CN115177606A - Application of aurintricarboxylic acid in preparation of anti-hepatitis B virus medicine - Google Patents
Application of aurintricarboxylic acid in preparation of anti-hepatitis B virus medicine Download PDFInfo
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- CN115177606A CN115177606A CN202110363716.8A CN202110363716A CN115177606A CN 115177606 A CN115177606 A CN 115177606A CN 202110363716 A CN202110363716 A CN 202110363716A CN 115177606 A CN115177606 A CN 115177606A
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- hbv
- ata
- hepatitis
- virus
- pharmaceutically acceptable
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- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
Abstract
The invention discloses application of aurintricarboxylic acid or pharmaceutically acceptable salt thereof in preparing a medicament for resisting hepatitis B virus. By ATA treatment of HBV infected and transfected hepatocytes, significant reduction in HBV DNA levels was found without alteration of viral proteins or transcripts. ATA treatment did not further reduce the levels of HBV DNA, viral proteins and transcripts in hepatocytes transfected with HBV RNase H inactivating mutants. In addition, ATA has inhibitory effect on HBV DNA synthesis in hepatocytes transfected with lamivudine-resistant HBV mutants. ATA is a potent inhibitor of HBV replication by disrupting viral polymerase RNase H activity.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of aurintricarboxylic acid in preparation of a hepatitis B virus resistant medicine.
Background
Hepatitis B Virus (HBV) infection is a global public health problem affecting hundreds of millions of people and is a high risk factor for cirrhosis and hepatocellular carcinoma. HBV is an icosahedral nanoparticle with a diameter of about 42nm, and the virion consists of an envelope containing HBsAg, glycoprotein and cellular fat, and a nucleocapsid containing e antigen (HbeAg), core antigen (HBcAg), circular double-stranded HBV-DNA and HBV-DNA polymerase (Pol), which are complete forms of virus and infectious (FIG. 1A). The HBV genome has a 3.2 kilobase pair partially double-stranded relaxed circular DNA (rcDNA), an incomplete (+) strand and an intact (-) strand covalently bonded to Pol at the 5' -end.
When HBV is infected, HBsAg pre-S1 is specifically combined with Na + -taurocholic acid cotransporter (NTCP) on the outer membrane of the hepatocyte substrate, and receptor-mediated virus is promoted to enter a host (figure 1C). HBV decovers after invading hepatocytes and then transports rcDNA into the hepatocyte nucleus and fully ligates to form covalently closed circular DNA (cccDNA). cccDNA is further transcribed and processed to produce pregenomic (pg) RNA and pronucleus viral RNA for subsequent translation of various viral proteins in the cytoplasm, including HBsAg, pol, x proteins, and HBcAg. At the same time, pgRNA and Pol are accumulated into the daughter nucleocapsid. HBV replication depends on the normal function of Pol, a multifunctional enzyme consisting of four domains, a Terminal Protein (TP), a spacer, reverse Transcriptase (RT)/DNA polymerase (HP), and RNase H (FIG. 1B). Importantly, replication of HBV is controlled by reverse transcriptase, using viral pgRNA template to synthesize (-) strand DNA, RNase H then cleaves heteroduplexes of RNA/DNA, HP synthesizes (-) strand DNA into (+) strand DNA, further forming rcDNA, which is either re-routed to the nucleus for cccDNA amplification or reassembled into budding virions.
HBsAg vaccines have long been shown to be effective in protecting vaccinees from HBV infection; however, once infected, cccDNA can persist throughout the life cycle of quiescent hepatocytes without affecting their viability; thus, current viral clearance is not efficient. At present, various anti-HBV drugs aiming at new targets are under development, including therapeutic vaccines, interfering RNA, toll-like receptor 7 agonists, cccDNA synthesis inhibitors and the like, but most of the drugs cannot effectively or completely eliminate cccDNA, and once the drugs are stopped, a great number of patients can rebound and the state of the disease can be repeated. In addition, long-term administration may also lead to the development of resistance variations. For example, lamivudine (also referred to as 3 TC) is a cytidine dideoxynucleoside derivative, a potent reverse transcriptase inhibitor during HBV replication. However, lamivudine treatment requires long-term oral administration, or recurrence, resulting in genotype mutations and loss of sensitivity. There are also a number of strategies for gene therapy of hepatitis b. Antisense nucleic acids have certain advantages in treating hepatitis B infection, however, they lack stability and have inaccurate effects, which limits their application prospects. RNAi is a highly efficient sequence-specific gene knockout technology that is rapidly developing in the field of infectious disease and malignant tumor gene therapy, however, RNAi-based therapy does not completely eliminate cccDNA pools. In conclusion, various treatment methods are different in HBV resistance, but all the treatment methods face the problems of low seroconversion rate of HBeAg, low clearance rate of HBsAg, easy relapse after drug withdrawal and the like. Therefore, the development of new anti-HBV drugs is imminent.
By comprehensively analyzing various defects in the antiviral drugs, although the anti-HBV drugs can effectively inhibit the replication of HBV and improve the life quality of patients, the problems of no response, drug resistance mutation and the like can increase the difficulty of subsequent treatment and increase the medical cost of long-term treatment. Therefore, the clinical medicine needs to be continuously created and the treatment concept needs to be changed, the immune mechanism of hepatitis B needs to be deeply researched, and the medicine which has good curative effect and safety and can enhance the curative effect of the existing anti-HBV medicine is screened from the existing medicine, so that better social benefit and economic benefit for reducing medical cost are generated, and the hepatitis B is finally changed into the disease which can be cured.
Aurin tricarboxylic acid (hereinafter referred to as ATA), 5- ((3-carboxy-4-hydroxyphenyl) (3-carboxy-4-oxo-2, 5-cyclohexadien-1-ylidene) methyl) -2-hydroxybenzoic acid (5- ((3-carboxy-4-hydroxyphenyl) (3-carboxy-4-oxo-2, 5-cyclohexadien-1-yliden) methyl) -2-hydroxybenzoic acid, is a heterogeneous mixture of non-sulfated, charged, electronegative aromatic polymers formed when salicylic acid is treated with formaldehyde, sulfuric acid, and sodium nitrite (see Cushman, et al, (1991) J.Med.Chem.34:329-337, cushman, et al, med.Chem.34: 337-342). Aurintricarboxylic acid has the following structure of formula I:
aurintricarboxylic acid (ATA) has low toxicity to various cells (see Balzarini, J., et al, (1986) Biochem Biophys Res Commun.136 (1), 64-71.). However, there has been no report on the potential inhibitory effect of ATA on HBV.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides the following technical scheme:
in a first aspect, there is provided the use of aurintricarboxylic acid, and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for inhibiting the replication of hepatitis b virus.
In a second aspect, there is provided the use of aurintricarboxylic acid, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting the proliferation of hepatitis b virus.
In a third aspect, the application of aurintricarboxylic acid or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting hepatitis B virus is provided.
Meanwhile, the invention also provides the application of the aurintricarboxylic acid or the pharmaceutically acceptable salt thereof in preparing the medicine for treating the viral hepatitis B.
According to an embodiment of the present invention, aurintricarboxylic acid or a pharmaceutically acceptable salt thereof can be used as the sole active ingredient of the drug, or can be further combined with other drugs, for example, interferons, including short-acting interferon and long-acting interferon; nucleoside analogs such as lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir, TAF and the like.
According to an embodiment of the invention, the medicament further comprises a pharmaceutically acceptable carrier.
Preferably, the carrier includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), sparingly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred.
Preferably, conventional cosolvent, buffering agent, pH regulator and the like can be added into the medicine.
Preferably, colorants, preservatives, flavors, flavorants, sweeteners or other materials are added to the medicament.
Preferably, the dosage form of the medicament comprises but is not limited to tablets, capsules, dripping pills, powder, solutions, suspensions, emulsions, granules, liposomes, buccal tablets, freeze-dried powder injections and the like. The above drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field. The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection and the like; or orally.
According to an embodiment of the invention, the medicament is for the treatment of patients who develop lamivudine resistance, i.e. in which hepatitis b virus develops viral variations with reduced sensitivity to lamivudine.
Preferably, in the above aspects, the hepatitis b virus is a lamivudine-resistant HBV mutant.
According to an embodiment of the invention, the pharmaceutically acceptable salt is selected from an alkali metal salt (e.g. sodium or potassium), an alkaline earth metal salt (e.g. calcium or magnesium), an ammonium salt, or a salt with an organic base providing a physiologically acceptable cation, for example a salt with: n-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexamethylenediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol.
The invention has the beneficial effects that:
the inventors performed ATA therapy on HBV infected and transfected hepatocytes and found that HBV DNA levels were significantly reduced without alteration of viral proteins or RNA transcripts. In contrast, ATA treatment did not further reduce HBV DNA, viral proteins and transcripts from hepatocytes transfected with RNase H mutants of HBV polymerase Pol protein. In addition, ATA has inhibitory effect on HBV DNA synthesis in hepatocytes transfected with lamivudine-resistant HBV mutants. Therefore, ATA is a potent inhibitor for inhibiting HBV replication by disrupting the RNase H activity of viral polymerase Pol. Compared with the existing medicines for treating the hepatitis B, the medicine can thoroughly eliminate the hepatitis B virus in the bodies of susceptible patients, has small toxic and side effects and different action sites, and the ATA has an inhibiting effect on DNA synthesis in liver cells of lamivudine-resistant HBV mutants. ATA is expected to become a new anti-HBV therapeutic drug.
Drawings
Figure 1 viral structure, protein domains of hbv and their life cycle in a host.
(A) Structural features of HBV particles. (B) description of HBV multi-functional domains. (C) Schematic representation of HBV infection and replication in host cells is shown.
Figure 2 ata inhibits replication of hepatitis b virus in hepatocytes.
(A) Differentiated hepatocytes were infected with HBV virus for 14 days in the presence or absence of 5 (low) or 10 μ M (high) ATA and virus production was measured by HBe ELISA at the indicated time points. (B) Huh-7 cells were transfected with the HBV replicon plasmid (HBV 1.5). After 24h of transfection, cells are treated with or without ATA for 48h, HBV DNA is extracted from HBV virus secreted from the culture medium, and qPCR detection is carried out by using HBV specific primers. (C) (B) separating HBV nucleocapsids in the cell lysate using Native Agarose Gel Electrophoresis (NAGE), and then performing Southern or Western blotting (NAGE) using HBV DNA probe or anti-HBc antibody. (D) Extracting the total RNA in the step (B), and detecting HBV transcripts by RT-qPCR. (E) HBV nucleocapsids in HepG2.2.15 cells were separated by Natural Agarose Gel Electrophoresis (NAGE), followed by Southern or Western blotting using HBV DNA probes or anti-HBc antibodies. (F) Extracting the total RNA in (E), and detecting the value of HBV transcripts relative to GAPDH by using RT-qPCR. * P <0.01; ns, not significant (two-sided, unpaired Student's t-test). Data are mean ± standard deviation (SEM) of three independent experiments. All Western blot data are representative of three independent experiments.
FIG. 3.ATA does not affect the replication of Pol RNase H mutant HBV in hepatocytes.
HBV polymerase Pol protein RNase H activity deficient type hepatitis B virus replicon plasmid (HBV 1.5. Delta. RNase H) was transfected into Huh-7 cells. Cells were treated with ATA for 48 hours 24 hours after transfection. (A) HBV nucleocapsids in the cell lysate are separated by Native Agarose Gel Electrophoresis (NAGE), and then Southern or Western blotting (NAGE) is performed using HBV DNA probes or anti-HBc antibodies. (B) Extracting the total RNA in (A), and detecting the value of HBV RNA transcript relative to GAPDH by RT-qPCR. ns was not significant (double-sided, unpaired Student's t-test). Data are mean ± standard deviation (SEM) of three independent experiments. All Western blot data are representative of three independent experiments.
Figure 4 ata inhibits lamivudine resistant HBV mutants.
Lamivudine-resistant HBV mutant plasmids (YIDD or YVDD) were transfected into Huh-7 cells. Cells were treated with or without 3TC or ATA for 48 hours 24 hours post-transfection. (A) HBV nucleocapsids in the cell lysate are separated by Native Agarose Gel Electrophoresis (NAGE), followed by Southern or Western blotting (NAGE) with HBV DNA probes or anti-HBc antibodies. (B) Extracting the total RNA in (A), and detecting the value of HBV RNA transcript relative to GAPDH by RT-qPCR. Data are mean ± standard deviation (SEM) of three independent experiments. All Western blot data are representative of three independent experiments.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Unless defined otherwise, or clear from the background, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Materials and methods:
1. a compound:
ATA is purchased from Sigma-Aldrich (St. Louis, MO, USA). ATA was dissolved in dimethyl sulfoxide (DMSO; sigma-Aldrich) to make 100mM stock solutions, stored at-20 ℃.
2. Cell culture and transfection:
HepaRG cells were cultured in William's E medium (Invitrogen, carlsbad, calif., USA) supplemented with 2mM L-glutamine, 200U/ml penicillin, 200. Mu.g/ml streptomycin, 10% Fetal Bovine Serum (FBS), 5. Mu.g/ml insulin, 20ng/ml epidermal growth factor, 50. Mu.M cortisol (Sigma-Aldrich), and 2. Mu.M DMSO simultaneously. Huh-7 or HepG2.2.15 cells were cultured in Dulbecco's modified Eagle's medium containing 10mM HEPES, 200U/ml penicillin, 200. Mu.g/ml streptomycin and 10% fetal bovine serum. Plasmids were transferred into Huh-7 or HepaRG cells using Fugene6 (Roche, shanghai, china) or Lipofectamine 2000 (Invitrogen) transfection reagents according to the instructions.
3. Hepatitis B virus infection
Infection inocula were prepared with 50-fold concentrated hepg2.2.15 cell culture supernatant. Specifically, the supernatant was freshly collected in 6% polyethylene glycol 8000 (PEG) 8k ) The inoculum is prepared by precipitating the virus particles in the presence. The virus particles were resuspended in phosphate buffer containing 25% fetal bovine serum. Infection of HepaRG cells with HBV (containing 1X 10) 7 Equivalent copies of HBV genome, inoculated 1X 10 5 Individual HepaRG cells), differentiated hepatocyte-like HepaRG cells as concentrated infection inoculum, were co-incubated with primary human hepatocytes and HepaRG cells. And (4) detecting the secretion of HBeAg, HBV DNA and RNA in a plurality of days after infection.
4. Native Agarose Gel Electrophoresis (NAGE) analysis
Whole nucleocapsid particles were separated from crude extracts of HBV replicating cells by agarose gel electrophoresis (see: liang, G et al, RNA edge of hepatitis B viruses trans-cripts by activation-induced cytidine deaminase. Proc Natl Acad Sci U S.2013, 110 (6), 2246-2251). The core-shell particles within the gel were denatured under alkaline conditions and transferred to nitrocellulose membranes (roche). The nucleocapsid DNA and the core protein were detected by Southern and Western blotting, respectively, using either a double-stranded HBV DNA probe or an anti-HBc antibody that spans the entire genome of the virus.
5. Expression vector
HBV replicon or P protein RNase H mutant HBV replicon plasmids (D737V) are described in the literature (Radziwill, G. Et al, mutation analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. J Virol 1990,64 (2), 613-20. DOI. The probe labeling and Southern blot signal were performed using the alkaline phosphatase direct labeling system (Amersham, little Chalfot, UK).
6. Quantitative polymerase chain reaction (qPCR) and Reverse Transcription (RT) PCR
Total RNA was isolated using Trizol reagent (Invitrogen) and treated 2 times with DNase I without RNase. Total RNA (1. Mu.g) was subjected to reverse transcription. The sample results were normalized to the GAPDH amplification results. The primers for detecting HBV RNA are forward 5 'CGGAAATATACATCGTTTTCCAT-3' and reverse 5 'AAGAGTCTCTCTATGTAAGACCTT-3', and the primers for detecting GAPDH RNA are forward 5 'TGCACCACAACTGCTTAGC-3' and reverse 5 'GGCATGGGACTGTGGTCATGAG-3'. rcDNA was purified from secreted virions or cytoplasmic nucleocapsids and total RNA was extracted from cells using Trizol. Purified RNA (1. Mu.g) was treated with DNase I (Invitrogen) and immediately reverse transcribed with Superscript III reverse transcriptase (Invitrogen) and oligo (dT) or random primers. And (3) finishing the real-time fluorescent quantitative PCR by adopting a delta Ct method.
7. Enzyme-linked immunosorbent assay (ELISA)
HBeAg enzyme linked immunosorbent assay is adopted.
8. Statistical analysis
Statistical analysis was performed using Graphpad Prism 6.0. Statistical comparisons between groups used a unpaired, two-tailed t-test, except where otherwise indicated.
Example 1: ATA inhibition of HBV replication
To investigate the inhibitory effect of ATA on HBV replication, HBV-infected hepatocytes HepaRG were first treated with various concentrations of ATA, and the HBeAg content was determined. HepaRG cells were infected with HBV virus without or with 5 (low) or 10 (high) μ M ATA for 14 days and the viral load (secreted HBeAg) in the supernatant was examined at various time points using HBe ELISA. As a result (fig. 2A), ATA inhibited replication of hepatitis b virus in HepaRG cells in a dose-dependent manner. ATA continued to inhibit HBeAg levels, and on day 14, 5 μ g/ml and 10 μ g/ml ATA treatment reduced HBeAg levels to 63.4% and 27.2% of untreated, respectively. Therefore, ATA has an inhibitory effect on replication of hepatitis b virus in hepatocytes.
The present invention explores the ATA mediated antiviral activity in HBV transfected hepatocytes. Hepatocyte-derived cell carcinoma Huh-7 cells were transfected with HBV replicon vectors for 24 hours, further treated with or without ATA for 48 hours, and then viral DNA in the supernatant containing secreted viral particles was detected using qPCR (fig. 2B). The results showed that HBV DNA levels remained at (36.5 ± 4.2)% and (23.4 ± 3.5)% when untreated, respectively, after ATA treatment at 5 and 10 μ M, indicating a significant decrease in HBV production in hepatocytes after ATA treatment (mean p < 0.01). In addition, in cell lysates incubated with different concentrations of ATA-treated HBV-transfected hepatocytes as described above, the HBV nucleocapsids were detected using NAGE, total RNA was extracted and HBV RNA transcripts were measured using RT-qPCR. The levels of nucleocapsid associated DNA (ncDNA) and cytoplasmic nucleocapsid core protein (HBcAg) were detected with HBV DNA probe and HBcAg antibody, respectively. In the NAGE assay, purified HBV particles were loaded directly onto the gel during electrophoresis to determine ncDNA and HBcAg by Southern and Western blotting using HBV DNA probes and anti-HBcAg antibodies, respectively. As shown in fig. 2C, ATA treatment significantly reduced HBV DNA levels, while HBcAg levels were not significantly different from those without ATA treatment. This result indicates that ATA has an inhibitory effect on viral DNA but not on viral RNA transcripts. This finding was confirmed when the transcription level of HBV was detected using RT-qPCR. As shown in FIG. 2D, HBV transcriptomes treated with 5 or 10. Mu.M ATA were (1.03. + -. 0.20) fold or (0.92. + -. 0.11) fold higher than those of the untreated group with no significant difference (p > 0.05).
The present invention further uses hepatocytes stably expressing HBV (HepG2.2.15 cells) to repeat the above-mentioned findings about the inhibitory mechanism of ATA against HBV replication. First, the levels of ncDNA and HBcAg were measured with or without ATA using the method of NAGE, and the results are shown in FIG. 2E. Consistent with the results of Huh-7 cells, there was observed a dose-dependent inhibition of ncDNA synthesis in HepG2.2.15 cells by ATA, but no inhibition of HBcAg expression. Furthermore, when 2.5, 5 and 10 μ M ATA were added, the HBV transcripts were (1.00. + -. 0.14), (0.89. + -. 0.08) and (0.95. + -. 0.04) fold respectively (FIG. 2F), as compared to the untreated group, confirming that ATA treatment did not damage the HBV transcripts.
Example 2 ATA inhibition of HBV Pol RNase H activity
The invention constructs a mutant HBV replicon plasmid HBV D737V mutant, and the plasmid expresses Pol protein with point mutation of D737V. This single amino acid substitution of Pol protein RNase H domain results in loss of RNase H activity, and HBV D737V mutant retains DNA polymerase activity, resulting in HBV replication arrest at RNA/DNA hybridization sites within the nucleocapsid. Huh-7 cells were transfected with wild-type HBV or HBV D737V mutant and further treated with different doses of ATA or not. The results are shown in FIG. 3A. ATA had an inhibitory effect on wild-type HBV DNA levels but not on HBcAg levels, validating the results of fig. 2C. ATA treatment had no effect on viral DNA levels in HBV D737V mutant. According to the literature, it is reported that the DNA (-) strand synthesis ability of the virus of HBV D737V mutant is weakened, while the (+) strand synthesis ability is affected. Therefore, it can be concluded that ATA does not affect the negative (-) strand HBV DNA synthesis by retaining the DNA polymerase activity of Pol. In contrast, ATA can destroy the RNase H activity of Pol, resulting in RNA/DNA hybridization and cessation of HBV replication. Thus, viral genome RC-DNA synthesis is hindered.
Meanwhile, viral transcript levels were measured from total RNA extracted from cell lysates (wild-type HBV or Huh-7 cells transfected with HBV D737V mutant) without or with ATA (FIG. 3B). In wild-type HBV transfected cells, treatment with 5 or 10 μ M ATA failed to inhibit viral transcription because HBV transcription levels remained (0.98 ± 0.19) fold (p > 0.05) and (0.96 ± 0.03) fold (p > 0.05), respectively, compared to no ATA treatment. In contrast, viral transcription of D737V HBV mutant transfected cells was reduced (0.47 ± 0.03) fold (p < 0.001) that of wild type HBV transfected cells, confirming that the virus stopped replication in the mutant transfected host. However, 5 or 10 μ M ATA treatment did not reduce viral transcription in D737V HBV mutant transfected cells, confirming that ATA treatment did not impair HBV transcription. As described above, ATA treatment does not significantly inhibit viral transcription of wild-type or RNase h-deficient mutant HBV. Therefore, ATA inhibits HBV replication by destroying the activity of the viral Pol RNase H and triggers the arrest of HBV at the RNA/DNA hybridization site, and is a potential new agent for the treatment of HBV infection.
Example 3 ATA inhibits viral replication of drug-resistant mutant HBV
Since ATA showed a unique mechanism to inhibit HBV Pol RNase H activity, YIDD, YVDD and YMDD (wild type) HBV were transfected into hepatocytes and the cells were treated with or without ATA or lamivudine in the next experiment. The YMDD motif is highly conserved in the polymerase of retroviruses, with M552I and M552V mutations found to be mainly resistant in HBV patients on long-term lamivudine treatment. The results are shown in FIG. 4A. While both ATA and lamivudine significantly inhibited wild-type HBV ncDNA levels, ATA treatment also significantly reduced viral DNA levels of YIDD and YVDD mutants. At the same time, ATA or lamivudine treatment did not alter HBcAg protein levels. Furthermore, HBV transcripts were unaffected after ATA or lamivudine treatment (fig. 4B). In conclusion, ATA can inhibit lamivudine-resistant HBV mutation, indicating that it is an effective anti-HBV genotype mutation agent.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (10)
1. The use of aurintricarboxylic acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting replication of hepatitis b virus.
2. The application of aurintricarboxylic acid or its pharmaceutically acceptable salt in preparing medicine for inhibiting hepatitis B virus proliferation is disclosed.
3. The application of aurintricarboxylic acid or its pharmaceutically acceptable salt in preparing medicine for resisting hepatitis B virus is disclosed.
4. The application of aurin tricarboxylic acid or pharmaceutically acceptable salt thereof in preparing a medicament for treating viral hepatitis B.
5. Use according to any one of claims 1 to 4, wherein aurintricarboxylic acid or a pharmaceutically acceptable salt thereof is used as the sole active ingredient in the medicament, or in combination with other medicaments.
6. The use of claim 5, wherein the other agent is an agent for the treatment of viral hepatitis B selected from the group consisting of interferons, including short-acting interferons and long-acting interferons; nucleoside analogs including lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir, TAF.
7. The use of any one of claims 1-4, wherein the medicament further comprises a pharmaceutically acceptable carrier.
8. The use according to claim 7, wherein the carrier comprises, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose acetate, etc.); among these, water-soluble carrier materials are preferred.
9. The use according to any one of claims 1 to 4, wherein the medicament is in a dosage form including, but not limited to, tablets, capsules, pills, powders, solutions, suspensions, emulsions, granules, liposomes, buccal tablets, lyophilized powder for injection, and the like; the preparation is used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection and intracavity injection; or orally.
10. The use according to any one of claims 1 to 4, wherein the hepatitis B virus is a lamivudine-resistant HBV mutant.
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