Application of mycophenolate in medicine for inhibiting coxsackie group B virus
By using mycophenolic ester to activate the immune signaling pathway and inhibit the replication and infection of the Coxsach B virus, the problem of poor treatment of the Coxsach B virus in the prior art was solved, and effective antiviral effects were achieved and Better security.
Patent Information
- Application Number
- CN202411467242.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2025-01-07
AI Technical Summary
The existing anti-Coxsackie Group B virus drugs have limited therapeutic effects, and the rapid virus mutation has led to huge challenges in vaccine development, and there is a lack of effective drugs to inhibit the Coxsackie Group B virus.
Mycophenolate Mofetil (MMF) is used as the active ingredient to upregulate the expression of JAK1 protein in the host protein and promote JAK1 phosphorylation, activate immune signaling pathways, enhance cells' resistance to the Coxsach B virus, and inhibit viral replication. and infection.
Mycophenolic acid has low cytotoxicity and high antiviral efficacy, which can effectively inhibit the replication and infection of the Coxsach B virus, reduce the inflammatory response caused by the virus, and improve the infection caused by the Coxsach B virus. Symptoms, a new antiviral drug option is provided.
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Figure CN119258072A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to application of mycophenolate mofetil in drugs for inhibiting Coxsackie B virus. Background Art
[0002] Coxsackie B4 virus (CV-B4) is a small, non-enveloped single-stranded RNA virus belonging to the Enteroviridae family. It can cause a variety of human diseases, including hand, foot and mouth disease, myocarditis, diabetes, etc. The infection rate of CV-B4 is high worldwide, especially in children and people with weak immune systems. Although the mortality rate of the virus is not high, infection may cause serious sequelae such as heart disease and nervous system damage. Existing antiviral drugs have limited therapeutic effects on CV-B4, mainly because these drugs cannot effectively prevent viral replication or because their side effects are large. In addition, the rapid mutation of the virus also poses a huge challenge to vaccine development. Therefore, the development of new anti-CV-B4 drugs is of great clinical significance. Mycophenolate Mofetil (MMF) is an immunosuppressant that is commonly used to prevent rejection reactions after organ transplantation. In recent years, studies have found that MMF also has antiviral potential. The present invention is based on the discovery that MMF has a good inhibitory effect on CV-B4, and aims to develop a new anti-CV-B4 drug.
[0003] In view of this, the present invention is proposed. Summary of the invention
[0004] One of the purposes of the present invention is to provide a use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting coxsackie group B virus, so as to solve the technical problem of the lack of a substance having a good inhibitory effect on coxsackie group B virus in the prior art.
[0005] The second object of the present invention is to provide a use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by coxsackie group B virus infection.
[0006] The third object of the present invention is to provide a use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product capable of improving symptoms caused by coxsackie group B virus infection.
[0007] A fourth object of the present invention is to provide a product.
[0008] In a first aspect, the present invention provides the use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance having mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting Coxsackie B virus.
[0009] Furthermore, the inhibition of Coxsackie B virus includes inhibiting the replication of Coxsackie B virus.
[0010] Furthermore, the inhibition of Coxsackie B virus includes exerting its effect by upregulating JAK1 protein expression in host proteins and / or promoting JAK1 protein phosphorylation.
[0011] In a second aspect, the present invention provides the use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance having mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by coxsackie group B virus infection.
[0012] In a third aspect, the present invention provides the use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance having mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product capable of improving symptoms caused by coxsackie group B virus infection.
[0013] Furthermore, the Coxsackie B group virus includes Coxsackie B4 virus and Coxsackie B2 virus.
[0014] Furthermore, the product includes a medicine.
[0015] In a fourth aspect, the present invention provides a product, the active ingredient of which is mycophenolate mofetil or a pharmaceutically acceptable salt thereof; the product has any of the following uses:
[0016] A1. Inhibit Coxsackie B virus;
[0017] A2. Treatment and / or prevention of symptoms caused by coxsackie B virus infection;
[0018] A3. Improve symptoms caused by Coxsackie B virus infection.
[0019] Furthermore, the product is a medicine.
[0020] Furthermore, the administration of the drug includes any one of oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, nasal administration or transdermal administration, or a combination of at least two thereof;
[0021] Preferably, the drug is administered once a day at a dosage of 20 to 50 mg / kg body weight each time.
[0022] The application of mycophenolate mofetil or its pharmaceutically acceptable salt or a substance with mycophenolate mofetil or its pharmaceutically acceptable salt as an active ingredient in the preparation of a product for inhibiting coxsackie B virus provided by the present invention is a new use of mycophenolate mofetil (MMF). Mycophenolate mofetil has lower cytotoxicity, higher antiviral efficacy, and better phase dosing effect, and can provide a new option for treating coxsackie B4 virus infection. The technical problem of the lack of substances with good inhibitory effect on coxsackie B virus in the prior art is solved.
[0023] On the other hand, it can be used as an antiviral drug to inhibit the replication and infection of Coxsackie B virus. By upregulating JAK1 protein expression and promoting JAK1 phosphorylation, mycophenolate activates immune signaling pathways and enhances cells' resistance to Coxsackie B virus. The new use of mycophenolate is expected to provide a new and effective means for treating Coxsackie B virus infection. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0025] Figure 1 This is a graph showing the toxic effects of different concentrations of mycophenolate mofetil on HELA cells provided in Example 1 of the present invention;
[0026] Figure 2 This is a graph showing the in vitro anti-Coxsackie B4 virus effect of different concentrations of mycophenolate provided in Example 2 of the present invention;
[0027] Figure 3 This is a graph showing the effect of mycophenolate mofetil added at different time points on virus-induced cytopathic effects provided in Example 3 of the present invention;
[0028] Figure 4 This is a graph showing the effect of mycophenolate mofetil added at different time points on the cell activity of virus-induced cytopathic effects provided in Example 3 of the present invention;
[0029] Figure 5 This is a diagram showing the effect of mycophenolate mofetil on the expression of antiviral mechanism proteins provided in Example 4 of the present invention;
[0030] Figure 6 This is a diagram showing the effect of mycophenolate mofetil on cellular gene levels under different treatments provided in Example 5 of the present invention;
[0031] Figure 7A statistical graph of cell gene expression levels under different treatments provided in Example 5 of the present invention;
[0032] Figure 8 This is a graph showing the in vitro anti-Coxsackie B2 virus effects of different concentrations of mycophenolate provided in Example 6 of the present invention. DETAILED DESCRIPTION
[0033] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.
[0034] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.
[0035] In one aspect, the present invention provides the use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting Coxsackie B virus.
[0036] The application is a new use of mycophenolate mofetil (MMF), which has lower cytotoxicity, higher antiviral efficacy, and better time-phase dosing effect, and can provide a new option for treating Coxsackie B4 virus infection, thereby solving the technical problem of the lack of substances with good inhibitory effects on Coxsackie B group virus in the prior art.
[0037] The experimental results showed that in cells treated with mycophenolate, the RNA and protein levels of CV-B4 decreased significantly, and the virus production decreased. The antiviral mechanism of mycophenolate involves multiple levels. On the one hand, mycophenolate can increase the expression of interferon. Interferon is an antiviral cytokine that can activate the body's natural immune response and inhibit the replication and spread of viruses. It can also increase the expression of antiviral-related metalloproteinases, directly degrade viral RNA, and prevent viral replication. In some specific embodiments, the inhibition of Coxsackie B virus includes inhibiting the replication of Coxsackie B virus. On the other hand, mycophenolate can upregulate the expression of JAK1 protein and promote the phosphorylation of JAK1, thereby activating the JAK-STAT signaling pathway. The JAK-STAT signaling pathway is an important signal transduction mechanism in cells and is crucial for regulating immune responses and antiviral defense. By activating this pathway, mycophenolate enhances the resistance of cells to CV-B4. In some specific embodiments, the inhibition of Coxsackie B virus includes exerting its effect by upregulating the expression of JAK1 protein in host proteins and / or promoting the phosphorylation of JAK1 protein.
[0038] According to another aspect of the present invention, there is also provided the use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by coxsackie group B virus infection.
[0039] Among them, diseases caused by Coxsackie B virus infection include herpangina, acute hemorrhagic conjunctivitis, hand, foot and mouth disease, pleurisy, myocarditis, pericarditis and hepatitis.
[0040] In addition to directly inhibiting viral replication, mycophenolate can also reduce the inflammatory response caused by coxsackie B virus. Studies have shown that the expression levels of inflammatory factors such as TNF-α and IL-6 are reduced in cells treated with mycophenolate, which helps to reduce tissue damage caused by viral infection. Comprehensively evaluate the antiviral effect of mycophenolate. According to another aspect of the present invention, there is also provided the use of mycophenolate or a pharmaceutically acceptable salt thereof or a substance with mycophenolate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product that can improve symptoms caused by coxsackie B virus infection.
[0041] Among them, symptoms caused by Coxsackie B virus infection include fever, sneezing, coughing and rash.
[0042] In some specific embodiments, the Coxsackie B group virus includes Coxsackie B4 virus and Coxsackie B2 virus.
[0043] In some specific embodiments, the product comprises a drug.
[0044] Mycophenolate has strong anti-Coxsackie B virus activity and can effectively inhibit viral replication and infection; secondly, the mechanism of action of mycophenolate is clear, and it inhibits the virus by activating the JAK-STAT signaling pathway and increasing interferon expression; finally, the safety of mycophenolate has been verified in other clinical applications, and it is expected that its application in the antiviral field will also have good safety. The research of the present invention provides an important theoretical basis and experimental basis for the development of new anti-Coxsackie B virus drugs. According to another aspect of the present invention, a product is also provided, the active ingredient of which is mycophenolate or a pharmaceutically acceptable salt thereof; the product has any of the following uses:
[0045] A1. Inhibit Coxsackie B virus;
[0046] A2. Treatment and / or prevention of symptoms caused by coxsackie B virus infection;
[0047] A3. Improve symptoms caused by Coxsackie B virus infection.
[0048] In some specific embodiments, the product is a drug.
[0049] In some specific embodiments, the administration of the drug includes any one or a combination of at least two of oral, intravenous, intramuscular, subcutaneous, sublingual, nasal or transdermal administration;
[0050] In some specific embodiments, the drug is administered at a dosage of 20 to 50 mg / kg body weight once a day.
[0051] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0052] Coxsackie B4 virus strain (COX-B4) was obtained from the National Institute of Respiratory Diseases, the First Affiliated Hospital of Guangzhou Medical University;
[0053] The Coxsackie B2 virus strain (COX-B4) was isolated by the Virus Research Laboratory of the Women and Children's Medical Center of Guangzhou Medical University;
[0054] Mycophenolate mofetil (MMF): purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number A4336.
[0055] Example 1 Detection of the cytotoxic effect of mycophenolate mofetil on HELA cells by CCK-8 method
[0056] HELA cells were cultured at 7×10 4Cells were seeded at a density of 100 μL / ml in a 96-well plate and incubated at 37°C with 5% CO 2 Culture in an incubator for 12 hours. When the cell density reaches 80%, take out the mycophenolate mother solution and dissolve it in room temperature and away from light. Dilute mycophenolate in multiple ratios to a concentration gradient (100 μM, 200 μM, 400 μM, 800 μM and 1600 μM) with virus maintenance solution. Aspirate the original culture medium in the 96-well plate, add different concentrations of mycophenolate to the 96-well plate, and add virus maintenance solution (DMEM containing 1% FBS) to the control group. Maintain 6 replicate wells for each concentration. Each HELA cell was cultured with the corresponding concentration of mycophenolate for 48 hours. Observe the toxic effects of the drug on the cells under a microscope (such as Figure 1 The CCK-8 solution was prepared according to the Cell Counting Kit-8 instruction manual with a DMEM:CCK-8 ratio of 10:1. The supernatant in the 96-well plate was aspirated and 100 μL of CCK-8 solution was added to each well. The plate was placed at 37°C and 5% CO. 2 The cells were incubated in an incubator for 30 min. Then, the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay to calculate the cell survival rate (cell survival rate = average absorbance of the drug-treated group / average absorbance of the normal control group). The concentration of the drug that caused half of the cells to die was defined as CC. 50 .
[0057] The cytotoxic effect of mycophenolate mofetil on HELA cells is manifested as: cell breakage and shedding, increase in intracytoplasmic particles, change in cell morphology, tending to be round, and a significant decrease in absorbance value.
[0058] Depend on Figure 1 It can be seen that after treatment with 1600 μM mycophenolate, the survival rate of HELA cells dropped below 50%. The data results show that mycophenolate acts on the CC of HELA cells. 50 The concentration of mycophenolate mofetil is within the safe and non-toxic range within 800 μM and is used in subsequent antiviral studies.
[0059] Example 2 Mycophenolate mofetil inhibits COX-B4 in vitro
[0060] HELA cells were cultured at 7×10 4 Cells were seeded at a density of 100 μL / ml in a 96-well plate and incubated at 37°C with 5% CO 2 Incubate incubator for 12 hours. When cell density reaches 80%, take out the mycophenolate mofetil stock solution and dissolve it at room temperature away from light. The CC of mycophenolate mofetil on each cell is measured in the previous step. 50Data, in this experiment, the maximum concentration for HELA cells was set to 100 μM. Mycophenolate mofetil was diluted to a concentration gradient (1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM and 200 μM) by virus maintenance solution, and mixed with 200 TCID50 COX-B4 in equal volumes to obtain a mixture of mycophenolate mofetil and 100 TCID50 COX-B4 at concentrations of 0.78125 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM, the culture medium of the 96-well plate was aspirated, and 100 μL of the mixture was added to each well according to the concentration gradient, the control group was added with virus maintenance solution (DMEM containing 1% FBS), and the virus control was added with 100 TCID COX-B4 as a positive control. Six duplicate wells were set for each mode of action to avoid deviation between wells, and the plates were placed at 37°C, 5% CO 2 Incubate incubator for 48 hours. Since the virus used in this experiment carries eGFP fluorescence, the number of fluorescence observed under a fluorescence microscope can be used to indicate the virus replication. If the drug has an antiviral effect, the number of fluorescence in the well corresponding to the drug will be less than that of the virus control group. Figure 2 As shown, the scale bar is 70 μm, where Mock is the control group, Cox-B4 is the virus control group, and MMF is the mixed group of mycophenolate mofetil and Cox-B4.
[0061] Depend on Figure 2 It can be seen that compared with the virus control, the fluorescence number in the wells of the MMF groups of different concentrations was less than that of the virus control group, and mycophenolate mofetil of various concentrations could significantly inhibit the proliferation of COX-B4. The half effective concentration of mycophenolate mofetil to inhibit COX-B4 replication was 0.3064 μM.
[0062] Example 3 The effect of mycophenolate mofetil on inhibiting COX-B4 in vitro
[0063] HELA cells were cultured at 7×10 4 Cells were seeded at a density of 100 μL / ml in a 96-well plate and incubated at 37°C with 5% CO 2 Culture in an incubator for 12 hours. When the cell density reaches 80%, take out the mycophenolate mother solution and dissolve it in the dark at room temperature. Dilute the mother solution to 10 μM with virus maintenance solution. Add mycophenolate 24 hours before COX-B4 infection, when the cells are infected (0 hours), and 4 hours after the cells are infected. Add 100 TCID COX-B4 to the virus control as a positive control. Set 6 duplicate wells for each mode of action to avoid deviation between wells. Set a corresponding virus control for each drug addition time. Place at 37°C and 5% CO 2Incubate in an incubator for 48 hours. The number of fluorescence observed under a fluorescence microscope indicates the replication of the virus. If the drug has an antiviral effect, the number of fluorescence in the well corresponding to the drug will be less than that of the virus control group.
[0064] The cell activity was tested by CCK-8 kit, and CCK-8 was added to the cells in a dark environment and incubated at 37°C for 1 hour. The absorbance at 450nm was detected by enzyme-linked immunosorbent assay to reflect the therapeutic effect of mycophenolate mofetil on virus-induced cytopathic effects at different time points. The specific results are as follows Figure 3 As shown, Mock is the control group, Cox-B4 is the virus control group, -24h is 24h before COX-B4 infection, 0h is when COX-B4 infection occurs, and 4h is 4h after COX-B4 infection. SPSS13.0 was used to calculate the difference in cell viability. The specific results are shown in Figure 4 As shown. It can be seen that mycophenolate mofetil has a significant inhibitory effect on COX-B4 when added at three time points: 24 hours before COX-B4 infection, during infection, and 4 hours after infection. The recovery of cell status is statistically different from that of the virus control group. Among them, the best effect is achieved when mycophenolate mofetil is added 4 hours after the addition of the virus, which suggests that mycophenolate mofetil has great potential as a therapeutic drug.
[0065] Example 4 WB test experiment of mycophenolate mofetil inhibiting Coxsackie B4 virus
[0066] In this example, the regulation of protein expression in mycophenolate-treated cells was tested. In this Western Blot experiment, Hela cells were selected as mycophenolate-treated cells. Hela cells were cultured at 10×10 4 Cells were seeded at a density of 100 cells / ml (10 mL) in a 10 cm culture dish and incubated at 37 °C and 5% CO 2 Culture in an incubator for 12 hours. When the cell density grows to 80%, prepare 10 ml of 20 μM mycophenolate solution with mycophenolate mother solution through virus maintenance solution. Take 5 mL of the prepared mycophenolate solution and mix it with 200 TCID50 COX-B4 virus to obtain a mixture of 10 μM mycophenolate and 100 TCID50 COX-B4 virus as the drug treatment group. Take 5 mL of the prepared mycophenolate solution and dilute it to 10 μM through virus maintenance solution as the drug control group. The virus control uses 100 TCID50 COX-B4 virus. The blank control group is added with virus maintenance solution (DMEM containing 1% FBS). Aspirate the culture medium in the 10 cm culture dish, and add 10 mL of each of the four groups: blank control group, drug control group, virus control group, and drug treatment group to 4 10 cm culture dishes at the same time. Place at 37 ° C and 5% CO 2Culture in the incubator for 48 hours, discard the supernatant, wash with PBS three times, add 300 μL of cell lysis buffer (RIPA strong: PI: PMSF = 100: 1: 2) to each culture dish, place on ice for lysis for 10 minutes, and shake continuously to keep the lysis uniform. Then use a low-temperature high-speed centrifuge at 10000rpm at 4 degrees for 30 minutes, and take the supernatant as the protein sample. According to the instructions of the BCA kit, the protein concentration is quantitatively normalized, and then an equal volume of 2*loading buffer is added and boiled in a 100℃ water bath for 10 minutes to obtain a Western Blot sample.
[0067] The results were observed as Figure 5 As shown, from left to right, the strips are blank control, drug control group, virus control group, and drug treatment group. It can be seen that mycophenolate mofetil upregulates the expression of JAK1 protein and promotes JAK1 phosphorylation, and downregulates the upregulation of IFN-α, IFN-β1, IFN-γ, and IL-8 caused by viral infection, providing a possible mechanism of action of mycophenolate mofetil, which inhibits viral replication by promoting the activation of JAK1.
[0068] Example 5: PCR test of intracellular factors after mycophenolate mofetil action
[0069] Hela cells were cultured at 10×10 4 Cells were seeded at a density of 100 cells / ml (10 mL) in a 10 cm culture dish and incubated at 37°C and 5% CO 2 Culture in an incubator for 12 hours. When the cell density reaches 80%, prepare 10 ml of 20 μM mycophenolate solution with mycophenolate mother solution through virus maintenance solution. Take 5 mL of mycophenolate solution and mix it with 200 TCID50 COX-B4 virus to obtain a mixture of 10 μM mycophenolate and 100 TCID50 COX-B4 virus as the drug treatment group. Take 5 mL of mycophenolate solution and dilute it to 10 μM through virus maintenance solution as the drug control group. The virus control uses 100 TCID50 COX-B4 virus. The blank control group is added with virus maintenance solution (DMEM containing 1% FBS). Aspirate the culture medium in the 10 cm culture dish, and add 10 mL of each of the four groups: blank control group, drug control group, virus control group, and drug treatment group to 4 10 cm culture dishes at the same time. Place at 37 ° C and 5% CO 2 Culture in an incubator for 48 h, discard the supernatant, wash with PBS three times, extract total nucleic acid using the traditional Trizol method, and add the kit BeyoFast TMThe reaction solution and program were configured under the guidance of SYBRGreenOne-StepqRT-PC RKit, and amplification was performed according to the 20uL system. The experimental results were statistically analyzed using GraphPadPrism8.0. COX-B4 VP1 gene front primer: 5-GGAACCT AGCGATACCATGCA-3-, rear primer: 5-ATGCGTCTGAACTGGAACGT-3-. GAPDH front primer: 5-GAAGGTCGGTGTGAACGGAT-3-, rear primer: 5-AGTGA TGGCATGGACTGTGG-3-. The results are shown in Figure 2. Figure 6 and Figure 7 As shown, NC is the blank control, MMF is the drug control group, B4 is the virus control group, B4+MMF is the drug treatment group, and the internal reference gene is GAPDH.
[0070] Depend on Figure 6 and Figure 7 It can be seen that in the virus control group (B4), the interferon series and antiviral-related metalloproteinases were decreased, which was not conducive to the antiviral activity of the cells. However, after the addition of mycophenolate mofetil (B4+MMF), the decreased interferon and antiviral factors were alleviated, providing new insights into the antiviral effect of mycophenolate mofetil.
[0071] Example 6 Mycophenolate mofetil inhibits Coxsackie B2 virus in vitro
[0072] HELA cells were cultured at 7×10 4 Cells were seeded at a density of 100 μL / ml in a 96-well plate and incubated at 37°C with 5% CO 2 Incubate incubator for 12 hours. When cell density reaches 80%, take out the mycophenolate mofetil stock solution and dissolve it at room temperature away from light. The CC of mycophenolate mofetil on each cell is measured in the previous step. 50 Data, in this experiment, the maximum concentration of Hela cells was set to 100μM. Mycophenolate was diluted to a concentration gradient (6.25μM, 12.5μM, 25μM, 50μM, 100μM and 200μM) by virus maintenance solution, and mixed with 200TCID50 COX-B2 in equal volumes to obtain a mixture of mycophenolate and 100TCID50COX-B1 at concentrations of 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, and 100TCID50COX-B1. The 96-well plate culture medium was aspirated, and 100μL of the mixture was added to each well according to the concentration gradient. The control group was added with virus maintenance solution (DMEM containing 1% FBS), and the virus control was added with 100TCID COX-B2 as a positive control. Six duplicate wells were set for each mode of action to avoid deviation between wells, and 37, 5% CO 2Incubate incubator for 48 hours. Since the virus used in this experiment carries eGFP fluorescence, the number of fluorescence observed under a fluorescence microscope can be used to indicate the virus replication. If the drug has an antiviral effect, the number of fluorescence in the well corresponding to the drug will be less than that of the virus control group. Figure 2 As shown, the scale bar is 70 μm, where Mock is the control group, COX-B2 is the virus control group, and the MMF group is the mycophenolate mofetil and COX-B2 mixed group.
[0073] Depend on Figure 8 It can be seen that compared with the virus control, the fluorescence numbers in the wells of the MMF groups with different concentrations were all less than that of the virus control group, and mycophenolate mofetil at various concentrations could significantly inhibit the proliferation of COX-B2.
[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. Use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting Coxsackie B virus.
2. The use according to claim 1, characterized in that: The inhibition of Coxsackie B virus includes inhibiting the replication of Coxsackie B virus.
3. The use according to claim 2, characterized in that: The inhibition of Coxsackie B virus includes exerting its effect by upregulating the expression of JAK1 protein in the host protein and / or promoting the phosphorylation of JAK1 protein.
4. Use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by coxsackie group B virus infection.
5. Use of mycophenolate mofetil or a pharmaceutically acceptable salt thereof or a substance with mycophenolate mofetil or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product capable of improving symptoms caused by coxsackie group B virus infection.
6. The use according to any one of claims 1 to 5, characterized in that: The Coxsackie B group virus includes Coxsackie B4 virus.
7. The use according to claim 6, characterized in that: The products include pharmaceuticals.
8. A product, the active ingredient of which is mycophenolate mofetil or a pharmaceutically acceptable salt thereof; the product has any of the following uses: A1. Inhibit Coxsackie B virus; A2. Treatment and / or prevention of symptoms caused by coxsackie B virus infection; A3. Improve symptoms caused by Coxsackie B virus infection.
9. The product according to claim 8, characterized in that The product described is a drug.
10. The product according to claim 7, characterized in that The administration of the drug includes any one of oral, intravenous, intramuscular, subcutaneous, sublingual, nasal or transdermal administration, or a combination of at least two thereof; Preferably, the drug is administered once a day at a dosage of 20 to 50 mg / kg body weight each time.