Application of AU15330 in preparation of medicine for treating hepatitis B

Abstract

The invention provides an application of AU15330 in preparing a medicine for treating hepatitis B or inhibiting HBV (Hepatitis B Virus). The application proves that AU15330 inhibits the transcriptional activity of cccDNA by inhibiting the accessibility of HBV cccDNA chromatin, so that the replication ability of HBV is reduced. The invention provides a new medicine for treating related diseases such as hepatitis B and hepatocellular carcinoma, and the provided target also provides a new choice for development of other similar medicines.

Description

Technical Field

[0001] The present application belongs to the field of hepatitis B treatment. Specifically, the present application provides the use of AU15330 in the preparation of a drug for treating hepatitis B. Background Art

[0002] Hepatitis B is a worldwide epidemic. Therefore, the task of preventing and treating hepatitis B is still very arduous and is a major issue faced by preventive medicine, social medicine and health administrative departments.

[0003] At present, the main drugs used in clinical treatment of hepatitis B are interferon and nucleoside analogs. However, interferon is only effective for less than 30% of patients, and has large side effects, so its application is subject to certain restrictions; nucleoside analogs have a long course of treatment, are prone to viral resistance and virological rebound after drug withdrawal. Therefore, in order to further improve the treatment effect of patients with current hepatitis B, it is necessary to fully understand the replication and regulation mechanism of the virus in the body and develop new treatment methods and strategies, which are of great significance to the treatment and prevention of hepatitis B. Summary of the invention

[0004] In view of the above situation, on the one hand, the present application provides the use of AU15330 in the preparation of drugs for treating hepatitis B.

[0005] On the other hand, the present application provides the use of AU15330 in the preparation of drugs for inhibiting HBV replication and transcription.

[0006] On the other hand, the present application provides a drug for treating and inhibiting HBV replication and transcription, wherein the drug comprises AU15330.

[0007] Furthermore, AU15330 is the only active ingredient in the drug.

[0008] Furthermore, the drug also contains other ingredients for treating hepatitis B or inhibiting HBV replication and transcription.

[0009] The other ingredients for treating hepatitis B or inhibiting HBV may be selected from nucleoside drugs, interferon drugs or Chinese and Western medicines for protecting the liver.

[0010] Furthermore, the drug inhibits HBV transcription.

[0011] Furthermore, the drug inhibits HBV replication.

[0012] Furthermore, the drug inhibits the accessibility of HBV cccDNA chromatin and thereby inhibits the transcriptional activity of cccDNA, thereby reducing the replication ability of HBV.

[0013] Furthermore, the medicine is an injection or an oral preparation.

[0014] The available dosage forms of the present application include, but are not limited to, injections such as injection solutions, powder injections, etc., and oral preparations such as tablets, capsules, oral solutions, etc.

[0015] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0016] Those skilled in the art can select suitable excipients for the drugs involved in this application based on general knowledge in the pharmaceutical field. Available excipients include but are not limited to solvents, co-solvents, pH regulators, osmotic pressure regulators, surfactants, thickeners, dispersants, viscosity regulators, antioxidants, masking agents, sweeteners, fillers, coating agents, capsule shells, adhesives, lubricants, etc.

[0017] AU15330 described in the present application is a proteolytic targeting chimera (PROTAC) degrader of SWI / SNF ATPase subunits SMARC2 and SMARC4, and its CAS number is 2380274-50-8.

[0018] In addition to demonstrating the inhibitory effect of AU15330 on HBV, this application also uses a variety of in vivo and in vitro HBV models to detect indicators related to viral activity, clarifying the role of AU15330 in inhibiting HBV replication and transcription; by analyzing the accessibility of cccDNA microchromatin and the epigenetic panorama, in-depth research is conducted on the mechanism of action of AU15330 in inhibiting HBV replication.

[0019] This application clarifies the use of AU15330 in the preparation of drugs for treating hepatitis B / drugs for inhibiting HBV replication, provides a new theoretical basis for the discovery of new targets and the treatment of chronic hepatitis B, and will also lay the foundation for exploring molecular targets and drug development for the prevention and treatment of hepatitis B, hepatocellular carcinoma and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the experimental results for evaluating the regulatory effect of AU15330 on HBV cccDNA transcription in HepAD38 cells; Part A is the experimental scheme; Part B shows the mRNA level; Part C shows the cccDNA level; Part D shows preCoremRNA / cccDNA; Part E shows HBV RNA and HBV DNA levels; Parts F and G show HBcAg levels (green).

[0021] Figure 2The experimental results of evaluating the regulatory effect of AU15330 on HBV cccDNA transcription in HepG2-NTCP cells. Part A is the experimental scheme; Part B shows the mRNA level; Part C shows the cccDNA level; Part D shows preCoremRNA / cccDNA; Part E shows HBV RNA level; Parts F and G show HBcAg level (green) and BRG1 (red).

[0022] Figure 3 The effect of AU15330 on HBV replication products in HepG2.2.15 cells and the experimental results of HBV cccDNA accessibility analysis using transposase chromatin accessibility analysis (Mnase-qPCR) were analyzed. Part A shows the mRNA level; Part B shows the HBc and BRG1 levels; Part C shows the MNase-qPCR analysis results; Part D shows the HBV promoter activity level. DETAILED DESCRIPTION

[0023] Example 1 Evaluation of the regulatory effect of AU15330 on HBV cccDNA transcription in HepAD38 cells

[0024] HepAD38 cells were cultured to confluence in 35 mm culture dishes, and then tetracycline was removed from the culture medium to induce HBV replication and cccDNA formation. After 10 days, tetracycline and lamivudine (3TC, 10mM) were added to the culture medium to shut down the transcription of viral pgRNA from the integrated HBV genome and prevent viral DNA replication. After 4 days, one group of cells continued to be cultured in a medium containing tetracycline and 3TC, while another group of cells was treated with AU15330 (5mM) for 4 days in the presence of tetracycline and 3TC. The DMSO concentration in all experimental groups was standardized to 0.1%.

[0025] Perform the following tests:

[0026] Total RNA was extracted and subjected to q-PCR analysis of preCore and RT-qPCR analysis of pgRNA and total HBV RNA; HBVHirt DNA was extracted and qPCR was performed to quantify cccDNA. The relative levels of cccDNA-based transcription were further determined by normalizing the preCore mRNA levels in each treatment group to the cccDNA levels in the DMSO control group.

[0027] HepAD38 cells were induced in tetracycline-free medium for 10 days to establish cccDNA, and then treated with DMSO or AU15330 (5mM) every 2 days for 4 days in the presence of 3TC (10mM) to analyze HBV RNA and HBcAg levels. Among them, 3.5kb preCore mRNA and pgRNA and 2.4 / 2.1kb HBs mRNA were labeled. 28S and 18S rRNA were used as loading controls. HBV cytoplasmic core DNA replication intermediates were detected by Southern blotting. The nuclei were stained blue with DAPI dye.

[0028] The results are as follows Figure 1 As shown: Part B shows that AU15330 significantly inhibits the levels of preCore, pgRNA, and total HBV RNA; Part C shows that AU15330 does not affect the level of HBV cccDNA; Part D shows that AU15330 significantly inhibits the transcriptional activity of HBV cccDNA; Part E shows that AU15330 significantly inhibits the levels of HBV RNA and DNA; Part F shows that AU15330 significantly inhibits the level of HBc by immunofluorescence staining; Part G shows that AU15330 significantly inhibits the level of HBc protein. This was also confirmed by Northern blot and Southern blot analysis of HBV replication products at the RNA and DNA levels.

[0029] Example 2 Evaluation of the regulatory effect of AU15330 on HBV cccDNA transcription in HepG2-NTCP cells

[0030] AU15330 antiviral evaluation was validated in the HBV infection system. HepG2-NTCP cells were infected with HBV particles (10 7 After 24 hours, the virus-containing medium was removed and the cells were cultured for an additional 7 days in the presence of AU15330.

[0031] Perform the following tests:

[0032] Total RNA was extracted and qRT-PCR of preCore and pgRNA and RT-qPCR of total HBV RNA were performed; HBV Hirt DNA was extracted and qPCR quantification of cccDNA was performed; the relative level of cccDNA-based transcription was further determined by normalizing the preCore mRNA level in each treatment group to the cccDNA level of the DMSO control group.

[0033] After 1 day post-infection with HBV (dpi), cells were treated with DMSO or AU15330 (5 mM) and collected at 7 dpi to analyze the levels of HBV RNA (E), HBcAg, and BRG1. 3.5 kb preCore mRNA, pgRNA, and 2.4 / 2.1 kb surface mRNA were labeled. 28S and 18S rRNA were used as loading controls. Nuclei were stained with DAPI dye (blue).

[0034] The results are as follows Figure 2 As shown, in the infection system, part B shows that AU15330 significantly inhibits the levels of preCore, pgRNA, and total HBV RNA; part C shows that AU15330 does not affect the level of HBV cccDNA; part D shows that AU15330 significantly inhibits the transcription activity of HBV cccDNA; part E shows that AU15330 significantly inhibits the level of HBV RNA; part F immunofluorescence staining shows that AU15330 significantly inhibits the level of HBc; part G shows that AU15330 significantly inhibits the level of HBc protein, which is further confirmed by Northern blot analysis of HBV replication products at the RNA level.

[0035] Example 3 Effect of AU15330 on HBV replication products in HepG2.2.15 cells

[0036] HepG2.2.15 cells were treated with DMSO or AU15330 (5 mM) every two days for a total of 4 days and used for RT-qPCR analysis of preCore mRNA, pgRNA and total HBV RNA levels as well as HBc levels.

[0037] The results are as follows Figure 3 As shown in parts A and B, the results showed that AU15330 also significantly reduced the RNA level (shown in part A) and protein level (shown in part B) of HBV replication products in HepG2.2.15 cells, indicating that AU15330 not only inhibits the transcriptional activity of HBV cccDNA, but also plays a role in inhibiting the entire replication cycle of HBV.

[0038] Example 4 Analyzing the accessibility of HBV cccDNA and HBV promoter activity using transposase chromatin accessibility analysis (Mnase-qPCR) and dual luciferase activity detection

[0039] After 1 day of HBV infection, HepG2-NTCP cells were treated with DMSO or AU15330 (5 mM) and collected at 7 dpi for MNase-qPCR analysis. HepG2 cells seeded in 96-well plates were co-transfected with 100 ng of Xp / Cp / S1p / S2p-Luc, and DMSO control AU15330 (5 mM) was added to analyze HBV promoter activity.

[0040] The results are as follows Figure 3 As shown in parts C and D of the figure, the results show that part C shows that AU15330 leads to reduced accessibility of almost all regions except the enhancer II region. Consistent with the chromatin accessibility data, part D shows that AU15330 leads to decreased HBV promoter activity. These results indicate that AU15330 regulates the microchromatin accessibility of cccDNA to reduce the transcriptional activity of cccDNA, thereby inhibiting HBV replication.

Claims

1. Application of AU15330 in the preparation of drugs for treating hepatitis B.

2. Application of AU15330 in the preparation of drugs for inhibiting HBV.

3. A drug for treating and inhibiting HBV, characterized in that: The drug contains AU15330.

4. The use according to claim 1 or 2 or the medicine according to claim 3, wherein AU15330 is the only active ingredient.

5. The use according to claim 1 or 2 or the medicine according to claim 3, wherein the medicine further comprises other ingredients for treating hepatitis B or inhibiting HBV.

6. The use according to claim 2, wherein the drug inhibits HBV transcription.

7. The use according to claim 2, wherein the drug inhibits HBV replication.

8. The use according to claim 2, wherein the drug inhibits the accessibility of HBV cccDNA chromatin and thereby inhibits the transcriptional activity of cccDNA, thereby reducing the replication ability of HBV.

9. The use according to claim 1 or 2 or the medicine according to claim 3, wherein the medicine is an injection or an oral preparation.

10. The use according to claim 1 or 2 or the medicine according to claim 3, further comprising a pharmaceutically acceptable excipient.

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