Use of ciprofibrate in the preparation of a drug for anti-hepatitis B virus

Abstract

The present invention relates to the use of ciprofibrate in the preparation of a drug for anti-hepatitis B virus, belonging to the field of biomedical technology. The present invention provides the use of ciprofibrate in the preparation of a drug for anti-hepatitis B virus. Ciprofibrate reduces the expression levels of hepatitis B surface antigen HBsAg, hepatitis B e antigen HBeAg, and hepatitis B core antigen HBcAg in human primary cells infected with hepatitis B virus, inhibits hepatitis B virus from infecting cells, inhibits the proliferation and replication of hepatitis B virus, can protect neonatal hepatocytes from being reinfected, has an anti-hepatitis B virus effect, and thus prevents and treats diseases caused by hepatitis B virus. Ciprofibrate, as a lipid-regulating drug, has minor side effects, does not require subcutaneous injection, and has high patient compliance.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the application of ciprofibrate in the preparation of a drug for anti-hepatitis B virus. Background Art

[0002] Hepatitis B virus (HBV) infection and its related diseases affect more than 250 million people worldwide. Nearly 820,000 people die each year from complications of liver failure, cirrhosis, and hepatocellular carcinoma (HCC) caused by persistent HBV infection. HBV infection remains a major global public health problem. Existing treatment drugs include pegylated interferon α and third-generation nucleotide analogs. Pegylated interferon α exerts its antiviral effect by regulating immune responses or directly inhibiting viral replication in hepatocytes. The third-generation nucleotide analogs can inhibit viral reverse transcription by targeting HBV polymerase and inhibit HBV replication in host hepatocytes.

[0003] However, pegylated interferon α needs to be administered by injection, with poor patient compliance, and interferon has relatively large side effects. Although the third-generation nucleotide analogs can reduce the activity of HBV polymerase, it is difficult to completely eliminate the virus, and the vast majority of patients need to take the drugs for life. Although pegylated interferon α and the third-generation nucleotide analogs can inhibit the expression of HBV antigens, they cannot completely eliminate them and cannot protect newly generated hepatocytes from infection.

[0004] Existing drugs for treating diseases caused by HBV mainly clear the virus by inhibiting HBV replication. These drugs only act on HBV in infected hepatocytes and cannot protect healthy hepatocytes from re-infection by HBV. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a new drug for inhibiting hepatitis B virus infection, namely the application of ciprofibrate in the preparation of a drug for anti-hepatitis B virus.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides the application of ciprofibrate in the preparation of a drug for anti-hepatitis B virus.

[0008] Further, the drug is a drug for inhibiting hepatitis B virus infection.

[0009] Further, the drug is a drug for inhibiting hepatitis B virus replication and / or proliferation.

[0010] In a specific embodiment of the present invention, the drug is a drug that reduces the expression level of at least one of hepatitis B surface antigen HBsAg, hepatitis B e antigen HBeAg, and hepatitis B core antigen HBcAg.

[0011] Further, the drug further contains a pharmaceutically acceptable additive or / and excipient or / and carrier.

[0012] Further, the drug is a tablet, granule, or liquid preparation.

[0013] In a second aspect, the present invention provides the use of ciprofibrate in the preparation of a drug for preventing and / or treating diseases caused by hepatitis B virus.

[0014] Further, the drug is a drug for preventing and / or treating hepatitis B.

[0015] Further, the drug is a drug for preventing and / or treating liver failure, liver cirrhosis, and hepatocellular carcinoma and their complications caused by hepatitis B virus.

[0016] Further, the drug is an anti-hepatitis B virus drug.

[0017] Further, the drug is a drug for inhibiting hepatitis B virus infection.

[0018] Further, the drug is a drug for inhibiting hepatitis B virus replication and / or proliferation.

[0019] In a specific embodiment of the present invention, the drug is a drug that reduces the expression level of at least one of hepatitis B surface antigen HBsAg, hepatitis B e antigen HBeAg, and hepatitis B core antigen HBcAg.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The existing anti-hepatitis B virus drugs mainly clear hepatitis B virus HBV by inhibiting virus replication. These drugs only act on the hepatitis B virus in the infected hepatocytes and cannot protect healthy hepatocytes from being reinfected by hepatitis B virus. The present invention has found through research that ciprofibrate reduces the expression levels of hepatitis B surface antigen HBsAg, hepatitis B e antigen HBeAg, and hepatitis B core antigen HBcAg in human primary cells infected with hepatitis B virus, inhibits hepatitis B virus from infecting cells, inhibits the replication and proliferation of hepatitis B virus, can protect neonatal hepatocytes from being reinfected, has an anti-hepatitis B virus effect, and thus prevents and treats diseases caused by hepatitis B virus. Ciprofibrate is a peroxisome proliferator-activated receptor agonist, has few side effects as a lipid-lowering drug, does not require subcutaneous injection, and has high patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1For the binding of different small molecule compound probes to hepatitis B surface antigen L-HBsAg protein.

[0022] Figure 2 For the effect of ciprofibrate on the contents of HBsAg and HBeAg after human primary cells are infected with HBV virus. Among them, A is HBsAg; B is HBeAg.

[0023] Figure 3 For the immunofluorescence staining map of human primary cells after human primary cells are infected with HBV virus and treated with ciprofibrate.

[0024] Figure 4 For the dose-effect curve of ciprofibrate inhibiting the growth of HepG2-NTCP cells.

[0025] Figure 5 For the contents of HBsAg and HBeAg when ciprofibrate is added while HepG2-NTCP cells are infected with HBV virus. Among them, A is HBeAg; B is HBsAg.

[0026] Figure 6 For the contents of HBsAg and HBeAg when ciprofibrate is added 48 hours after HepG2-NTCP cells are infected with HBV virus. Among them, A is HBeAg; B is HBsAg. Specific implementation manners

[0027] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0028] Recovery medium (manufacturer: Shenzhen Liwo Company, product number: LV-Rec001), purification medium (manufacturer: Shenzhen Liwo Company, product number: LV-PHIK00108), plating medium (manufacturer: Shenzhen Liwo Company, product number: LV-WEP001), maintenance medium (manufacturer: Shenzhen Liwo Company, product number: LV-WEM00), differentiation medium (manufacturer: TAKARA, product number: Y30051).

[0029] Example 1 Binding of small molecule compound probe to hepatitis B surface antigen L-HBsAg protein

[0030] I. Experimental method

[0031] The small molecule compound probes are respectively: sofalcone, prednisone, dexamethasone, isotretinoin, rhein, eupatoriopicrin B, amlodipine, finasteride, 4-hydroxycinnamic acid, ciprofibrate, testosterone, flumequine and cryptochlorogenic acid.

[0032] 1. Mix each small molecule compound probe with hepatitis B surface antigen L-HBsAg protein in phosphate buffered saline PBS (1 μg / mL) to prepare a 20 μL sample reagent containing 40 μmol small molecule compound probe and 1 μg hepatitis B surface antigen L-HBsAg protein. Incubate gently with shaking at 21 °C for 1 h to obtain the incubated sample.

[0033] 2. Mix NaVc (sodium acetate, solvent is double-distilled water ddH2O), THPTA (tris(3-hydroxypropyltriazolylmethyl)amine, solvent is dimethyl sulfoxide DMSO), CuSO4 (copper sulfate, solvent is double-distilled water ddH2O) and TAMRA-azide (tetramethylrhodamine azide, solvent is DMSO) to prepare a click chemical reaction mixture containing 1 mmol / L NaVc, 100 mmol / L THPTA, 1 mmol / L CuSO4 and 50 mmol / L TAMRA-azide. Add 33 μL of the click chemical reaction mixture to each incubated sample in step 1 and incubate at room temperature 25 °C for 1 - 2 h to obtain the sample after click chemical reaction.

[0034] 3. Precipitate the sample after click chemical reaction in step 2 with pre-cooled acetone (-20 °C) overnight (16 h) and collect the precipitate.

[0035] 4. Centrifuge the precipitate in step 3 at 12000 rpm for 10 min at 4 °C, discard the supernatant, collect the precipitate and let the acetone evaporate.

[0036] 5. Dissolve the precipitate treated in step 4 in 40 μL of 1×loading buffer, load it onto an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel and separate by electrophoresis, and visualize by fluorescence scanning in a laser scanner (manufacturer: Azure Biosystems, model: Sapphire RGBNIR). Stain the SDS-PAGE gel with Coomassie Brilliant Blue for 10 min, wash with water overnight and take a picture the next day.

[0037] II. Experimental Results

[0038] As Figure 1 shown, ciprofibrate binds specifically to L-HBsAg protein, and other small molecule compound probes cannot bind specifically to L-HBsAg protein.

[0039] Example 2 Effect of Ciprofibrate on the Infection of Human Primary Cells with Hepatitis B Virus HBV

[0040] I. Experimental Method

[0041] 1. Prepare two tubes of recovery medium and pre-warm it to 37°C to obtain pre-warmed recovery medium. Take out two tubes of cryopreserved human primary hepatocytes from liquid nitrogen and quickly thaw them in a 37°C water bath by rotating clockwise rapidly until only a small ice ball remains to obtain thawed human primary hepatocytes. Use a 1000 μl pipette to gently mix the thawed human primary hepatocytes and then aspirate them, and add them drop by drop into the pre-warmed recovery medium to obtain recovery medium containing human primary hepatocytes.

[0042] 2. Gently rotate and shake the recovery medium containing human primary hepatocytes in step 2, place it in an incubator and let it stand for 30 min, then centrifuge it at 50 g for 5 min at room temperature (25°C) to collect the cells.

[0043] 3. Resuspend the cells processed in step 2 with 2 ml of purification medium, slowly add 1 ml of plating medium (with obvious stratification) along the tube wall, centrifuge it at 800 g for 20 min at 4°C (the centrifuge speed is set as: acceleration is 9, deceleration is 1), take the turbid band (viable cell layer) between the purification medium and the plating medium, and transfer it to a new 15 ml centrifuge tube.

[0044] 4. Add plating medium with a volume twice that of the viable cell layer to the cells processed in step 3, mix well, then centrifuge it at 50 g at room temperature for 5 min, discard the supernatant, and collect the cells.

[0045] 5. Rinse the 24-well plate coated with rat tail collagen with sterile PBS, resuspend the cells collected in step 4 with 5 ml of plating medium, plate 2000 cells per well, and after plating for 24 h, change the medium to maintenance medium (PHH) and culture for 24 h.

[0046] 6. Perform HBV virus infection:

[0047] Using differentiation medium (IPS) as a solvent, dilute polyethylene glycol 8000 with a mass concentration of 40% (polyethylene glycol with an average molecular weight of 8000) to polyethylene glycol 8000 with a mass concentration of 4% to prepare IPS containing 4% polyethylene glycol 8000. Calculate the volume of HBV virus required according to the number of wells to be infected (20 μl / well), add the HBV virus to the IPS containing 4% polyethylene glycol 8000, and gently mix to prepare an infection system.

[0048] Using the infection system as a solvent, prepare ciprofibrate with concentrations of 0.25 mM and 0.5 mM (drug addition group) and Myrcludex B with a concentration of 0.2 μM (positive control group), and use the infection system without adding drugs as a negative control, add 500 μl / well to the cells cultured in step 5.

[0049] 7. At 24 h after infection in step 6, replace the ciprofibrate, Myrcludex and the infection system with maintenance medium, culture for 24 h, then replace the fresh maintenance medium every 48 h, and collect the medium; detect the contents of hepatitis B surface antigen HBsAg and hepatitis B e antigen HBeAg in the medium using the Architect system and the chemiluminescent microparticle immunoassay (CMIA) kit (manufacturer: Abbott Laboratories).

[0050] 8. On the 10th day of infection in step 7, collect the cells and perform immunofluorescence staining:

[0051] (1) Wash the cells once with sterile PBS, fix with 4% paraformaldehyde by mass concentration for 15 min, and wash the cells 3 times with 500 μl PBS.

[0052] (2) Mix the cells treated in step (1) with 0.5% TritonX-100 by volume concentration (polyethylene glycol octylphenyl ether, solvent is PBS), permeabilize the membrane at room temperature (25 °C) for 15 min, and then wash the cells 3 times with 500 μl PBS.

[0053] (3) Block the cells treated in step (2) with ready-to-use normal goat serum at room temperature (25 °C) for 60 min, wash the cells 3 times with 500 μl / well PBS, 5 min each time.

[0054] (4) Gently add the primary antibody of hepatitis B core antigen HBcAg into the wells treated in step (3), incubate overnight (12 h) at 4 °C, and wash the cells 3 times with 500 μl / well PBS, 5 min each time.

[0055] (5) Incubate the cells treated in step (4) with mouse secondary antibody in the dark on a shaker at room temperature for 1 h; after incubation, wash the cells 3 times with 500 μl / well PBS in the dark, 5 min each time.

[0056] (6) Add dapi (4’,6-diamidino-2-phenylindole) to cover the cell surface treated in step (5) for nuclear counterstaining, and take pictures with an inverted microscope IX73.

[0057] II. Experimental Results

[0058] (1) As shown in Figure 2 A and Figure 2 B, by detecting the antigen expression levels in the ciprofibrate addition group and the control group, it can be seen that compared with the control group, the antigen expression level in the ciprofibrate addition group is significantly reduced.

[0059] (2) As shown in Figure 3As shown, by comparing the immunofluorescence of the ciprofibrate-treated group and the control group, it can be seen that the fluorescence of HBcAg decreased in the ciprofibrate-treated group.

[0060] Ciprofibrate inhibits HBV infection of primary human cells at the HBV infection stage, thereby reducing the expression levels of HBsAg and HBeAg.

[0061] Example 3 The IC of ciprofibrate inhibiting the growth of HepG2-NTCP cells (a liver cancer cell line overexpressing sodium taurocholate cotransporting polypeptide (NTCP)-1) 50

[0062] I. Experimental methods

[0063] 1. Seed HepG2-NTCP cells into 96-well plates at 8000 cells per well, and culture the cells in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum by volume concentration in a cell incubator at 37°C and 5% CO2 for 24 h.

[0064] 2. Add ciprofibrate at final concentrations of 5 mM, 1 mM, 200 μM, 40 μM, 8 μM, 1.6 μM, 320 nM, 64 nM, and 12.8 nM to the wells in step 1, and replace one column with fresh DEME medium containing 10% fetal bovine serum by volume concentration as the experimental control well.

[0065] 3. Place the cells treated in step 2 into the incubator for culture. After 24 h, add 10 μl of CCK8 reagent (Cell Counting Kit-8 cell counting reagent) to each well, incubate in the incubator at 37°C and 5% CO2 for 1 h, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance.

[0066] II. Experimental results

[0067] As Figure 4 shown, the IC of ciprofibrate inhibiting the growth of HepG2-NTCP cells 50 is 1.704 mM. This example studied the reasonable use concentration of ciprofibrate. The use concentrations of ciprofibrate at 500 μM and 250 μM do not affect cell proliferation.

[0068] Example 4 The effects of ciprofibrate on HepG2-NTCP cells before and after HBV infection

[0069] I. Experimental methods

[0070] 1. Plate HepG2-NTCP cells into a 24-well plate moistened with rat tail collagen at a cell density of 0.4 M per well. Shake to ensure uniform cell spreading. Culture the cells in DEME medium containing 10% fetal bovine serum in a cell incubator at 37°C and 5% CO2. After the cells adhere, replace the medium with differentiation medium (IPS) and culture for 24 h.

[0071] 2. The HepG2-NTCP cells cultured in step 1 were infected with HBV virus using the same infection method as in Example 2, wherein the drug-dosing group was divided into two groups:

[0072] (1) Group A was infected with HBV virus and ciprofibrate was added at the same time: the infection system was used as the solvent, and ciprofibrate at concentrations of 0.25 mM and 0.5 mM was prepared as the drug-adding group, 500 μl / well, and added to the HepG2-NTCP cells cultured in step 2. After 24 hours of infection, the solvent of 0.25 mM and 0.5 mM ciprofibrate and 0.2 μM Myrcludex was replaced with IPS medium, and the cells were cultured with drugs for 24 hours; the solvent of 0.25 mM and 0.5 mM ciprofibrate and 0.2 μM Myrcludex was replaced with PHH medium, and the cells were cultured with drugs. The drugs were added in the form of liquid replacement every 48 hours, and the culture medium was collected;

[0073] (2) Group B was infected with HBV virus and then ciprofibrate was added: 500 μl / well of the infection system was added to the HepG2-NTCP cells cultured in step 2. After 24 hours of infection, the infection system was replaced with IPS culture medium and cultured for 24 hours; ciprofibrate with PHH as solvent was added at concentrations of 0.25 mM and 0.5 mM, respectively, as the drug-addition group, 500 μl / well, and the drug was added in the form of liquid replacement every 48 hours, and the culture medium was collected.

[0074] 3. The contents of hepatitis B surface antigen HBsAg and hepatitis B e antigen HBeAg in the culture medium of step 2 were detected using the Architect system and chemiluminescent microparticle immunoassay (CMIA) kit (Abbott Laboratories).

[0075] 2. Experimental Results

[0076] like Figure 5 A and Figure 5 As shown in B, in group A, drugs were added simultaneously with HBV infection, and the levels of HBsAg and HBeAg in the supernatant of the 0.25 mM and 0.5 mM ciprofibrate groups were lower than those in the control group.

[0077] like Figure 6 A and Figure 6As shown in B, the drugs were added to Group B 48 hours after HBV infection. The content of HBeAg in the supernatant of the ciprofibrate 0.25 mM group and 0.5 mM group decreased. The content of HBsAg in the supernatant of the ciprofibrate 0.5 mM group decreased compared with the control group, while the content of HBsAg in the ciprofibrate 0.25 mM group did not show a significant decrease compared with the control group.

[0078] In summary, ciprofibrate inhibits viral biosynthesis, inhibits virus release, and enhances the host's antiviral ability.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of ciprofibrate in the preparation of a drug for anti-hepatitis B virus.

2. The application according to claim 1, characterized in that, The drug is a drug for inhibiting hepatitis B virus infection.

3. The application according to claim 1, wherein The drug is a drug for inhibiting hepatitis B virus replication and / or proliferation.

4. The application according to claim 1, characterized in that, The drug is a drug for reducing the expression level of at least one of hepatitis B surface antigen HBsAg, hepatitis B e antigen HBeAg and hepatitis B core antigen HBcAg.

5. The application according to any one of claims 1 to 4, characterized in that, The drug further contains pharmaceutically acceptable additives or / and excipients.

6. The application according to claim 5, characterized in that, The drug is a tablet, granule or liquid preparation.

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