Application of obakla mesylate in the preparation of anti-BKV drugs

Abstract

The present invention provides the use of obatoclax mesylate in the preparation of an anti-BKV drug. In Vero E6 cells, the half-cytotoxic concentration of obatoclax is 7.26 μM, which has low cytotoxicity. It inhibits the expression of both the BKV early gene large T antigen and the late gene VP1, and significantly inhibits the replication of BKV in cells and in the cell supernatant. The half-inhibitory concentration of BKV in cells is 0.2 μM, and the half-inhibitory concentration of BKV in the cell supernatant is 0.02 μM. The inhibitory effect is more obvious as the drug concentration increases. Obatoclax mesylate has high effectiveness and safety and has broad application prospects in drugs for anti-BKV and for treating diseases related to BKV virus infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of obatoclax mesylate in the preparation of anti-BKV (BK polyomavirus) drugs. Background Art

[0002] BKV is a small, non-enveloped DNA virus. BKV infection is ubiquitous in the human population, with a seroprevalence of approximately 80% worldwide. BKV infection typically occurs in early childhood, often within the first ten years of age; in immunocompetent individuals, it is usually asymptomatic or presents with mild symptoms similar to respiratory infections. However, BKV infection can reactivate and cause serious illness in immunosuppressive settings, such as in HIV infection and after transplantation. Kidney transplant recipients (KTRs) are the patient group most frequently experiencing BKV reactivation, with 19.5% of kidney transplant recipients developing BK viremia post-transplant, and a proportion of these recipients will go on to develop polyomavirus-associated nephropathy (BKVAN), which is significantly associated with the risk of graft loss. In addition, hemorrhagic cystitis (HC) is a well-recognized BKV-related complication in hematopoietic stem cell transplant recipients (HSCT).

[0003] Few controlled studies are available to guide the management of BK viremia and BKVAN in renal transplant recipients. Following identification of BK viremia or BKVAN, the typical clinical approach is to gradually reduce immunosuppression with serial monitoring of BK viremia using polymerase chain reaction (PCR). However, even with reduced immunosuppression, some patients continue to experience BKV viremia and BKVAN; furthermore, reduction in immunosuppression carries the risk of triggering acute rejection.

[0004] Currently, there are no specific antiviral drugs for BKV. Although fluoroquinolones, cidofovir, and leflunomide are clinically used to combat BKV infection, the efficacy of these drugs lacks evidence from large-scale prospective studies. Although virus-specific immune control is the ultimate goal of treatment and a durable cure, antiviral therapies are urgently needed to reduce or prevent BKV disease and thus shorten the time required to establish virus-specific immunity.

[0005] Obatoclax Mesylate is a pan-BCL-2 family protein inhibitor and a BH3 mimetic with anticancer and broad-spectrum antiparasitic activity. In the treatment of advanced malignancies, obatoclax directly induces apoptosis in cultured acute myeloid leukemia (AML) cells and primary patient samples, and exhibits antitumor activity in mice bearing solid tumors and myeloma cell xenografts. Recent studies have revealed that obatoclax possesses specific antiviral properties, exhibiting inhibitory effects against influenza, Zika, and coronaviruses. However, its inhibitory effects on other viral species remain unknown. Summary of the Invention

[0006] In order to overcome at least one problem existing in the prior art, the present invention provides an effective anti-BKV drug and treatment method, specifically providing the use of obatoclax mesylate in the preparation of anti-BKV drugs. Obatoclax mesylate can inhibit the expression of early and late BKV genes and inhibit the replication of BKV.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention is to provide the use of obaklav mesylate or its derivatives, or a pharmaceutical composition containing obaklav mesylate or its derivatives, in the preparation of an anti-BKV drug. The structural formula of the obaklav mesylate is shown in formula (I):

[0009]

[0010] Furthermore, the anti-BKV drug includes one of the following drugs: a drug for treating and / or preventing diseases caused by BKV replication, and a drug for treating and / or preventing diseases caused by BKV early and late gene expression.

[0011] Furthermore, the anti-BKV drug includes one of the following drugs: a drug that inhibits BKV replication, a drug that inhibits the expression of BKV early and late genes. Specifically, the BKV early and late genes include the BKV early gene large T antigen and the late gene VP1.

[0012] Furthermore, the anti-BKV drug is selected from at least one of the following drugs: a drug for treating and / or preventing viremia caused by BKV, a drug for treating and / or preventing hemorrhagic cystitis caused by BKV, and a drug for treating and / or preventing viral nephropathy caused by BKV.

[0013] Furthermore, the effective concentration of the obaclava mesylate or its derivative is 0.05 to 3 μM, preferably 0.125 to 2.0 μM.

[0014] Furthermore, for Vero E6 cells, the half-maximal cytotoxic concentration of the obaclava mesylate or its derivatives is 7.26 μM.

[0015] Furthermore, for Vero E6 cells, the intracellular half-maximal inhibitory concentration of BKV was 0.2 μM.

[0016] Furthermore, for Vero E6 cells, the half-maximal inhibitory concentration of BKV in the cell supernatant was 0.02 μM.

[0017] Furthermore, for Huh7 cells, the half-maximal cytotoxic concentration of the obaclava mesylate or its derivatives is 1.05 μM.

[0018] Furthermore, the derivatives include pharmaceutically acceptable salts or esters of obaclava mesylate, or compounds with equivalent functions formed by reducing or adding groups in the structure of formula (I).

[0019] Furthermore, the dosage forms of the drug include: tablets, powders, granules, capsules, emulsions, injections, etc.

[0020] Furthermore, the administration of the drug includes: oral, intravenous, intramuscular, nasal, oral mucosa, lungs and respiratory tract, skin, etc.

[0021] Furthermore, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. It is understood that the type and amount of the selected carrier or excipient are also adaptively adjusted based on the different pharmaceutical dosage forms and administration methods. The above-mentioned carriers or excipients are all conventional reagents commonly used in the art.

[0022] Furthermore, the pharmaceutical composition also contains other drugs capable of resisting BKV.

[0023] Compared with the prior art, the present invention adopts the above technical solution to achieve the following beneficial effects:

[0024] Obatoclax mesylate effectively inhibits BKV early and late gene expression and viral replication. Its half-maximal cytotoxic concentration (CC50) in Vero E6 cells was 7.26 μM, indicating low cytotoxicity. It inhibited the expression of both the BKV early gene large T-antigen and the late gene VP1, and significantly inhibited BKV replication in cells and cell supernatants. The inhibitory effect became more pronounced with increasing drug concentration. Obatoclax mesylate exhibits high efficacy and safety, and holds broad promise as a drug for the treatment of BKV and BKV-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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:

[0026] Figure 1 This is a schematic diagram of the results of a screening experiment for obaclasulfonate in one embodiment of the present invention;

[0027] Figure 2 1 is a graph showing the experimental results of the cytotoxicity of obaclava mesylate in one embodiment of the present invention.

[0028] Figure 3 This is a schematic diagram of the results of obaclava mesylate inhibiting the expression of BKV early and late genes in one embodiment of the present invention.

[0029] Figure 4 This is a schematic diagram of the results of obaclava mesylate inhibiting BKV replication in cells in one embodiment of the present invention.

[0030] Figure 5 This is a schematic diagram of the results of obaclava mesylate inhibiting BKV replication in cell supernatant in one embodiment of the present invention.

[0031] Figure 6 This is a schematic diagram of the effect of obaclava mesylate on HBV replication in liver cancer cells Huh7 according to one embodiment of the present invention. DETAILED DESCRIPTION

[0032] 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 understood that the described embodiments are only some of the embodiments of the present invention, and are not 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. The equipment used in each step of the following examples is conventional equipment. If no corresponding national standards exist, the steps are performed in accordance with generally accepted international standards, conventional conditions, or the 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 technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention.

[0033] 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.

[0034] Obatoclax mesylate (purchased from Target Mol, CAS 803712-79-0) used in the following examples has an English name of Obatoclax Mesylate and a molecular formula of C 21 H 23 N3O4S, the specific molecular structure is shown in formula (I):

[0035]

[0036] In the following examples, unless otherwise described, conventional techniques such as cell biology will be used in the examples, which are well known to those skilled in the art and are fully described in reference books such as Bruce Alberts' "Molecular Biology of the Cell", 5th edition (2002). Alternatively, the methods can be carried out according to the instructions provided by the reagent manufacturers.

[0037] Example 1 - Screening to obtain Opaquelae mesylate

[0038] The early gene region of the prBKV / VP1 expression plasmid was deleted and replaced with the secretory Nanoluciferase gene to construct the expression of prBKV / T-Nluc, as shown in the schematic diagram. Figure 1As shown in part A (for the specific construction method of prBKV / VP1, please refer to patent CN202310508053.3 - A polyomavirus BKV recombinant expression vector and its application); prBKV / T-Nluc and Cre recombinase expression plasmid were co-transfected into VERO E6 cells (purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences) in a 1:1 ratio, and then drug treatment was added, and drug screening was performed by detecting the luciferase intensity in the supernatant.

[0039] Luciferase assay:

[0040] The luciferase activity in the supernatant was measured using the Nano-Glo Luciferase Assay System (N1120, Promega, Madison, USA). Luciferase Assay Substrate and Nano- Luciferase Assay Buffer (1:50) was mixed and then reacted with an equal amount of supernatant. After 5 minutes of reaction, the luciferase activity was detected using a luminometer (Promega). The background (medium only) was subtracted from the measured relative light unit value, and the luminescence intensity was expressed in logarithmic units. The test results are shown in Figure 2. Figure 1 As shown in part B, it shows that obaclava mesylate reduces the luciferase intensity in the supernatant in a concentration-dependent manner.

[0041] Example 2 - Cytotoxicity Experiment of Obaclala Methanesulfonate

[0042] This example tests the toxic effects of different concentrations of a compound (obaklav mesylate) on Vero E6 cells and Huh7 cells (both purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences). The specific procedures include:

[0043] (1) Vero E6 and Huh7 cells (1×10 4 The culture plate was placed in an incubator and cultured for 12 h;

[0044] (2) Different concentrations of the test compound (0.31 μM, 0.63 μM, 1.25 μM, 2.5 μM, 10.0 μM, 20.0 μM, 40.0 μM) were added to the culture wells. The control group was the solvent DMSO. After 72 hours, the effect of the compound on cell proliferation was detected.

[0045] (3) Use the Tongren CCK8 endpoint assay kit (ck04). Discard the culture medium in the wells and add 100 μL of serum-free culture medium containing 10% CCK8 solution to each well; place the culture plate in a 37°C incubator and incubate for 1 to 4 hours;

[0046] (4) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0047] The experimental results of Vero E6 cells are as follows Figure 2 As shown, the half cytotoxic concentration (CC50) of obaclave mesylate for Vero E6 is 7.26 μM, indicating low cytotoxicity and high safety. The half cytotoxic concentration (CC50) of obaclave mesylate for Huh7 is 1.05 μM.

[0048] Example 3 - Obaclala mesylate inhibits the expression of BKV early and late genes

[0049] This example uses Western Blot experiments to observe the effects of different concentrations of a compound (obaklav mesylate) on the expression of BKV early and late genes in cells. The specific process includes:

[0050] (1) Vero E6 cell suspension (2×10 5 / well), and the culture plate was placed in an incubator for 12 h;

[0051] (2) Each well was transfected with 0.5 μg prBKV / VP1 and 0.5 μg pCMV-Cre using Lip 3000. After culturing at 37°C for 8 h, different concentrations of compounds (0.125 μM, 0.25 μM, 0.5 μM, 1.0 μM, 2.0 μM) were added. The control group was treated with an equal amount of DMSO. Samples were collected after 72 h. The prBKV / VP1 expression plasmid was constructed using the BKV Dunlop strain (GenBank: V01108.1) sequence as a template (for specific construction methods, see patent CN202310508053.3 - A polyomavirus BKV recombinant expression vector and its application).

[0052] (3) Lyse the cells using SDS lysis buffer, collect the cell lysate, add protein loading buffer, and denature at 100°C for 10 min.

[0053] (4) SDS-polyacrylamide gel electrophoresis was performed, the membrane was transferred, the membrane was blocked, and the cells were incubated with antibodies [anti-β-actin (Sigma-Aldrich), anti-SV40 Large T Antigen (15729S, Cell Signaling Technology, Beverly, USA) and anti-BKV VP1 (MAB3204, Abnova)]. After incubation with the corresponding secondary antibodies, the cells were developed using ECL Blotting Substrate (Millpore).

[0054] The experimental results are as follows Figure 3 As shown, obakla mesylate inhibits the expression of both the early gene large T antigen (T-antigen) and the late gene VP1, and the inhibitory effect becomes more obvious as the concentration increases.

[0055] Example 4 - Obaklav Mesylate Inhibits BKV Replication

[0056] This example uses Southern blot experiments to observe the effects of different concentrations of a compound (obaklav mesylate) on BKV virus replication in cells. The specific process includes:

[0057] (1) Vero E6 cell suspension (5×10 5 The culture dish was placed in an incubator for pre-culture for 12 h, and the cells grew adherently.

[0058] (2) Each well was transfected with 1 μg prBKV / VP1 and 1 μg pCMV-Cre using Lip 3000. After culturing at 37°C for 8 h, different concentrations of compounds (0.125 μM, 0.25 μM, 0.5 μM, 1.0 μM, 2.0 μM) were added. The control group was treated with an equal amount of DMSO. Samples were collected after 72 h. The prBKV / VP1 expression plasmid was constructed using the BKV Dunlop strain (GenBank: V01108.1) sequence as a template (for specific construction methods, see patent CN202310508053.3 - A polyomavirus BKV recombinant expression vector and its application).

[0059] (3) Extraction of intracellular BKV DNA

[0060] 1) Cell lysis: discard the supernatant of the transfected cells, wash twice with pre-cooled PBS, add 200 μL of Hirtsolution (10 mM Tris, 10 mM EDTA and 0.6% SDS), and lyse at room temperature for 10 min.

[0061] 2) Precipitate cell proteins and DNA: Add 100 μL of 5 M NaCl, shake gently for 2 minutes, scrape the cells, transfer to an EP tube, and incubate at 4°C overnight.

[0062] 3) Proteinase K digestion to remove viral capsid: Centrifuge at 14,000 rpm at 4°C for 40 min, discard the precipitate, and add proteinase K to the supernatant and incubate at 37°C for 8 h.

[0063] 4) Phenol / chloroform extraction to precipitate viral DNA: Extract twice with equal volumes of phenol / chloroform. Add 2 μL of 20 mg / mL glycogen and an equal volume of isopropanol, mix well, and precipitate overnight at -20°C. Centrifuge at 15,000 g for 15 minutes, discard the supernatant, and wash the pellet twice with 75% ethanol, discarding all the ethanol. Allow to stand until any residual ethanol evaporates, then dissolve in 20 μL of sterile distilled water.

[0064] (4) Southern blot detection of BKV replication

[0065] 1) Enzyme digestion: The extracted intracellular BKV DNA was digested with BamHI and DpnI at 37°C for 2 hours.

[0066] 2) Agarose electrophoresis: The digested BKV DNA was subjected to 1% agarose gel electrophoresis (80 V, 1.5 h).

[0067] 3) Denaturation: Place the gel after electrophoresis in freshly prepared denaturing solution (0.5 M NaOH and 1.5 M NaCl) and denature at room temperature for 1 hour with shaking.

[0068] 4) Neutralization: Discard the denaturing solution and pour in neutralizing solution (1.5 M NaCl and 1 M Tris-HCl, pH 7.4) for neutralization. Neutralize twice by shaking at room temperature for 30 minutes each time.

[0069] 5) Transfer: Use downward capillary transfer. The transfer system, from bottom to top, consists of: absorbent paper, Parafilm membrane, two layers of 3mm filter paper, nylon membrane, agarose gel containing DNA, two layers of 3mm filter paper, and a salt bridge with both ends immersed in 20× SSC buffer (3M NaCl and 0.3M sodium citrate). Transfer at room temperature for at least 8 hours.

[0070] 6) DNA crosslinking: After transfer, remove the nylon membrane, soak the membrane in 2×SSC for 5 min, drain the excess liquid, place the nylon membrane between two pieces of filter paper, and crosslink under UV for 90 s.

[0071] 7) Prehybridization: Place the membrane in a hybridization tube, add 5 ml of hybridization solution, and prehybridize at 42°C for 30 minutes.

[0072] 8) Probe Denaturation and Hybridization: Denature an appropriate amount of probe in a 100°C metal bath for 5 minutes. Immediately place the denatured probe on ice for 5 minutes. Recover the prehybridization solution and replace it with 5 ml of fresh hybridization solution. Add the denatured probe and hybridize at 42°C for 6-8 hours.

[0073] 9) Wash to remove unbound probe: Wash twice with 2×SSC at room temperature for 5 minutes each time. Then wash twice with 0.5×SSC at 68°C for 15 minutes each time.

[0074] 10) Blocking: Dilute 10× Blocking solution to 1× blocking working solution with maleic acid buffer, add appropriate amount of blocking solution, and block at 37°C for 30 min.

[0075] 11) Antibody incubation: Dilute anti-DIG AP antibody with the above 1× blocking buffer (antibody diluted 1:10,000), discard the blocking buffer and replace with an appropriate amount of antibody incubation buffer, incubate at 37°C for 30 min.

[0076] 12) Wash away unbound antibodies: Wash the membrane twice with washing buffer at 37°C for 15 min each time.

[0077] 13) Color Development: First, equilibrate the membrane with detection buffer for 5 minutes. Simultaneously, dilute and prepare 1× CSPD colorimetric solution with detection buffer. Place the equilibrated nylon membrane onto a Parafilm membrane, front side up. Evenly add the colorimetric solution. Cover with another layer of Parafilm. Remove any bubbles and excess colorimetric solution from the front of the nylon membrane. Incubate at room temperature, protected from light, for 5 minutes. Detect the cumulative signal using a chemiluminescence detector and save the result.

[0078] The experimental results are as follows Figure 4 As shown, obaklavam mesylate significantly inhibited BKV replication, with the inhibitory effect increasing with increasing drug concentration. The half-maximal inhibitory concentration (IC50) was 0.2 μM as indicated in the figure. The experimental results demonstrate that obaklavam mesylate has a strong inhibitory effect on BKV replication and can be used as a potential therapeutic drug against BKV.

[0079] Example 5 - Opaquelae Mesylate Inhibits Viral Amounts in Supernatant

[0080] In this example, the real-time quantitative PCR (qPCR) method is used to detect the supernatant of drug-treated cells to verify the level of viral DNA in the supernatant, which includes the following steps:

[0081] (1) Removal of unencapsulated viral DNA: 200 μL of the cell supernatant culture medium after drug treatment in Example 4 was treated with 0.1 mg / mL DNase I and 10 mM MgCl2 at 37°C for 2 h.

[0082] (2) DNA extraction: Viral DNA was extracted according to the kit instructions (Sangon Biotech, B518267).

[0083] (3) Real-time quantitative PCR was performed using the Tiangen SYBR Green I chimeric fluorescence method (Tiangen, FP205). Amplification primers specific for the late protein VP1 were used: VP1 upstream primer: 5'-GCAGCTCCCAAAAAGCCAAA-3'; downstream primer: 5'-CTGGGTTTAGGAAGCATTCTA-3'. A standard curve was established using 10-fold serial dilutions of the plasmid BKV Dunlop-PUC19, and the amount of BKV DNA in the test components was calculated based on the standard curve.

[0084] The experimental results are as follows Figure 5 As shown, obaklavam mesylate has a significant inhibitory effect on BKV viral DNA in the supernatant. At the same time, the inhibitory effect becomes more obvious with the increase of drug concentration. The half inhibitory concentration (IC50) is shown to be 0.02 μM.

[0085] Example 6 - Effect of Obakla Methanesulfonate on Hepatitis B Virus (HBV) in Huh7 Liver Cancer Cells This example used an ELISA experiment to observe the effects of different concentrations of the compound on HBV in cells. The specific process was as follows:

[0086] (1) Huh7 cell suspensions were pre-cultured in a 6-well plate in an incubator for 12 hours to allow adherent growth. Each well of Huh7 cells was first transfected with 6 μL of Turbofect transfection reagent and 3 μg of p1.3×HBV plasmid. After 12 hours, different concentrations of the compound of formula (I) were added. After 72 hours, the cell supernatant was collected.

[0087] (2) ELISA detection of HBsAg and HBeAg levels in the supernatant

[0088] The ELISA kit of Shanghai Kehua Biotechnology Co., Ltd. was used to detect the HBsAg and HBeAg levels in the cell supernatant. The relative absorbance value (OD450) at 450nm can reflect the relative level of the antigen. The specific method is briefly described as follows: the cell supernatant collected in step (1) was diluted 20 times with PBS and added to the ELISA enzyme-linked plate, 50uL per well, and then the enzyme reaction solution was added, and the plate was placed at 37℃ for 30min. The reaction solution was then discarded and washed 5 times with washing solution. Then, reaction solutions A and B were added, and the plate was placed at 37℃ for 10min. The stop solution was added and the absorbance value at 450nm was read using a spectrometer. The test results are as follows: Figure 6 As shown, obaclava mesylate had no significant effect on HBV antigen expression, which indicates that obaclava mesylate has no inhibitory effect on HBV.

[0089] The present invention mainly evaluates the anti-BKV activity of obaklav mesylate represented by chemical formula (I) through in vitro experiments; the in vitro experiments mainly include the inhibition of the expression of early and late BKV genes, the inhibition of intracellular viral DNA replication, the inhibition of the viral DNA content in the supernatant, and the determination of the drug's cytotoxicity, etc., which solves the current problem of the urgent need for effective anti-BKV drugs and provides a new use of obaklav mesylate in anti-BKV drugs.

[0090] 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 obaklav mesylate or a pharmaceutical composition containing obaklav mesylate in the preparation of an anti-BKV drug, characterized in that: The structural formula of the obaclava mesylate is shown in formula (I):

2. The use according to claim 1, characterized in that The anti-BKV drugs include: drugs used for treating and / or preventing diseases caused by BKV replication.

3. The use according to claim 1, characterized in that The anti-BKV drugs include drugs that inhibit the replication of BKV.

4. The use according to any one of claims 1 to 3, characterized in that The anti-BKV drug is selected from at least one of the following drugs: a drug for treating and / or preventing viremia caused by BKV, a drug for treating and / or preventing hemorrhagic cystitis caused by BKV, and a drug for treating and / or preventing viral nephropathy caused by BKV.

5. The use according to claim 1, characterized in that The effective concentration of the obaclava mesylate is 0.125 μM to 2.0 μM.

6. The use according to claim 1, characterized in that For Vero E6 cells, the half-maximal cytotoxic concentration of the obaklav mesylate is 7.26 μM; or, for Vero E6 cells, the half-maximal inhibitory concentration of BKV in the cells is 0.2 μM; or, for Vero E6 cells, the half-maximal inhibitory concentration of BKV in the cell supernatant is 0.02 μM; or, for Huh7 cells, the half-maximal cytotoxic concentration of the obaklav mesylate is 1.05 μM.

7. The use according to claim 1, characterized in that The pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient.

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