equal volume of methyl acetate every day. It can be seen that after administration, the kidney size decreased and the kidney weight index decreased, indicating that YS-121 can inhibit the development of kidney cysts in PKD mice.

[0136] Figure 2a and Figure 2b The control group and the treatment group Ksp-Cre; Pkd1 flox / flox The figure shows the results of HE staining of the kidneys of mice, where the mice in the treatment group were subcutaneously injected with the compound of formula (I) dissolved in methyl acetate at a concentration of 0.3 mg / ml for 7 consecutive days from the 4th day after birth to the 11th day after birth, with a dose of 2 mg / kg; the mice in the control group were injected with an equal volume of methyl acetate every day. It can be seen that the area of ​​cysts in the kidneys of mice after administration was reduced, and the morphology of renal tissue was significantly improved. This shows that YS-121 can inhibit the development of renal cysts in PKD mice.

[0137] Figure 3 This is a toxicity test of YS-121 on normal MDCK cells. The cell growth activity of MDCK cells after YS-121 treatment was observed by CCK8 detection. As shown in the figure, YS-121 concentration in the range of 0.1-100 μmol / l had no effect on the normal growth of MDCK.

[0138] Figure 4 The inhibitory effect of YS-121 on forsklin-induced MDCK cell cysts. Forsklin, a cAMP agonist, was used to induce cysts in MDCK dog kidney cells, which was used as a cell model of PKD cysts. The cells were then cultured with medium containing different concentrations of YS-121. It can be observed that YS-121 has an inhibitory effect on forsklin-induced MDCK cysts, and this inhibitory effect is dose-dependent.

[0139] Figure 5 The growth curve of MDCK cell vesicles is shown in Figure 1. The vesicle diameter is counted to measure the cyst size. The vesicle diameter is counted from the 4th day of drug administration to the 12th day to observe the vesicle growth. It can be seen that YS-121 has a significant inhibitory effect on cysts, and this inhibitory effect increases with the increase of dose.

[0140] Figure 6a-6b and Figure 7a-Figure 7b PKD mice were given methyl acetate (control) and YS-121 (administered) on the fourth day after the onset of the disease, and kidney sections were collected on the 11th day for immunohistochemical staining. PCNA and Ki67 are both proliferation markers, and their expression reflects the proliferation ability of cells. It can be seen that after the administration of YS-121, the expression levels of PCNA and Ki67 proteins in kidney tissues were significantly downregulated. Therefore, YS-121 is likely to alleviate the disease pathology of PKD by inhibiting proliferation.

[0141] Figure 8a-8b pH + / - and pN - / -Immunofluorescence experiments were performed to detect the DNA replication efficiency after the cells were administered with different concentrations of YS-121. It can be seen that after treatment with compound YS-121, the fluorescence intensity of PCNA was reduced, and the DNA replication efficiency in the S phase was also reduced.

[0142] In summary, through the above experimental comparison, the compound YS-121 shown in formula (I) can significantly reduce the kidney weight ratio and cyst index of ADPKD mice, improve renal tissue morphology, significantly reduce the damage of renal tissue morphology in polycystic kidney mice, and significantly improve the size of cell vesicles in vitro. At the same time, this drug can significantly inhibit the proliferation ability of PKD renal tubular tissue and reduce the replication efficiency of DNA. It shows that the compound can improve autosomal dominant polycystic kidney disease and provide valuable reference significance for the clinical prevention and treatment of ADPKD.

[0143] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0144] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. Use of a dual inhibitor YS-121 or a pharmaceutically acceptable derivative thereof in the preparation of a medicament for preventing and / or treating autosomal dominant polycystic kidney disease, wherein the dual inhibitor YS-121 has a structure as shown in formula (I): Formula (I) in, The pharmaceutically acceptable derivative is selected from at least one of a pharmaceutically acceptable salt, a polymorph, a co-crystal, a radiolabeled form and a combination thereof.

2. The use according to claim 1, characterized in that: The dual inhibitor YS-121 is a dual inhibitor of mPGES-1 and 5-lipoxygenase.

3. The use according to claim 1, characterized in that: The medicament further comprises at least one pharmaceutically acceptable carrier.

4. The use according to claim 1, characterized in that: The medicament further comprises at least one pharmaceutically acceptable excipient.

5. The use according to claim 1, characterized in that: The medicament further comprises at least one pharmaceutically acceptable auxiliary agent.

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