Application of Tomatine in Anti-Hepatitis Virus

Abstract

The present invention provides the use of tomatidine in anti-hepatitis virus treatment. This invention discovers for the first time a novel anti-hepatitis virus substance, tomatidine, which can inhibit the entry of hepatitis B virus (HBV) and, to a certain extent, reduce its replication. Cytotoxicity experiments show that tomatidine at viral inhibitory concentrations produces virtually no adverse reactions. Tomatidine can target different stages of the HBV life cycle, consistent with the clinical approach of combining drugs for antiviral treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of tomatidine in resisting hepatitis viruses. Background Art

[0002] Chronic infection with hepatitis B virus (HBV) remains a global public health problem. Chronic hepatitis B (CHB) can persist throughout life, causing varying degrees of liver damage and ultimately leading to cirrhosis and liver tumors. Therefore, studying the pathogenesis of CHB and developing effective antiviral treatments are of great scientific and social significance.

[0003] HBV belongs to the Hepadnaviridae family. Its mature particles contain a relaxed circular DNA genome (rcDNA) of approximately 3.2 kb. During de novo infection, HBV specifically binds to its receptor, sodium taurocholate cotransporting polypeptide (NTCP), and enters hepatocytes via clathrin-mediated endocytosis. Within the cytoplasm, HBV undergoes endosomal transport and endosomal escape, ultimately shedding its envelope. Guided by a nuclear localization sequence, the exposed nucleocapsid, driven by motor proteins, approaches the nucleus along microtubules. Subsequently, the nucleocapsid disassembles, allowing the rcDNA to enter the nucleus, completing HBV entry and establishing de novo infection. In the nucleus of infected hepatocytes, rcDNA is repaired to covalently closed circular DNA (cccDNA), which is the original template for HBV transcription and replication; in the cytoplasm, the viral pregenomic RNA is packaged into the nucleocapsid, and viral DNA synthesis is initiated through reverse transcription. Part of the mature viral nucleocapsid particles obtain envelope proteins through the multivesicular body pathway and are secreted outside the hepatocytes, while the other part is transported to the cell nucleus again to replenish the cccDNA pool, completing the viral replication cycle in the cell.

[0004] Currently, clinically used antiviral drugs include pegylated interferon (Peg-IFN-α) and nucleoside (acid) analogs (NAs). Peg-IFN-α, approved by the US FDA in 2002, overcomes the shortcomings of conventional IFN-α, such as its short half-life, repeated injections, and low sustained viral response. It enhances innate immunity and triggers T cell-mediated immune responses. It has demonstrated excellent therapeutic efficacy in HBeAg-positive CHB patients, with higher HBeAg seroconversion rates and HBsAg clearance, and reduced incidence of cirrhosis and liver tumors. The main NAs used in first-line clinical practice include entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF). As polymerase reverse transcriptase inhibitors, they have advantages over first-generation drugs such as lamivudine (LAM) in terms of lower drug resistance and nephrotoxicity. They can effectively inhibit HBV DNA replication and significantly improve liver tissue pathology.

[0005] The combined use of antiviral drugs with different mechanisms and targeting different stages of the HBV life cycle is currently a powerful means to improve treatment efficacy. However, the drugs currently used in clinical practice only target the replication process, and there is a lack of effective means for other steps in the life cycle. Therefore, the development of new anti-HBV drugs still deserves further exploration and research. The new anti-HBV drugs currently under development include: Myrcludex B (myristoylated HBV L protein 2-49 peptide) and cyclosporin A (CsA). Myrcludex B interacts with the hepatocyte-specific receptor NTCP, inhibiting the specific infection of HBV and hepatitis delta virus (HDV) to hepatocytes, and has been approved for marketing in Europe; the HBV entry inhibitor Myrcludex B is a synthetic lipopeptide derived from the HBV pre-surface antigen 1 (preS1) domain. After binding to NTCP, Myrcludex B can not only effectively prevent HBV from spreading between hepatocytes, but also may inhibit the expansion of the cccDNA pool (Volz T, et al. 2013; J Hepatol 58(5):861–867). CsA is often used as an immunosuppressant in organ transplantation. CsA and its analogues can potentially inhibit the activity of NTCP transporter, thereby blocking HBV from entering hepatocytes (Nkongolo S, et al. 2014; J Hepatol 60(4):723–731).

[0006] Although a variety of anti-HBV drugs are involved in clinical practice, they have the following disadvantages: (1) Peg-IFN-α has disadvantages such as inconvenient administration and large side effects, and it does not have a good therapeutic effect on all CHB patients, especially when HBsAg is very high, the therapeutic effect is not ideal, that is, Peg-IFN-α does not have a good therapeutic effect on CHB patients with very high HBsAg, and has certain adverse reactions on various systems in the body. Therefore, not all CHB patients are suitable for Peg-IFN-α treatment; (2) NAs can only inhibit viral replication, but cannot completely eliminate the replication template cccDNA in infected liver cells. There is still a risk of viral rebound and replication after drug withdrawal; (3) Myrcludex B, which is in the clinical trial stage, can block the spread of HBV between liver cells and inhibit the amplification of the cccDNA pool, but its HBsAg clearance effect is not ideal; and because Myrcludex The interaction between B and NTCP impairs sodium-dependent bile acid uptake, thereby causing an increase in bile acid levels; (4) CsA and its analogs impair sodium-dependent bile acid uptake, thereby inducing various adverse reactions.

[0007] Since both Peg-IFN-α and Myrcludex B have adverse reactions to varying degrees, it is necessary to find new antiviral drugs that do not affect the host's own physiological processes and have fewer side effects; since Peg-IFN-α has a poor therapeutic effect on patients with CHB with high levels of HBsAg, not all CHB patients are sensitive to Peg-IFN-α treatment, so universally applicable antiviral drugs are needed; since NAs only inhibit the HBV DNA replication process and cannot reduce the cccDNA pool, multi-functional antiviral drugs targeting multiple links in the viral life cycle are needed.

[0008] Tomatidine is a natural steroidal alkaloid from unripe green tomatoes. It is the aglycone of tomatine. Tomatine is mainly found in the fruits, stems and leaves of unripe green tomatoes (up to 500 mg / kg). As the tomatoes mature, the content usually decreases by 99%, and in red tomatoes it is less than 5 mg / kg. Tomatidine is a metabolite of tomatine and has a variety of beneficial biological effects, including anti-inflammatory, lipid-lowering, improving muscle health and extending the lifespan of nematodes. To date, it has been confirmed that tomatidine has inhibitory effects on coronavirus, arteritis virus, porcine encephalomyocarditis virus, Seneca virus, Israeli acute paralysis virus, dengue virus, chikungunya virus, porcine epidemic diarrhea virus, etc. However, to date, there has been no research on the anti-hepatitis virus effect of tomatidine and its mechanism of action. Summary of the Invention

[0009] To overcome at least one problem existing in the prior art, the present invention has discovered for the first time a new anti-hepatitis virus substance - tomatidine, which can inhibit the entry of viruses (specifically HBV) and reduce viral (specifically HBV) replication to a certain extent. The above-mentioned tomatidine can target different stages of the HBV life cycle, providing a new idea and new option for the clinical treatment of hepatitis virus infection with combination drugs.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] The first aspect of the present invention is to provide the use of tomatidine or a pharmaceutically acceptable salt thereof in the preparation of anti-hepatitis virus drugs.

[0012] Furthermore, the tomatidine is a natural small molecule steroid compound, and its molecular formula is as follows:

[0013]

[0014] Furthermore, the hepatitis virus includes: hepatitis B virus and hepatitis D virus. In a specific verification test scheme, the hepatitis virus is hepatitis B virus.

[0015] Furthermore, the medicine is a medicine for preventing and / or treating hepatitis virus infection.

[0016] Furthermore, the drug is an antiviral drug targeting early infection of hepatitis virus and / or replication of hepatitis virus. The above-mentioned drug includes a virus entry inhibitor and / or a virus replication inhibitor, that is, the tomatidine or a pharmaceutically acceptable salt thereof is used to prepare a hepatitis virus entry inhibitor and / or a hepatitis virus replication inhibitor. It is understandable that the above-mentioned drug may contain other ingredients, or tomatidine or a pharmaceutically acceptable salt thereof may be used as a single component directly as a hepatitis virus entry inhibitor and / or a hepatitis virus replication inhibitor. Preferably, the drug is a hepatitis B virus entry inhibitor.

[0017] Furthermore, for HepG2-NTCP cells, the median toxic concentration (CC50) of tomatidine or a pharmaceutically acceptable salt thereof is 948.2 μM. In one embodiment, for cells, the administration concentration of tomatidine or a pharmaceutically acceptable salt thereof is 3 to 10 μM, which is within the safe and effective range.

[0018] Furthermore, the hepatitis virus is hepatitis B virus, and the mechanism of action of the tomatidine or a pharmaceutically acceptable salt thereof is at least one of the following: inhibiting the secretion of HBeAg after viral infection, inhibiting the expression of core protein after viral infection, inhibiting viral replication, inhibiting viral entry, and inhibiting early viral infection (i.e., inhibiting de novo viral infection).

[0019] Furthermore, the administration of tomatidine or a pharmaceutically acceptable salt thereof is selected from at least one of the following: pre-infection treatment, treatment during infection, and post-infection treatment. Preferably, pre-infection treatment; or, co-treatment before and during infection.

[0020] Furthermore, the tomatidine or a pharmaceutically acceptable salt thereof is administered before infection, and the anti-hepatitis virus drug is a drug for preventing hepatitis B virus infection.

[0021] Furthermore, the drug contains a pharmaceutically acceptable carrier.

[0022] Furthermore, the dosage forms of the drug include: tablets, powders, granules, capsules, emulsions, and injections.

[0023] Furthermore, the drug can be used alone or in combination, for example, it can be used in combination with existing anti-hepatitis virus drugs.

[0024] It is understandable that the type and amount of the selected carrier or excipient are also adaptively adjusted based on the different drug dosage forms and administration methods. The above-mentioned carriers or excipients are all conventional reagents commonly used in the art.

[0025] The above-mentioned "pharmaceutically acceptable salts" are acid addition salts formed by the compound with an acid, or base addition salts formed by the compound with a base; wherein the acids include but are not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, tartaric acid, salicylic acid, citric acid, malic acid, methanesulfonic acid, p-toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid, and succinic acid; the base addition salts include salts derived from inorganic bases and salts derived from organic non-toxic bases, the salts derived from inorganic bases include but are not limited to: aluminum, ammonium, potassium, sodium, iron, copper, calcium, ferrous, magnesium, lithium, and manganese, the salts derived from organic non-toxic bases include but are not limited to: primary, secondary, and tertiary amine salts, substituted amines including naturally substituted amines, cyclic amines, ethanolamine, N-ethylmorpholine, N-ethylpiperidine, and glucosamine.

[0026] The aforementioned "pharmaceutically acceptable carrier" is one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "Compatibility" herein refers to the ability of the components of the composition to blend with the compound of the present invention, and with each other, without significantly reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers, wetting agents (e.g., sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0027] Furthermore, in the application, a replication-deficient recombinant rHBV replicon model was constructed for studying early entry inhibitors, which included the following steps: selecting single restriction sites on the pCMV-ayw1.1 (GHT) plasmid: Nde I (CA^TATG) and EcoR I (G^AATTC); I was double-enzyme digested to obtain the target vector fragment; payw1.1-GHT plasmid was used as a template, and PCR was performed with two pairs of primers: core-GTG-F1, core-GTG-R1 and core-GTG-F2, core-GTG-R2, respectively. The reaction conditions were denaturation at 98°C for 2 min, followed by 98°C for 15 s; 65°C for 20 s; 72°C for 30 s / 90 s, and 72°C for 5 min after 30 cycles to obtain PCR products; a cloning kit based on DNA sequence homology recombination was used to transform the ligation product of the PCR product and the target vector fragment into DH5α competent cells, and the cells were cultured and amplified; the amplified bacterial solution was sent for sequencing, and the correct clone was expanded and cultured according to the sequencing results. A plasmid extraction kit was used to extract the plasmid according to its operating instructions to obtain a replication-defective recombinant rHBV replicon model.

[0028] Furthermore, the primers used for PCR amplification are as follows, wherein: the bold italic underlined parts are restriction enzyme cutting sites, and the underlined parts are mutation sites:

[0029] core-GTG-F1:5'-GCAGTACATCAAGTGTAT CATATG CCAAGTACGCCCCCTA-3';

[0030] core-GTG-R1: 5'-ATAAGGGTCGATGTCCA C GCCCCAAAGCCACCCAA-3';

[0031] core-GTG-F2: 5'-TTGGGTGGCTTTGGGGGC G TGGACATCGACCCTTAT-3′;

[0032] core-GTG-R2: 5'-TGGTGGAAGGTTGTG GAATTC CACTGCATGGCCTGAGGAT-3'.

[0033] The replication-deficient recombinant rHBV replicon model constructed above is a recombinant HBV (rHBV) with a deletion mutation in the HBV core protein gene. Specifically, the ATG encoding the core protein gene has been mutated to GTG within the backbone of the HBV replicon payw1.1-GHT (this replicon deletes the ATG encoding the pre-core protein gene and mutates it to TG, resulting in the plasmid itself not secreting HBeAg). This model is capable of rescuing HBV viral replication under conditions of core protein trans-complementation. The resulting recombinant progeny virus (rHBV) possesses only one-time infection characteristics and, lacking the core gene, is incapable of active replication within infected cells.

[0034] Compared with the prior art, the present invention adopts the above technical solution to achieve the following beneficial effects:

[0035] This study, published in the journal Nature Communications, for the first time, discovered and validated the anti-hepatitis virus effect of tomatidine. Specifically, tomatidine inhibits both viral entry and replication, specifically hepatitis B virus (HBV), with a particularly significant inhibitory effect on entry. Combined with cytotoxicity assay results, tomatidine at viral inhibitory concentrations produces virtually no adverse reactions. Tomatidine can simultaneously target multiple steps in the viral life cycle, aligning well with the clinical approach of combined antiviral therapy.

[0036] Preliminary studies of the specific embodiments of the present invention have demonstrated that: (1) in the HepG2-NTCP infection model, by detecting the level of hepatitis B virus E antigen (HBeAg) in the supernatant and the expression of HBV core protein (core) in the cells, Tomatidine treatment significantly inhibited HBV de novo infection. This antiviral effect is mainly reflected in the inhibition of the early entry process of HBV, while it has only a weak inhibitory effect on late replication. (2) Since the cccDNA template encoding HBeAg is partly derived from the repair of rcDNA in early infection and partly from the supplementation of the cccDNA pool in the subsequent replication process, in order to further prove the inhibition of Tomatidine on the entry process, firstly, a time course experiment of HBV infection was designed, namely, Tomatidine pre-infection pretreatment, infection treatment, co-treatment before and during infection, and post-infection treatment. It was found that pre-infection pretreatment and co-treatment before and during infection had the most obvious inhibitory effect, followed by infection treatment, and post-infection treatment only showed a slight decrease in HBeAg on the 7th day, which may be due to the weak inhibition of replication by Tomatidine. Secondly, a HBV strain was constructed. A recombinant HBV (rHBV) with a core protein gene deletion mutation was co-transfected into Huh7 cells with this plasmid and a plasmid encoding the wild-type core protein. Due to the complementation of the core protein, rHBV replicated in large quantities and was secreted into the cell supernatant. The obtained rHBV was used to infect HepG2-NTCP. Due to the absence of core, rHBV did not replicate, eliminating the effect on replication. This experiment also proved that tomatidine treatment also inhibited rHBV de novo infection. (3) Based on the fact that HBV and hepatitis delta virus (HDV) have the same early infection molecular mechanism, it is reasonable to infer that tomatidine should also be able to inhibit HDV infection of hepatocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0038] Figure 1 This is a schematic diagram of the results of the cytotoxicity test of Tomatidine on HepG2-NTCP cells in one embodiment of the present invention;

[0039] Figure 2 This is a schematic diagram of the results of studying the effect of Tomatidine on de novo HBV infection in the HepG2-NTCP model in one embodiment of the present invention;

[0040] Figure 3This is a schematic diagram showing the effect of Tomatidine on HBV replication and the expression and secretion of HBeAg and HBsAg in one embodiment of the present invention;

[0041] Figure 4 This is a schematic diagram showing the results of a time course experiment of Tomatidine inhibiting de novo HBV infection in one embodiment of the present invention;

[0042] Figure 5 This is a schematic diagram showing the results of Tomatidine inhibiting rHBV de novo infection in one embodiment of the present invention;

[0043] Figure 6 This is a schematic diagram showing the results that Tomatidine does not inhibit lentivirus and adenovirus infection in one embodiment of the present invention. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. It should be noted that the described embodiments are only some of the embodiments of the present invention, and are not intended to be exhaustive. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national standards. Experimental materials in the following examples where the sources are not specified are all commercially available raw materials. The equipment used in each step of the following examples is conventional equipment. If there are no corresponding national standards, the steps are carried out in accordance with generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise specified, all parts are by weight, and all percentages are by mass. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention.

[0045] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0046] In the following examples, the cell lines used include: HepG2-NTCP cell line was kindly donated by Professor Yuan Zhenghong of Fudan University; HepAD38 was preserved in the inventor's laboratory and can induce (Tet-off) HBV virus replication and secrete infectious virus particles; Huh7 cell line was preserved in the inventor's laboratory.

[0047] In the following examples, the reagents used include: DMEM high glucose medium, Opti-MEM medium, Gibco fetal bovine serum, penicillin-streptomycin double antibody, Puromycine, and G418 were all purchased from Thermo Fisher; CCK8 kit was purchased from Beyotime; Tomatidine and Doxycycline were purchased from MCE; PEG8000, type I rat tail collagen, and dimethyl sulfoxide (DMSO) were purchased from Sigma; HBeAg detection kit was purchased from Kehua; Southern blot kit was purchased from Thermo Fisher; anti-core antibody was purchased from Changdao; anti-GFP and anti-β-actin antibodies were purchased from Proteintech; goat serum was purchased from Thermo Fisher; Alexa Fluor TM 555-labeled goat anti-rabbit fluorescent secondary antibody was purchased from ThermoFisher; HRP-labeled goat anti-mouse and goat anti-rabbit IgG H&L were purchased from Abcam; DAPI, 4% PFA, and Polyberene were purchased from Beyotime; TritonX-100 was purchased from Sangon; BamHI, EcoRI endonucleases, and Buffer were purchased from New England Biolabs; PCR high-fidelity DNA polymerase, dNTPs, and Buffer were purchased from TaKaRa; seamless cloning enzyme was purchased from Beyotime; gel recovery kit was purchased from Novatomic; plasmid extraction kit was purchased from Macherey-Nagel; and DNA transfection reagent Neofect was purchased from Codein Technology Co., Ltd.

[0048] In the following examples, the plasmids and strains used include: payw1.3 encodes 1.3 copies of the HBV genome and was constructed by the inventor's laboratory; payw1.1-GHT was constructed by the inventor's laboratory, the latter of which is driven by a CMV promoter to produce 1.1 copies of the HBV genome, and the start codon ATG of the pre-core protein gene (pre-core) at the 5' end of the replicon is mutated to TG, so it cannot produce and secrete HBeAg; pcDNA3.1-core plasmid was constructed by the inventor's laboratory; payw1.1-GHT and pcDNA3.1-core plasmids were constructed by the inventor's laboratory; DH5α competent cells were purchased from Qingke Company.

[0049] In the following examples, the collection and preparation of wild-type HBV includes the following steps:

[0050] 1) Cell culture: HepAD38 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% double-antibody, supplemented with 1 μg / mL Puromycine, 1 μg / mL Doxycycline, and 400 μg / mL G418, and cultured in a 37°C cell culture incubator with 5% CO2 and saturated humidity.

[0051] 2) Virus collection: When cells have expanded to the desired number, remove the antibiotics and add 2.5% DMSO. Collect the cell supernatant every three days and store temporarily in a 4°C refrigerator for a total of five collections.

[0052] 3) Virus Concentration: The cell supernatant was centrifuged at 2000 r / min at 4°C for 5 min to remove the cell pellet, which was then filtered through a 0.45 μM filter membrane. PEG8000 was added to a final concentration of 8%, and the cells were refrigerated at 4°C overnight to precipitate. On the second day, the supernatant was centrifuged at 2000 r / min at 4°C for 30 min, discarded, and the pellet retained. The pellet was resuspended in Opti-MEM at a volume of 1 / 100-1 / 200 and tumbled on a 4°C invertor overnight. On the third day, the concentrated supernatant was centrifuged at 12000 g / min at 4°C for 30 min. The supernatant was the concentrated HBV virions.

[0053] In the following examples, 5 mg of tomatidine powder was dissolved in 1.203 mL of DMSO to prepare a 10 mM stock solution. A portion was further diluted 10-fold with DMSO to a 1 mM stock solution and stored at -80°C. Upon use, the two stock solutions were diluted with DMEM complete medium to the desired working solution concentration and added to the cultured cells.

[0054] In the following examples, HepG2-NTCP cells were infected with wild-type HBV and recombinant rHBV to discover and verify a novel small molecule anti-HBV infection compound, Tomatidine (specifically, a viral entry inhibitor).

[0055] Example 1 - Tomatidine cytotoxicity assay

[0056] This example detects the cytotoxicity of Tomatidine, which includes the following experimental steps:

[0057] 1) Cell culture and plating: HepG2-NTCP cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double-antibody in a 37°C cell culture incubator with 5% CO2 and saturated humidity. When the density reached 90-95%, the cells were digested and plated at 2-3×10 cells per well. 5 Cells were plated in 48-well plates pretreated with collagen and treated accordingly after 24 h.

[0058] 2) Drug addition: Tomatidine was added to the cells at final concentrations of 1, 3, 10, 30, 100, 300, 1000, and 3000 μM, with three biological replicates for each concentration. 0.1% DMSO was used as a control group without drug addition for 48 h.

[0059] 3) Cytotoxicity assay: The assay was performed according to the instructions of the CCK8 kit, and a blank control group without cells was set up.

[0060] The above CCK8 experimental results are as follows Figure 1 As shown: Tomatidine is a natural small molecule steroid compound, which has almost no cytotoxicity at low concentrations. After being treated on HepG2-NTCP for 48 hours, the half cytotoxic concentration CC50 is 948.2 μM.

[0061] Example 2 - Detection of Tomatidine Inhibiting Early Wild-Type HBV Infection

[0062] This example studies the effect of Tomatidine on de novo HBV infection in the HepG2-NTCP model, which includes the following experimental steps:

[0063] 1) HepG2-NTCP cell culture and plating: same as in Example 1;

[0064] 2) HBV infection: Before infection, cells were pretreated with tomatidine at concentrations of 0 μM, 3 μM, and 10 μM for 10 h. An infection system was then prepared with 500 MOI of HBV virions, 2.5% DMSO, 4% PEG8000, and DMEM complete medium to a volume of 200 μL. Tomatidine at the aforementioned concentrations was added, and the cells were incubated at 37°C for 12 h. The supernatant was discarded, the cells were washed five times with PBS, and the medium was replaced with DMEM complete medium containing 2.5% DMSO. The supernatant was collected every two days for HBeAg detection. On day 9 after infection, the cells were fixed with 4% PFA and used for immunofluorescence detection of HBV core protein.

[0065] 3) HBeAg detection: supernatants were collected on days 3, 5, 7, and 9 after infection and tested by ELISA according to the kit instructions;

[0066] 4) HBV core protein detection: After 9 days of infection, cells were fixed and blocked and permeabilized with 0.2% Triton X-100 diluted with 20% goat serum for 30 minutes. The cells were washed three times with PBS, and anti-core antibody was added, followed by incubation at 4°C overnight. The cells were washed three times with PBS, and goat anti-rabbit fluorescent secondary antibody was added, followed by incubation at room temperature for 1 hour. The cells were washed three times with PBS, and DAPI was added to stain the nuclei for 5 minutes at room temperature. The cells were washed three times with PBS, and observed under a fluorescence microscope.

[0067] Tomatidine was added at concentrations of 0 μM, 3 μM, and 10 μM before and during infection. The results of HBeAg levels in the supernatant and core expression in the cells were shown in Figure 2. Figure 2 As shown: Tomatidine inhibits the secretion of HBeAg in the supernatant after HBV infection, and Tomatidine inhibits the expression of core protein in cells after HBV infection.

[0068] Example 3-Effect of Tomatidine on HBV Replication and Protein Expression and Secretion

[0069] In this example, in order to exclude the effect of tomatidine on HBV replication and protein expression and secretion, HBV DNA, HBeAg and HBsAg were detected by southern blot and ELISA on HepAD38 and Huh7 transfected with payw1.3 system, respectively. The specific steps included: (1) HepAD38 cells stably expressing HBV genome were plated at 1.5×10 6 The cells were plated in a collagen-pretreated 6-well plate. After the cells adhered to the plate on the next day, tomatidine was added at final concentrations of 1 μM, 3 μM, and 10 μM, respectively. 2 μM ETV was used as a control and the cells were treated for 72 h. HBV DNA replication was detected by Southern blot. (2) Huh7 cells with a confluence of 90-95% were plated at 2-3 × 10 cells per well. 5 The cells were plated in a 48-well plate and transfected with payw1.3 after adherence on the next day. The medium was changed after 8-12 hours and tomatidine was added to the final concentrations of 1μM, 3μM, and 10μM, respectively. 2μM ETV was used as a control and the treatment was continued for 72 hours. The supernatant was collected and diluted 10-fold with PBS. HBeAg and HBsAg were detected by ELISA.

[0070] The above test results are as follows Figure 3 As shown: Tomatidine has a weak inhibitory effect on HBV replication, but does not affect the expression and secretion of HBeAg and HBsAg themselves.

[0071] Example 4 - Time course experiment of HBV infection

[0072] This example verifies whether Tomatidine is an inhibitor of early entry, and includes the following experimental steps:

[0073] In the HepG2-NTCP de novo infection model, part of the cccDNA template encoding HBeAg comes from the repair of rcDNA in the early infection, and the other part comes from the supplementation of the cccDNA pool during the subsequent replication process. Since Tomatidine will weakly inhibit HBV replication (verification results of Example 3), in order to prove that Tomatidine is an inhibitor of early entry, this example designed a time course experiment of HBV infection, namely, Tomatidine pretreatment before infection, treatment during infection, co-treatment before and during infection, and treatment after infection, specifically: 10 μM Tomatidine treatment for 10 hours only before HBV infection, which is pretreatment before infection; 10 μM Tomatidine treatment for 12 hours only during HBV infection, which is treatment during infection; 10 μM Tomatidine treatment for 22 hours before and during infection, which is co-treatment before and during infection; 10 μM Tomatidine treatment for 9 days after HBV infection, which is treatment after infection. The time points of Tomatidine treatment of cells are as follows: Figure 4 As shown in the upper left part of the figure, the supernatants were collected on days 3, 5, 7, and 9 after infection, and HBeAg was detected by ELISA according to the kit instructions.

[0074] Test results such as Figure 4 As shown in the lower left and right parts of the image, the inhibitory effect of Tomatidine pre-treatment on HBV infection is significantly better than drug intervention during viral infection. That is, pre-treatment before infection and co-treatment before and during infection have the most obvious inhibitory effects, followed by treatment during infection. However, HBeAg only slightly decreased on the 7th day after infection, which may be due to the weak inhibition of replication by Tomatidine. The above results suggest that the main antiviral mechanism of Tomatidine is located in the early steps of viral infection.

[0075] Example 5 - Detection of Tomatidine Inhibiting Early rHBV Infection

[0076] This example constructs a replication-deficient recombinant rHBV replicon to further verify whether Tomatidine is an early entry inhibitor, which includes the following experimental steps:

[0077] (1) Construction of HBV Core protein-deficient (payw1.1-GHT-C-null) mutant plasmid

[0078] 1) Selection of vector restriction enzyme cutting sites and fragment primer design (the bold italic underlined part is the restriction enzyme cutting site, and the underlined part is the mutation site)

[0079] Select single restriction enzyme sites on the pCMV-ayw1.1(GHT) plasmid: Nde I (CA^TATG) and EcoR I (G^AATTC)

[0080] The primers were designed as follows (synthesized by Shanghai Boshang Biotechnology Co., Ltd.):

[0081]

[0082] core-GTG-R1: 5'-ATAAGGGTCGATGTCCA C GCCCCAAAGCCACCCAA-3'

[0083] core-GTG-F2: 5'-TTGGGTGGCTTTGGGGGC G TGGACATCGACCCTTAT-3'

[0084]

[0085] 2) Enzyme Digestion: 2 μg of payw1.1-GHT plasmid was digested with Nde I and EcoR I, resulting in a 50 μL digestion system. Incubate at 37°C for 3 hours to allow for full reaction. The target vector fragment was then isolated by agarose gel electrophoresis, excised, and recovered using a gel recovery kit.

[0086] 3) Overlapping PCR: Using the payw1.1-GHT plasmid as a template, PCR was performed with two pairs of primers: core-GTG-F1 and core-GTG-R1, and core-GTG-F2 and core-GTG-R2. The reaction conditions were denaturation at 98°C for 2 min, followed by 30 cycles of 98°C for 15 s, 65°C for 20 s, and 72°C for 30 s / 90 s, followed by 72°C for 5 min. PrimerStar (TaKaRa) was used for the PCR.

[0087] 4) Replace the corresponding sequence in the payw1.1-GHT plasmid with the PCR fragment: Recover the PCR product using a gel extraction kit. Using a DNA sequence homology recombination cloning kit (Seamles cloning kit, Beyotime Biotechnology), incubate the vector and fragment at 50°C for 30 minutes. Finally, transform 20 μL of the ligation product into DH5α competent cells. Single clones were selected and transferred to LB medium supplemented with antibiotics and cultured at 37°C with shaking for 8–12 hours for amplification.

[0088] 5) Plasmid Extraction and Storage: 500 μL of the amplified bacterial solution was collected for sequencing. Based on the sequencing results, the correct clones were inoculated into a large flask at a ratio of 1:1000 for expansion. Plasmid extraction was performed using a plasmid extraction kit according to the instructions. After measuring the concentration and purity of the extracted plasmid, the extracted plasmid was labeled and stored at -20°C.

[0089] (2) Collection and preparation of recombinant virus (rHBV)

[0090] 1) Cell culture: Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double-antibody in a 37° C. cell culture incubator with 5% CO 2 and saturated humidity.

[0091] 2) Cell transfection and rHBV virion collection: When the cell density reaches approximately 70%, the payw1.1-GHT-C-null and pcDNA3.1-core plasmids are prepared in a 1:1 transfection system and added to the Huh7 cell supernatant. The medium is replaced after 8-12 hours, and the supernatant is collected after 4 days of culture to obtain the rHBV virions.

[0092] 3) rHBV virus concentration: the same as the above-mentioned wild-type HBV concentration step.

[0093] (3) Detection of Tomatidine's inhibition of early rHBV infection

[0094] The detection method was the same as in Example 2, "Detection of Tomatidine Inhibiting Early Wild-Type HBV Infection." Because the rHBV genome lacks the gene encoding the core protein, it cannot replicate and replenish the cccDNA pool. The cccDNA, the expression template for HBeAg, is formed solely from the repair of incoming rcDNA. Because cccDNA is randomly lost during cell division, HBeAg levels produced by rHBV infection are lower and do not continue to rise compared to wild-type HBV infection. Therefore, HBeAg was only detected in the supernatant on day 5 after infection.

[0095] The above-mentioned payw1.1-GHT plasmid does not produce HBeAg, but can initiate the translation of HBeAg from the 3' end gene under the conditions of HBV cccDNA circularization. Therefore, the production of HBeAg can be used to indicate the formation of viral cccDNA in transfected cells. Based on the above-mentioned plasmid backbone, the HBV mutant plasmid payw1.1-GHT-C-null was further constructed, which can rescue HBV viral replication under the conditions of core protein transcomplementation. The resulting progeny recombinant virus (rHBV) has only one-time infection characteristics. Since it does not have the core gene, it cannot form active replication in infected cells.

[0096] HBeAg test results Figure 5 As shown: In rHBV-infected HepG2-NTCP cells, tomatidine co-treatment at 3μM and 10μM before and during infection also inhibited rHBV de novo infection, with statistically significant inhibition (P<0.01). In the above-mentioned replication-deficient recombinant rHBV replicon model system, the infected cells lack core-related viral replication, so HBeAg is derived solely from cccDNA formed by rHBV de novo infection. These results confirm that tomatidine can inhibit the early stage of HBV infection.

[0097] Example 6 - Tomatidine does not inhibit lentivirus (LV) and adenovirus (ADV) infection

[0098] This example uses Tomatidine to verify the inhibitory effect on other viral infections and finds that it does not inhibit the infection of lentivirus (LV) and adenovirus (ADV). Specifically, the steps include:

[0099] (1) HepG2-NTCP cell culture and plating: same as in Example 1;

[0100] (2) LV / ADV infection: Before infection, cells were pretreated with tomatidine at concentrations of 0 μM, 3 μM, and 10 μM for 10 h. HepG2-NTCP was infected with LV / ADV carrying the target gene or negative control at an MOI of 200. Polybrene at a final concentration of 10 μg / mL was added during LV infection to enhance the infection effect. After 24 h, the virus was removed and DMEM complete medium was added to continue culture for 24 h.

[0101] (3) LV / ADV infection detection: 48 h after infection, the GFP expression level was observed under a fluorescence microscope; and western blot samples were collected to detect the expression level of the target protein.

[0102] The above test results are as follows Figure 6 As shown: Tomatidine does not inhibit the infection of LV and ADV.

[0103] The above examples show that tomatidine has inhibitory effects on both HBV entry and replication. To eliminate the effect of tomatidine on replication, the present invention, on the one hand, provides a time-course experiment of tomatidine dosing, and on the other hand, constructs a replication-deficient recombinant rHBV replicon model. This model provides an effective method for studying early entry inhibitors. The above experiments verify that tomatidine can inhibit the early stage of HBV infection and can be used as a viral entry inhibitor to prevent and treat hepatitis B virus infection.

[0104] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. Use of tomatidine or a pharmaceutically acceptable salt thereof in the preparation of an anti-hepatitis virus drug, characterized in that: The drug is an antiviral drug targeting early infection of hepatitis virus and / or replication of hepatitis virus, and the hepatitis virus is hepatitis B virus.

2. The use according to claim 1, characterized in that For HepG2-NTCP cells, the half toxic concentration CC50 of the tomatidine or a pharmaceutically acceptable salt thereof is 948.2 μM.

3. The use according to claim 1, characterized in that The mechanism of action of the tomatidine or a pharmaceutically acceptable salt thereof is at least one of the following: inhibiting the secretion of HBeAg after viral infection, inhibiting the expression of core protein after viral infection, inhibiting viral entry, and inhibiting early viral infection.

4. The use according to claim 1, characterized in that The administration mode of the tomatidine or pharmaceutically acceptable salt thereof is selected from at least one of the following: pre-infection treatment, treatment during infection, and post-infection treatment.

5. The use according to claim 4, characterized in that The administration mode of the tomatidine or pharmaceutically acceptable salt thereof is pretreatment before infection, and the anti-hepatitis virus drug is a drug for preventing hepatitis B virus infection.

6. The use according to claim 1, characterized in that The drug comprises a pharmaceutically acceptable carrier.

7. The use according to claim 1, characterized in that The dosage forms of the medicine include: tablets, powders, granules, capsules, emulsions and injections.

8. The use according to claim 1, characterized in that The medicine is used alone or in combination.

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