Application of alzovudine in resisting viral hepatitis B

Abstract

The invention discloses an application of alzovudine in resisting viral hepatitis B, and belongs to the technical field of medicine application. The alzovudine is used for treating and resisting viral hepatitis B. An in-vivo and in-vitro combined efficacy verification system is established, a multi-genotype and drug-resistant mutation HBV model is covered, experimental results are stable and repeatable, and screening and evaluation of novel nucleoside anti-HBV drugs can be effectively guided. The invention reveals that the FNC has the comprehensive technical advantages of broad-spectrum virus resistance, drug-resistant mutation resistance, strong antigen removal capability, high safety and the like in the treatment of hepatitis B virus infection, breaks through the bottleneck that the existing NAs has limited curative effect and the antigen is difficult to remove, and provides a new candidate drug and a technical route for functional cure of chronic hepatitis B.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology, specifically relating to the application of azvudine in the treatment of hepatitis B. Background Technology

[0002] Hepatitis B virus (HBV) infection is a major contributing factor to chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. According to the World Health Organization, approximately one-third of the global population has been infected with HBV, and currently about 290 million people are chronically infected. As a high-prevalence area for HBV, my country has approximately 90 million patients with chronic hepatitis B (CHB). The liver function damage, liver cancer incidence, and medical burden caused by long-term infection have become a serious public health problem.

[0003] The main clinical strategy for treating chronic hepatitis B (CHB) is to inhibit viral replication using nucleotide analogues (NAs), such as entecavir, tenofovir, and lamivudine. These drugs can significantly reduce serum HBV DNA levels, but they are ineffective at clearing covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg), requiring long-term or even lifelong medication. Long-term medication not only increases the risk of drug tolerance and adverse reactions but also limits the achievement of clinical functional cure.

[0004] For chronic hepatitis B (CHB) caused by HBV infection, current clinical treatments mainly rely on non-reactive proteins (NAs) and interferon to suppress viral replication and modulate the immune system. While these approaches have shown some efficacy in slowing disease progression and reducing viral load, they still have limitations such as low cure rates and high relapse rates, and cannot fundamentally eliminate the virus. NAs, which inhibit HBV polymerase activity, such as lamivudine, adefovir dipivoxil, entecavir, and tenofovir, work by incorporating into the viral DNA chain to block replication, thereby effectively inhibiting serum HBV DNA levels and improving liver function. However, this mechanism cannot completely eliminate viral cccDNA, nor can it significantly reduce HBsAg and HBeAg levels. Furthermore, long-term use can easily lead to drug-resistant mutant strains (such as rtM204V and rtL180M), requiring patients to take medication long-term or even for life, and viral rebound is common after discontinuation. Therefore, developing innovative drugs with novel mechanisms of action that combine the ability to inhibit HBV replication and clear antigens has become a key focus of anti-HBV research.

[0005] Azvudine (FNC) is a novel deoxycytidine analog initially developed as an antiretroviral drug for the treatment of HIV infection. Recent studies have revealed that FNC can be converted to its active triphosphate form (FNC-TP) within host cells via multi-step phosphorylation, competitively incorporating into the viral nucleic acid chain and causing chain termination, thereby blocking viral replication. Furthermore, FNC can exert broad-spectrum antiviral effects by modulating the host immune response and interferon signaling pathway. Existing studies have confirmed that FNC shows significant inhibitory effects in various RNA and DNA virus infection models, with good safety profiles. However, no published literature or patents report its pharmacological effects and application potential in HBV infection. Considering that HBV replication depends on reverse transcription and that drug-resistant strains are less sensitive to existing NAs, FNC's unique chemical structure and mechanism of action hold promise for overcoming drug resistance and exerting a synergistic advantage in HBV replication inhibition and antigen clearance. Therefore, exploring the application of FNC in the treatment of hepatitis B virus and verifying its inhibitory effects on HBV replication and viral antigen expression has significant scientific importance and potential clinical translational value. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide the application of azvudine in the treatment of hepatitis B, and to propose a new use of FNC in anti-HBV drugs, providing new drug candidates and treatment strategies for the functional cure of CHB.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of azvudine in the treatment of hepatitis B, wherein the azvudine is used to treat hepatitis B.

[0008] Furthermore, the safe concentration of the azvudine is 100-300 nmol / L.

[0009] Furthermore, the safe concentration of the azvudine is 100, 200, or 300 nmol / L.

[0010] The beneficial effects of this invention are as follows: 1. This invention provides the first systematic verification of the significant antiviral activity of azvudine (FNC) in HBV infection. FNC significantly reduces HBV DNA levels without significant cytotoxicity and dose-dependently inhibits HBsAg and HBeAg secretion. Compared with existing NAs, FNC not only effectively inhibits viral replication but also exhibits higher efficiency in antigen clearance, providing a new drug direction for functional cure of HBV.

[0011] 2. This invention demonstrates that FNC possesses broad-spectrum anti-HBV properties across genotypes and against drug-resistant mutations. In both D-genotype and C-genotype entecavir resistance models, FNC can significantly inhibit HBV DNA replication and antigen expression, overcoming the limitations of existing nucleoside analogues in reducing efficacy against drug-resistant strains.

[0012] 3. In a mouse model of chronic AAV-HBV replication established by high-pressure hydrodynamic injection, FNC exhibited good in vivo efficacy and tissue protection. After four weeks of continuous gavage administration, the serum HBV DNA load, HBsAg, and HBeAg levels in mice decreased significantly, and the HBcAg positive signal in liver tissue was significantly reduced, suggesting that FNC can simultaneously inhibit viral replication and improve liver tissue damage. Furthermore, in terms of serological virology, the positive control TDF did not show significant efficacy against the antigen, while FNC effectively inhibited HBsAg and HBeAg; in terms of hepatological virology, the positive control TDF showed some efficacy against HBcAg, but its inhibitory effect was far less than that of FNC.

[0013] 4. As a broad-spectrum antiviral drug with high safety, well-defined pharmacokinetic characteristics, and mature formulation, FNC showed no significant cellular or organ toxicity within its effective dosage range. The comprehensive pharmaceutical and clinical safety data for FNC provide a direct and feasible basis for its redevelopment and repositioning in the anti-HBV field.

[0014] 5. This invention establishes an in vitro-in vivo efficacy verification system covering multi-genotype and drug-resistant HBV models. The experimental results are stable and reproducible, effectively guiding the screening and evaluation of novel nucleoside analog anti-HBV drugs. This invention reveals that FNC possesses comprehensive technical advantages in the treatment of hepatitis B virus infection, including broad-spectrum antiviral activity, resistance to drug-resistant mutations, strong antigen clearance ability, and high safety. It overcomes the bottlenecks of limited efficacy and poor antigen clearance of existing nucleoside analogs, providing new candidate drugs and technical pathways for the functional cure of chronic hepatitis B.

[0015] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0016] To make the purpose, technical solution, and beneficial effects of the invention clearer, the following figures are provided for illustration: Figure 1 Here is the chemical structural formula of the FNC of this invention; Figure 2 This is a graph showing the effect of FNC on the viability of HepG2.2.15 cells according to the present invention; Figure 3 This is a diagram showing the effect of FNC on the viability of HepG2.A64 cells in this invention; Figure 4 This is a graph showing the effect of the FNC of the present invention on the HBV DNA replication level in HepG2.2.15 cells; Figure 5 The figure shows the effect of the FNC of the present invention on the levels of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells; Figure 6 This diagram illustrates the inhibitory effect of the FNC of the present invention on HBV DNA levels in HepG2.A64 (entecavir-resistant strain); Figure 7 The figure shows the effect of the present invention's TDF on the levels of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells; Figure 8 This diagram illustrates the inhibitory effect of the FNC of the present invention on HBV DNA and HBsAg in HepG2.A64 cells. Figure 9 This is a diagram showing the effect of TDF of the present invention on HBV DNA and HBsAg in HepG2.A64 cells; Figure 10 This is a flowchart of the in vivo antiviral experiment of FNC in this invention; Figure 11 This is a diagram showing the effect of the FNC of the present invention on the serum HBV DNA level in mice; Figure 12 This is a graph showing the effect of the FNC of the present invention on the serum HBsAg level in mice; Figure 13 This is a graph showing the effect of the FNC of the present invention on the serum HBeAg level in mice; Figure 14 This diagram illustrates the effect of the FNC of this invention on HBcAg expression in mouse liver tissue. Detailed Implementation

[0017] To address the limitations of existing anti-HBV treatments, low viral antigen clearance rates, and a lack of novel nucleoside analogue candidates, this invention designs and establishes a systematic in vitro anti-HBV activity evaluation and in vivo efficacy verification system. This system systematically evaluates the anti-HBV potential and mechanism of action of azvudine FNC from multiple dimensions, including viral replication, antigen expression, drug resistance, and safety.

[0018] Azvudine: FNC; Hepatitis B virus: HBV.

[0019] like Figure 1-14 As shown, this invention discloses the application of azvudine in the treatment of hepatitis B, and provides in vitro and in vivo validation: I. In vitro experiments P1. HepG2.2.15 cells (stable replicating wild-type HBV of genotype D prevalent in Europe and the United States) and HepG2.A64 cells (stable replicating entecavir-resistant HBV of genotype C from Chinese patients) were selected as in vitro antiviral activity verification models to take into account different HBV genotypes and drug resistance characteristics, so as to ensure the broad applicability and clinical relevance of experimental results. P2. To ensure the scientific reliability of the efficacy evaluation of anti-hepatitis B virus drugs, cytotoxicity tests were performed on FNCs. HepG2.2.15 and HepG2.A64 cells with good growth status were selected, digested and counted, and seeded at 2×104 cells / well in 48-well plates. After 12 h of incubation in a P3 incubator, different concentrations of FNC (0, 100, 200, 300, 400, 500, 1000, 2000 nmol / L) were administered. After 5 days of incubation, the supernatant was collected, 100 μL of diluted CCK-8 reagent was added, and the mixture was incubated in an incubator for 1 h. The OD value was measured at 450 nm to determine that the non-cytotoxic concentrations of FNC were 100, 200, and 300 nmol / L, providing a safe dosage basis for subsequent antiviral experiments. P4. The intervention concentrations of the positive control TDF were selected as 2.5, 5, and 10 μmol / L. FNC and TDF were applied to HepG2.2.15 and HepG2.A64 cells, respectively, to carry out anti-HBV experiments.

[0020] Anti-hepatitis B virus activity test: S1. PCR-fluorescent probe "one-tube method" was used to detect the level of hepatitis B virus DNA in cell culture supernatant, and the inhibitory effects of FNC and TDF on viral replication were evaluated at the molecular level. The specific method is as follows: ① Take HepG2.2.15 and HepG2.A64 cells in logarithmic growth phase and seed them in 48-well plates at 2×104 cells / well. After incubation for about 12 hours until the cells adhere stably, start drug administration. Use complete medium containing 10% fetal bovine serum to serially dilute FNC and TDF stock solutions to working solution. The final DMSO volume fraction of each group should be ≤0.1%. Then change the drug-containing medium according to the following groups. Each group had 3 replicates: 1) Blank control group: Added an equal volume of medium containing 0.1% DMSO, without FNC; 2) Low-dose FNC group: Added FNC medium with a final concentration of 100 nmol / L; 3) Medium-dose FNC group: Added FNC medium with a final concentration of 200 nmol / L; 4) High-dose FNC group: Added FNC medium with a final concentration of 300 nmol / L. Each group had 3 replicates: 1) Blank control group: Added an equal volume of medium containing 0.1% DMSO, without TDF; 2) Low-dose TDF group: Added FNC medium with a final concentration of 2.5 μmol / L; 3) Medium-dose TDF group: Added FNC medium with a final concentration of 5 μmol / L; 4) High-dose TDF group: Added FNC medium with a final concentration of 10 μmol / L. ② Cells were cultured under the above conditions, and cell supernatant was collected on day 5 after drug administration according to the experimental design. HBV DNA was detected using a fluorescent probe "one-tube method" kit (PCR-mix was prepared according to the instructions, nucleic acid release agent and sample were added, lysed, and then added to the PCR-mix, total volume 39 µL; amplification conditions: 37℃ for 2 min (UDG reaction), 95℃ for 3 min (pre-denaturation), followed by 45 cycles: fluorescence acquisition at 95℃ for 10 s, 60℃ for 35 s, and finally cooling at 25℃ for 10 s). The HBV DNA content of the sample was calculated from the standard curve. S2. ELISA was used to detect the secretion levels of HBsAg and HBeAg to evaluate the effects of FNC and TDF on viral antigen expression. The specific method was as follows: ① Take HepG2.2.15 and HepG2.A64 cells in logarithmic growth phase and seed them at 2×10⁴ cells / well in 48-well plates. After incubation for about 12 hours until the cells are stably attached, start drug administration. Use complete medium containing 10% fetal bovine serum to serially dilute FNC and TDF stock solutions to working solution; the final DMSO volume fraction of each group should be ≤0.1%; change the drug-containing medium according to the following groups: Each group had 3 replicates: 1) Blank control group: an equal volume of medium containing 0.1% DMSO, without glycyrrhizin A; 2) Low-dose FNC group: FNC medium with a final concentration of 100 nmol / L; 3) Medium-dose FNC group: FNC medium with a final concentration of 200 nmol / L; 4) High-dose FNC group: FNC medium with a final concentration of 300 nmol / L. Each group had 3 replicates: 1) Blank control group: Added an equal volume of medium containing 0.1% DMSO, without TDF; 2) Low-dose TDF group: Added FNC medium with a final concentration of 2.5 μmol / L; 3) Medium-dose TDF group: Added FNC medium with a final concentration of 5 μmol / L; 4) High-dose TDF group: Added FNC medium with a final concentration of 10 μmol / L. Cells were cultured under the above conditions, and cell supernatant was collected on day 5 after drug administration according to the experimental design. 50 µL of sample was added to each well of the coated plate, with 2 wells each for negative, positive and blank control. After incubation at 37°C for 60 min, 50 µL of enzyme conjugate was added to each well, and the plate was incubated at 37°C in the dark for 30 min. The plate was washed 5 times and allowed to stand for 1 min each time. 50 µL of substrate A and B solutions were added to each well, and the plate was developed at 37°C in the dark for 30 min. 50 µL of stop solution was added, and the absorbance was measured at 450 nm. The relative expression levels of HBsAg and HBeAg were calculated. S3. By statistically analyzing the experimental data obtained in S1 and S2, the dose dependence and inhibitory efficiency of FNC and TDF were determined. After the experimental data of each group were corrected by the blank control group, they were normalized with the mean of the control group as 1, and the relative changes of FNC and TDF under different concentrations were calculated.

[0021] like Figure 2 As shown, the CCK-8 assay was used to detect the effect of different concentrations of FNC on the viability of HepG2.2.15 cells at 1d, 3d, and 5d. The ordinate represents cell viability, and the abscissa represents the FNC treatment concentration. When the FNC concentration was in the range of 0-300 nmol / L, cell viability did not decrease significantly compared with the control group, indicating that the compound had no significant toxicity to cells within this range. Concentration data are expressed as mean ± standard deviation (mean ± SD). (*p<0.05, **p<0.01 vs. blank control group) like Figure 3 As shown, the CCK-8 assay was used to detect the effect of different concentrations of FNC on the viability of HepG2.A64 cells at 1, 3, and 5 days. The ordinate represents cell viability, and the abscissa represents the FNC treatment concentration. When the FNC concentration was in the range of 0-500 nmol / L, cell viability did not decrease significantly compared with the control group, indicating that the compound had no significant toxicity to cells within this range. Concentration data are expressed as mean ± standard deviation (mean ± SD). (**p < 0.01 vs. blank control group) like Figure 4 The effect of different concentrations of FNC (100, 200, 300 nmol / L) on the HBV DNA level in the cell culture supernatant after treatment for 1, 3, and 5 days was shown. The results indicated that longer treatment times resulted in better inhibition. Data are expressed as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. control group) like Figure 5The image shows the effect of different concentrations of FNC (100, 200, 300 nmol / L) on the levels of HBV DNA, HBsAg, and HBeAg in the cell culture supernatant after treatment with these concentrations for 5 days. Data are presented as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. control group) like Figure 6 The effect of different concentrations of FNC (100, 200, 300 nmol / L) on the HBV DNA level in the cell culture supernatant after treatment for 1, 3, and 5 days was shown. The results indicated that longer treatment times resulted in better inhibition. Data are presented as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. control group) like Figure 7 The effect of treating HepG2.2.15 cells with tenofovir (TDF, 2.5, 5, 10 μmol / L) as a positive control for 5 days on the levels of HBV DNA, HBsAg, and HBeAg in the cell culture supernatant is shown. Data are expressed as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. blank control group) like Figure 8 The effect of different concentrations of FNC (100, 200, 300 nmol / L) on the levels of HBV DNA and HBsAg in the cell culture supernatant after treatment with these concentrations for 5 days is shown in the figure. Data are expressed as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. control group) like Figure 9 The effect of treating HepG2.A64 cells with tenofovir (TDF, 2.5, 5, 10 μmol / L) as a positive control for 5 days on the levels of HBV DNA and HBsAg in the cell culture supernatant is shown. Data are expressed as mean ± standard deviation (mean ± SD). (*p<0.05, **p<0.01 vs. blank control group) With increasing FNC concentration, the HBV DNA content in the cell culture supernatant decreased significantly in a concentration-dependent manner. Simultaneously, FNC significantly reduced HBsAg and HBeAg secretion levels, demonstrating a dual inhibitory effect on viral replication and antigen expression. Compared with the positive control drug tenofovir disoproxil fumarate (TDF), FNC showed significantly greater antigen-inhibiting effects.

[0022] In summary, the in vivo experiments of this invention have verified that FNC can significantly reduce HBV replication levels and antigen expression without affecting cell viability, indicating that it has excellent in vitro antiviral efficacy and broad-spectrum inhibitory characteristics.

[0023] II. In vivo experiments S1. Using a high-pressure hydrodynamic method, 20 μg of pAAV-HBV1.2 HBV plasmid was injected via the tail vein to establish an HBV-infected mouse model. Three days later, blood was collected from the orbital cavity to monitor HBsAg, HBeAg, and HBV DNA. S2, Modeling successful (serum HBV DNA level ≥5×10⁻⁶) 4 Mice with IU / mL concentration and positive for HBsAg and HBeAg were randomly divided into a normal saline group (NS, administered by gavage), a low / high dose FNC group (administered by gavage of the corresponding concentration of FNC), and a positive control TDF group (TF, administered by gavage). The mice were administered the drugs by gavage for 4 weeks, and serum virological changes were dynamically monitored by taking blood samples from the orbital cavity every week.

[0024] Figure 10 To establish a mouse model of HBV infection with full immune response using high-pressure hydrodynamics, mice with successful modeling were randomly divided into a control group, a low / high dose FNC group, and a positive control TDF group. Blood samples were taken from the orbital cavity weekly to monitor virological changes. After four consecutive weeks of administration, the mice were sacrificed, and serum and liver were collected for later use.

[0025] like Figure 11 As shown, serum HBV DNA levels in mice at different time points were compared. Results indicated that FNC continuously reduced HBV DNA levels with prolonged administration, with higher doses of FNC showing a more significant effect. Data are expressed as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. saline group) like Figure 12 As shown, FNC significantly reduced HBsAg levels during the 1-4 week dosing period, with the inhibitory effect gradually increasing over time. The positive control TDF did not show a significant inhibitory effect on HBsAg. Data are presented as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. saline group) like Figure 13 As shown, FNC significantly reduced HBeAg levels during the 1-4 week administration period, with the inhibitory effect gradually increasing over time. The positive control TDF did not show a significant inhibitory effect on HBeAg. Data are presented as mean ± standard deviation (mean ± SD). (*p < 0.05, **p < 0.01 vs. saline group) like Figure 14As shown, the fields of view are displayed at different magnifications of 5× and 20×. Liver tissue HBcAg staining was used to evaluate the level of HBV expression in the liver. The NS group showed strong HBcAg positive signal; both FNC (low and high doses) significantly reduced the HBcAg staining intensity, suggesting that viral replication in the liver was suppressed. A similar improvement was observed in the TDF group.

[0026] Serological test results showed that FNC could significantly reduce serum HBV DNA, HBsAg and HBeAg levels starting from the second week of administration, and these levels gradually decreased with the duration of treatment.

[0027] Figure 11-13 The results showed that, in terms of serological virology, the positive control TDF did not show significant efficacy against the antigen, while FNC effectively inhibited HBsAg and HBeAg. In terms of hepatological virology, although the positive control TDF showed some efficacy against HBcAg, it was far less effective than the inhibitory effect of FNC. Furthermore, the liver tissue cells of mice treated with FNC were well-arranged, with normal nuclei, and no significant necrosis or inflammatory infiltration was observed.

[0028] Combined in vitro and in vivo results demonstrate that FNC significantly inhibits HBV replication and antigen secretion at both cellular and animal levels, and maintains its antiviral effect in drug-resistant strains. Its antiviral activity is dose-dependent and exhibits good safety, providing sufficient experimental evidence for its potential as a novel nucleoside analogue candidate drug against HBV.

[0029] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The application of azvudine in the treatment of hepatitis B virus infection, characterized by: Azvudine is used to treat hepatitis B.

2. The application of azvudine in the treatment of hepatitis B according to claim 1, characterized in that: The safe concentration of azvudine is 100-300 nmol / L.

3. The application of azvudine in the treatment of hepatitis B according to claim 2, characterized in that: The safe concentrations of azvudine are 100, 200, and 300 nmol / L.

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