Use of AU15330 in the manufacture of a medicament for the treatment of hepatitis B

Abstract

The application provides an application of AU15330 in preparation of a medicine for treating hepatitis B or inhibiting HBV. The application proves that AU15330 can inhibit the transcription activity of cccDNA by inhibiting the accessibility of HBV cccDNA chromatin, thereby reducing the replication ability of HBV. The application provides a new medicine for treating hepatitis B and related diseases such as hepatocellular carcinoma, and the target point also provides a new selection for the development of other such medicines.

Description

Technical Field

[0001] This application pertains to the field of hepatitis B treatment, specifically, this application provides the use of AU15330 in the preparation of a drug for treating hepatitis B. Background Technology

[0002] Hepatitis B is a globally prevalent infectious disease. Therefore, the prevention and control of hepatitis B remains a very challenging task, a major issue jointly faced by preventive medicine, social medicine, and health administration departments.

[0003] Currently, the main drugs used clinically to treat hepatitis B are interferon and nucleoside analogues. However, interferon is effective in less than 30% of patients and has significant side effects, limiting its application. Nucleoside analogues have long treatment courses and are prone to viral resistance and virological rebound after discontinuation. Therefore, to further improve the treatment outcomes for patients with active hepatitis B, it is necessary to fully understand the viral replication regulation mechanisms in the body and develop new treatment methods and strategies, which is of great significance for the treatment and prevention of hepatitis B. Summary of the Invention

[0004] In response to the above situation, this application provides the use of AU15330 in the preparation of drugs for treating hepatitis B.

[0005] On the other hand, this application provides the use of AU15330 in the preparation of drugs that inhibit HBV replication and transcription.

[0006] On the other hand, this application provides a medicament for treating the inhibition of HBV replication and transcription, said medicament comprising 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] Other components for treating hepatitis B or inhibiting HBV may include nucleoside analogues, interferon drugs, or hepatoprotective Chinese and Western medicines.

[0010] Furthermore, the drug inhibits HBV transcription.

[0011] Furthermore, the drug inhibits HBV replication.

[0012] Furthermore, the drug reduces HBV replication capacity by inhibiting the accessibility of HBV cccDNA chromatin, thereby inhibiting the transcriptional activity of cccDNA.

[0013] Furthermore, the drug is an injectable or oral preparation.

[0014] The applicable dosage forms of this application include, but are not limited to, injectable preparations such as injection solutions and powder injections, and oral preparations such as tablets, capsules, and oral solutions.

[0015] Furthermore, the drug 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. The types of excipients that can be used include, but are not limited to, solvents, cosolvents, pH adjusters, osmotic pressure adjusters, surfactants, thickeners, dispersants, viscosity modifiers, antioxidants, flavor maskers, sweeteners, fillers, coating agents, capsule shells, adhesives, lubricants, etc.

[0017] AU15330 described in this application is a proteolytic targeted chimeric (PROTAC) degrader of SWI / SNF ATPase subunits SMARC2 and SMARC4, with CAS number 2380274-50-8.

[0018] In addition to demonstrating the inhibitory effect of AU15330 on HBV, this application also uses various in vivo and in vitro HBV models to detect viral activity-related indicators, clarifying the role of AU15330 in inhibiting HBV replication and transcription; and through analysis of the accessibility of cccDNA microchromatin and the epigenetic panorama, it further investigates the mechanism of AU15330 in inhibiting HBV replication.

[0019] This application clarifies the application of AU15330 in the preparation of drugs for treating hepatitis B / inhibiting HBV replication, providing 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 developing drugs for the prevention and treatment of hepatitis B and hepatocellular carcinoma and other related diseases. Attached Figure Description

[0020] Figure 1 The results show the experimental effects of AU15330 on the regulation of HBV cccDNA transcription in HepAD38 cells. Part A shows the experimental protocol; Part B shows the mRNA level; Part C shows the cccDNA level; Part D shows the preCore mRNA / cccDNA ratio; Part E shows the HBV RNA and HBV DNA levels; and Parts F and G show the HBcAg level (green).

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

[0022] Figure 3 The effects of AU15330 on HBV replication products in HepG2.2.15 cells were investigated, and the accessibility of HBV cccDNA was analyzed using transposase chromatin accessibility assay (Mnase-qPCR). Part A shows mRNA levels; Part B shows HBc and BRG1 levels; Part C shows the Mnase-qPCR results; and Part D shows HBV promoter activity levels. Detailed Implementation

[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 medium to induce HBV replication and cccDNA formation. After 10 days, tetracycline and lamivudine (3TC, 10 mM) were added to the medium to shut down the transcription of viral pgRNA from the integrated HBV genome and to prevent viral DNA replication. After 4 days, one group of cells continued to be cultured in medium containing tetracycline and 3TC, while another group of cells was treated with AU15330 (5 mM) for 4 days in the presence of tetracycline and 3TC. DMSO concentrations in all experimental groups were normalized to 0.1%.

[0025] The following tests will be performed:

[0026] Total RNA was extracted and analyzed by q-PCR for preCore and by RT-qPCR for pgRNA and total HBV RNA. HBVHirt DNA was extracted and quantified by qPCR for cccDNA. The relative level of cccDNA-based transcription was further determined by normalizing the preCore mRNA level in each treatment group to the cccDNA level in the DMSO control group.

[0027] HepAD38 cells were induced for 10 days in tetracycline-free medium 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. 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 obtained by Southern blotting. Cell nuclei were stained blue with DAPI dye.

[0028] The results are as follows Figure 1 As shown: Part B shows that AU15330 significantly inhibited preCore, pgRNA, and total HBV RNA levels; Part C shows that AU15330 did not affect HBV cccDNA levels; Part D shows that AU15330 significantly inhibited HBV cccDNA transcriptional activity; Part E shows that AU15330 significantly inhibited HBV RNA and DNA levels; Part F shows that immunofluorescence staining significantly inhibited HBc levels; Part G shows that AU15330 significantly inhibited HBc protein levels. This was further confirmed by Northern and Southern blot analyses 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] Validation of AU15330's antiviral efficacy in HBV infection systems. HepG2-NTCP cells were infected with HBV particles (10-1 ml). 7 (One viral genome), after 24 hours, the virus-containing culture was removed, and the cells were cultured for another 7 days in the presence of AU15330.

[0031] The following tests will be performed:

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

[0033] One day after HBV infection (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. Cell 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 inhibited the levels of preCore, pgRNA, and total HBV RNA; Part C shows that AU15330 did not affect the level of HBV cccDNA; Part D shows that AU15330 significantly inhibited the transcriptional activity of HBV cccDNA; Part E shows that AU15330 significantly inhibited the level of HBV RNA; Part F shows that immunofluorescence staining significantly inhibited the level of HBc by AU15330; Part G shows that AU15330 significantly inhibited the level of HBc protein, which was 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 (5mM) every two days for a total of 4 days. The treatment was used for RT-qPCR analysis of preCore mRNA, pgRNA, total HBV RNA levels, and HBc levels.

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

[0038] Example 4 used transposase chromatin accessibility assay (Mnase-qPCR) and dual-luciferase activity assay to analyze the accessibility of HBV cccDNA and HBV promoter activity.

[0039] One day after 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, with DMSO control AU15330 (5 mM) added simultaneously, and HBV promoter activity was analyzed.

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

Claims

1. Application of AU15330 in the preparation of drugs that inhibit HBV.

2. In the application according to claim 1, AU15330 is the only active ingredient in the drug.

3. The application according to claim 1, wherein the drug further comprises other components that inhibit HBV.

4. The application according to claim 1, wherein the drug inhibits HBV transcription.

5. The application according to claim 1, wherein the drug inhibits HBV replication.

6. The application according to claim 1, wherein the drug reduces HBV replication capacity by inhibiting the accessibility of HBV cccDNA chromatin, thereby inhibiting the transcriptional activity of cccDNA.

7. The application according to claim 1, wherein the drug is an injectable or oral preparation.

8. The application according to claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients.

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