Use of rilpivirine in the preparation of a medicament against swine influenza virus subtype H1N1

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of rilpivirine in the preparation of drugs against H1N1 subtype swine influenza virus. The present invention discovers for the first time that the compound rilpivirine can effectively inhibit the proliferation of H1N1 subtype swine influenza virus and has relatively low toxicity to cells. It is experimentally proven that rilpivirine can effectively inhibit and kill H1N1 subtype swine influenza virus on the PK15 cell model in vitro experiments, can effectively inhibit the invasion of H1N1 subtype swine influenza virus, and has relatively low cytotoxicity. It can be used as a new type of drug against H1N1 subtype swine influenza virus, opening up a new drug use for rilpivirine, and also laying an experimental foundation and providing a new perspective for the development of highly efficient and specific drugs against H1N1 subtype swine influenza virus.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of rilpivirine in drugs against H1N1 subtype swine influenza virus. Background Art

[0002] The H1N1 subtype swine influenza virus is a novel H1N1 subtype swine influenza virus reassembled from human influenza virus and swine influenza virus. The PB2, PB1, and M genes of this virus are derived from the influenza A H1N1 virus, the PA, NP, and NS genes are derived from the swine-derived H3N2 subtype influenza virus, and the HA and NA genes are derived from the swine-derived H1N1 subtype influenza virus. It is a novel swine influenza virus reassortant virus. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the present invention provides the application of rilpivirine in the preparation of drugs against H1N1 subtype swine influenza virus.

[0004] Rilpivirine is a non-nucleoside reverse transcriptase inhibitor, which is mostly used in combination with other antiretroviral drugs and is extremely effective against HIV-1 infection. However, there has been no record of rilpivirine being used for the prevention or treatment of H1N1 subtype swine influenza virus so far.

[0005] The present invention first confirms that rilpivirine can effectively inhibit the proliferation of H1N1 subtype swine influenza virus and has relatively low toxicity to cells. Therefore, it has the prospect of being developed into a drug against H1N1 subtype swine influenza virus.

[0006] One of the objectives of the present invention is to provide the application of rilpivirine in the preparation of drugs against H1N1 subtype swine influenza virus.

[0007] Another objective of the present invention is to provide a pharmaceutical composition against H1N1 subtype swine influenza virus.

[0008] To achieve the above-mentioned invention objectives, the present invention relates to the following technical solutions:

[0009] In the first aspect of the present invention, there is provided the application of rilpivirine in the preparation of drugs against H1N1 subtype swine influenza virus. And thus, rilpivirine is effective for the prevention and / or treatment of diseases related to H1N1 subtype swine influenza virus.

[0010] "Drug against H1N1 subtype swine influenza virus" refers to a substance that has a significant inhibitory and killing effect on H1N1 subtype swine influenza virus and has good effects in directly killing the virus, adsorbing and blocking the virus, and blocking virus replication.

[0011] According to the present invention, rilpivirine can also be used in combination with other non-drug active ingredients.

[0012] In view of this, a second aspect of the present invention provides a pharmaceutical composition against H1N1 subtype swine influenza virus, which is composed of rilpivirine and at least one other non-pharmaceutical active ingredient.

[0013] Among them, rilpivirine is taken at a drug concentration not lower than the median effective concentration (EC 50 ), and the median effective concentration (EC 50 ) of rilpivirine against H1N1 subtype swine influenza virus is 5.16 μM; the therapeutic index of rilpivirine against H1N1 subtype swine influenza virus is 19.38. Of course, when rilpivirine is used in combination with other drugs or active ingredients that have the same applications as those mentioned in the present invention, such as inhibiting and / or killing or assisting in inhibiting and / or killing H1N1 subtype swine influenza virus, its drug concentration can theoretically be lower than the above-mentioned median effective concentration, but special exceptions are not excluded.

[0014] Compared with the prior art, the present invention has the following effects and advantages:

[0015] The present invention first discovers that the compound rilpivirine can effectively inhibit the proliferation of H1N1 subtype swine influenza virus and has relatively low toxicity to cells. It has been experimentally proven that the median cytotoxic concentration (CC 50 ) of rilpivirine against PK15 cells is ≥100 μM, while the median effective concentration (EC 50 ) against H1N1 subtype swine influenza virus is 5.16 μM; the therapeutic index of rilpivirine against H1N1 subtype swine influenza virus is 19.38, indicating its prospect of being developed into a drug against H1N1 subtype swine influenza virus, opening up a new drug use for rilpivirine, and also laying an experimental foundation and providing a new perspective for the development of highly efficient and specific drugs against H1N1 subtype swine influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0017] Figure 1 It is a diagram showing the effect of rilpivirine against H1N1 subtype swine influenza virus-infected cells in an embodiment of the present invention; Figure 1 A is the virus control group; Figure 1 B is the PK15 normal cell group; Figure 1 C is the infected cell drug test group (using 6.25 μM rilpivirine).

[0018] Figure 2 It is a diagram showing the median cytotoxic concentration (CC 50 ) of rilpivirine against PK15 cells in Example 2 of the present invention.

[0019] Figure 3 This is the graph of the 50% effective concentration (EC 50 ) of rilpivirine against H1N1 subtype swine influenza virus in Example 3 of the present invention.

[0020] Figure 4 This is the effect diagram of the direct killing effect of rilpivirine on H1N1 subtype swine influenza virus in Example 5 of the present invention.

[0021] Figure 5 This is the effect diagram of the blocking effect of rilpivirine on the adsorption of H1N1 subtype swine influenza virus in Example 5 of the present invention.

[0022] Figure 6 This is the effect diagram of the blocking effect of rilpivirine on the replication of H1N1 subtype swine influenza virus in Example 5 of the present invention. Detailed implementation manners

[0023] As introduced in the background art, in the prior art, there has been no report on the use of rilpivirine for the prevention and treatment of H1N1 subtype swine influenza virus so far.

[0024] In view of this, in a specific implementation manner of the present invention, there is provided the use of rilpivirine in the preparation of a drug against H1N1 subtype swine influenza virus. And thus, rilpivirine is effective for the prevention and / or treatment of diseases related to H1N1 subtype swine influenza virus. The active ingredient in the drug against H1N1 subtype swine influenza virus is rilpivirine. It should be noted that this application is publicly disclosed for the first time and is different from its known clinical medication uses. Its structural formula is as follows:

[0025]

[0026] Among them, in the drug against H1N1 subtype swine influenza virus, rilpivirine takes a drug concentration not lower than the 50% effective concentration (EC50). The 50% effective concentration (EC 50 ) of rilpivirine against H1N1 subtype swine influenza virus is 5.16 μM;

[0027] In the sense of the present invention, a "drug against H1N1 subtype swine influenza virus" refers to a substance in which the contained rilpivirine has an obvious inhibitory effect on H1N1 subtype swine influenza virus.

[0028] Therefore, in some embodiments of the present invention, there is provided the use of rilpivirine in the preparation of a drug for inhibiting and / or killing H1N1 subtype swine influenza virus; or, the use of rilpivirine in the preparation of a drug for inhibiting the proliferation of H1N1 subtype swine influenza virus; specifically, the inhibition of the proliferation of H1N1 subtype swine influenza virus means that it has good effects on the direct killing, adsorption blocking, and replication blocking of the virus.

[0029] According to the present invention, rilpivirine can also be used in combination with other non-pharmaceutical active ingredients.

[0030] Therefore, in another specific embodiment of the present invention, there is provided a pharmaceutical composition for anti-H1N1 subtype swine influenza virus, which is composed of rilpivirine and at least one other non-pharmaceutical active ingredient.

[0031] In the pharmaceutical composition, rilpivirine can be used as the sole active ingredient against H1N1 subtype swine influenza virus. When rilpivirine is administered in combination with at least one other pharmaceutical active ingredient, the effect of rilpivirine against H1N1 subtype swine influenza virus can be enhanced. Therefore, in some embodiments of the present invention, the pharmaceutical composition further comprises at least one other pharmaceutical active ingredient.

[0032] Specifically, the other pharmaceutical active ingredients include substances that can inhibit and / or kill H1N1 subtype swine influenza virus or assist in inhibiting and / or killing H1N1 subtype swine influenza virus.

[0033] In some embodiments of the present invention, rilpivirine is taken at a drug concentration not lower than the median effective concentration (EC 50 ). The median effective concentration (EC 50 ) of rilpivirine against H1N1 subtype swine influenza virus is 5.16 μM.

[0034] Specifically, the other non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

[0035] In some embodiments of the present invention, the dosage forms include liquid dosage forms, solid dosage forms, topical preparations, or sprays.

[0036] Specifically, the dosage forms include true solutions, colloids, particulate dosage forms, emulsion dosage forms, suspension dosage forms, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powder injections, inclusion compounds, implants, patches, liniments.

[0037] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0038] Example 1 Determination of virus TCID 50 assay

[0039] After digesting PK15 cells, inoculate them into a 96-well cell culture plate at a cell density of 1×10 5 cells / mL. Place it in a cell culture incubator at 37°C and 5% CO2. After culturing into a monolayer of cells, discard the cell growth medium in the wells. The virus diluent of H1N1 subtype swine influenza virus serially diluted 10-fold (the dilution degrees are 10-1 ~10 -10 ) Inoculate into a 96-well plate filled with a monolayer of cells, 100 μL per well, place it in an incubator at 37 °C and 5% CO2 for continued culture, and observe the CPE of the cells daily, and record in detail the number of wells with cytopathic changes. At the same time, set up a normal cell control group and a blank control group, with 3 replicates in each group. When no further cytopathic changes occur, perform a hemagglutination test using a 96-well V-shaped plate. Add 50 μL of cell suspension to each well, then add 50 μL of 0.75% chicken red blood cells, let it stand at room temperature for 20 minutes, check whether the chicken blood agglutinates, read and record the hemagglutination result, and calculate the virus TCID according to the Karber method 50 。

[0040] Table 1 TCID of H1N1 subtype swine influenza virus 50

[0041]

[0042] Note: TCID 50 , Tissue culture infective dose, the median tissue culture infective dose, also known as the 50% tissue culture infective dose; that is, the amount of virus required to cause cytopathic changes or death (cytopathic effect, CPE) in half of the cells in a culture plate well or test tube

[0043] Results Figure 1 As shown, morphological observations under the microscope found that virus dilutions at different concentrations caused cytopathic changes at 48 h. The refractivity of the cells changed, the monolayer structure was damaged, the cells showed necrosis, gradually became reticular and formed vacuoles, and some cells lysed and fell off in fragments. After 72 h, the cytopathic changes in each well no longer continued. The number of hemagglutination wells at different concentrations was counted, the hemagglutination ratio at different concentrations was calculated, and the TCID of H1N1 subtype swine influenza virus was calculated according to the Karber method 50 value:

[0044] LgTCID 50 =L - D(S - 0.5)

[0045] (L: logarithm of the highest dilution; D: difference between the logarithms of the dilutions; S: sum of the positive well ratios)

[0046] LgTCID 50 =L - D(S - 0.5)= - 1 - 1×(5.3 - 0.5)= - 5.8

[0047] TCID 50 =10 -5.8 / 0.1mL

[0048] That is, dilute the virus 105.8 Inoculation of 100 μL can cause cytopathic effect in 50% of the cells.

[0049] Example 2: Toxicity experiment of rilpivirine on PK15 cells

[0050] PK15 cells are susceptible cells to H1N1 subtype swine influenza virus. Therefore, first, the cytotoxicity of rilpivirine on PK15 cells was detected. The specific experimental steps are as follows:

[0051] (1) Inoculate 100 μL of cells (PK15 1×10 4 cells / well) in a 96-well plate.

[0052] (2) After culturing until a monolayer of PK15 is formed, proceed to the next step of drug addition analysis. Discard the culture medium, add 100 μL of serum-free DMEM containing different drug concentrations to each well (dilute the drug addition at a two-fold concentration gradient starting from an initial drug concentration of 200 μM), and perform 3 parallels for each concentration. At the same time, for the control wells: add 100 μL of serum-free DMEM culture medium. For the zero-adjustment well: do not seed cells.

[0053] (3) After culturing at 37 °C and 5% CO2 for 72 h, operate according to the CCK-8 kit instructions, and measure the OD value at 450 nm using an enzyme-linked immunosorbent assay reader.

[0054] (4) After continuing to culture at 37 °C and 5% CO2 for 1 h, measure the absorbance value at 450 nm. Set the A450nm of normally growing cells as 100% cell control.

[0055] (5) Analyze the data and calculate the half cytotoxic concentration (CC 50 ) value of rilpivirine using GraphPad Prism5. The results are as Figure 2 shown.

[0056] Results: Rilpivirine showed a dose-dependent relationship, that is, with the increase of the drug concentration, the cytopathic effect became more obvious. After statistical analysis, it was determined that the half toxic concentration of rilpivirine was ≥100 μM.

[0057] Example 3: Inhibition experiment of rilpivirine on H1N1 subtype swine influenza virus

[0058] (1) Inoculate 1×10 4 PK15 cells in each well of a 96-well plate and culture overnight in a 37 °C, 5% CO2 incubator;

[0059] (2) Discard the culture medium and add 100 μL of 10000 TCID 50Diluent of H1N1 subtype swine influenza virus (After the cells were confluent with serum-free DMEM, the virus diluent was added. According to the initial concentration of 50 μM, the drug was added with a two-fold concentration gradient dilution, and cultured in a 5% CO2 incubator;

[0060] (3) After 72 h, a hemagglutination test was performed using a 96-well V-shaped plate. 50 μL of cell suspension was added to each well, and then 50 μL of 0.75% chicken red blood cells was added. It was left standing at room temperature for 20 minutes to check whether the chicken blood agglutinated, and the hemagglutination result was read and recorded.

[0061] (4) Analyze the data, and the results are as Figure 3 shown. Then calculate the corresponding therapeutic index TI value according to the formula TI = CC 50 / EC 50 .

[0062] Results: Through the hemagglutination test results, the effective inhibition rate of the drug against H1N1 subtype swine influenza virus can be calculated. It can be seen from the results that within the safe concentration range of rilpivirine, its effective inhibition rate increases with the increase of the drug concentration, showing a certain dose-effect relationship. Through the analysis software, the half-maximal effective concentration (EC 50 ) of H1N1 subtype swine influenza virus is 5.16 μM. The therapeutic index of rilpivirine against H1N1 subtype swine influenza virus is 19.38.

[0063] Example 4 Effect of adding compound at different times on the replication of H1N1 subtype swine influenza virus

[0064] In vitro antiviral inhibition tests were carried out on rilpivirine using three different action modes: adding the drug first and then the virus, adding the virus first and then the drug, and pre-incubating the drug and the virus.

[0065] (1) Direct killing effect of the drug on the virus

[0066] An equal amount of 10000 TCID 50 virus solution was mixed evenly with different concentrations of drug diluent and pre-incubated at 37°C and 5% CO2 in an incubator for 2 h, then added to a confluent monolayer of 96-well cell culture plates. According to the initial concentration of 50 μM, the drug was added with a two-fold concentration gradient dilution, 100 μL / well for each drug concentration gradient, and incubated in the incubator for 2 h. The supernatant was discarded, and the cell maintenance medium was added for continued culture. In this experiment, normal cell control group, virus control group and blank control group were set up at the same time. Each concentration had 3 replicates. After 72 h, the hemagglutination test was performed to detect the results, and the EC 50 of the compound was obtained using GraphPad Prism5 software.

[0067] Results: Under the administration method of pre-incubating rilpivirine with H1N1 subtype swine influenza virus, through the analysis software, the effect of rilpivirine on H1N1 subtype swine influenza virus is asFigure 4 as shown. From Figure 4 it can be seen that under this mode of action, rilpivirine shows 100% complete inhibitory effect on H1N1 subtype swine influenza virus at concentrations greater than 6.25 μM within the safe concentration range, indicating that rilpivirine has a certain direct inactivating effect on H1N1 subtype swine influenza virus.

[0068] (2) Blocking effect of the drug on the adsorption of H1N1 subtype swine influenza virus

[0069] Inoculate the digested cells into the well plate at a cell density of 1×10 4 per well. After the cells grow into a monolayer, discard the supernatant. Dilute the drug with a two-fold concentration gradient starting from an initial concentration of 50 μM. Add 100 μL / well of the diluted drug solution at different concentration gradients to the 96-well cell culture plate with a monolayer of cells. Incubate in the incubator for 2 h in advance, then discard the supernatant, wash twice with PBS, and add an equal volume of 10,000 TCID 50 virus solution and culture in an incubator at 37 °C and 5% CO2. In this experiment, normal cell control group, virus control group and blank control group are set up at the same time. Each concentration is set with 3 replicates. After 72 h, perform hemagglutination test to detect the results and calculate the antiviral efficiency of the drug at different concentrations under this mode of action.

[0070] Results: Through the analysis software, the effect of rilpivirine on H1N1 subtype swine influenza virus is as Figure 5 shown. The results show that within the safe concentration range, the effective inhibition rate of rilpivirine on H1N1 subtype swine influenza virus can reach 100% at a concentration of 12.5 μM and above, and the effective inhibition rate at a concentration of 6.25 μM can reach 33.33%, indicating that rilpivirine can prevent the adsorption of H1N1 subtype swine influenza virus to cells.

[0071] (3) Blocking effect of the drug on the replication of H1N1 subtype swine influenza virus

[0072] Inoculate the digested cells into the well plate at a cell density of 1×10 4 per well. After the cells grow into a monolayer, discard the supernatant. Add an equal volume of 10,000 TCID 50 virus solution to the 96-well cell culture plate with a monolayer of cells. Incubate in an incubator at 37 °C and 5% CO2 for 1 h in advance, then discard the supernatant, wash the cells twice with PBS, dilute the drug with a two-fold concentration gradient starting from an initial concentration of 50 μM, and then add the diluted drug solution at different concentrations, 100 μL / well for each drug solution gradient. In this experiment, normal cell control group, virus control group and blank control group are set up at the same time. Each concentration is set with 3 replicates. Incubate in an incubator at 37 °C and 5% CO2, perform hemagglutination test to detect the results after 72 h, analyze the data and draw conclusions.

[0073] Result: Through the analysis software, the effect of rilpivirine on blocking the replication of H1N1 subtype swine influenza virus is as Figure 6 shown. The results show that within the safe concentration range, the effective inhibition rate of rilpivirine at a concentration of 6.25 μM and above against H1N1 subtype swine influenza virus is 100%, indicating that rilpivirine can effectively block the replication of H1N1 subtype swine influenza virus in PK15 cells.

[0074] In the application example of the present invention, using PK15 cells as a carrier, in a cell pathogenic model, in vitro antiviral inhibition studies were carried out using three different action modes: adding the drug first and then the virus, adding the virus first and then the drug, and the virus acting in advance and then adding the drug. The novel antiviral effect of rilpivirine was discovered, and it has a certain inhibitory effect on H1N1 subtype swine influenza virus.

[0075] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of rilpivirine in the preparation of a drug against swine influenza virus of H1N1 subtype.

2. The use according to claim 1, characterized in that, The active ingredient in the drug against H1N1 subtype swine influenza virus is rilpivirine.

3. The use according to claim 1, characterized in that, Rilpivirine is the sole active ingredient in the drug against H1N1 subtype swine influenza virus.

4. The use according to claim 1, characterized in that, In the said drug against H1N1 subtype swine influenza virus, the half-maximal effective concentration of rilpivirine is 5.16 μM.

5. The use according to claim 1, characterized in that, Use of rilpivirine in the preparation of a drug for inhibiting and / or killing H1N1 subtype swine influenza virus.

6. The use according to claim 1, characterized in that, Use of rilpivirine in the preparation of a drug for inhibiting the proliferation of H1N1 subtype swine influenza virus.

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