Application of small drug molecule Simeprevir in preparation of anti-neobunyavirus drug

Abstract

The invention provides an application of a drug small molecule Simeprevir in preparation of an anti-neobunyavirus drug, belongs to the field of drug preparation, provides a new application direction of the Simeprevir and a new direction of preparation of the anti-neobunyavirus drug by applying the drug small molecule Simeprevir (cimiprevir) in preparation of the anti-neobunyavirus drug, and particularly provides a new application direction of the Simeprevir (cimiprevir) and a new application direction of the anti-neobunyavirus drug in preparation of the anti-neobunyavirus drug in preparation of the anti-neobunyavirus drug in preparation of the anti-neobunyavirus drug in preparation of the anti-neobunyavirus drug in preparation of the anti-neobunyavirus drug. A new thought is provided for preventing and treating the new bunyavirus.

Description

Technical Field

[0001] The present invention belongs to the field of drug preparation, and particularly relates to the application of the drug small molecule Simeprevir in the preparation of drugs against novel bunyavirus, and also relates to an oral tablet and an intravenous injection for anti-novel bunyavirus containing Simeprevir. Background Art

[0002] Novel bunyavirus, specifically referring to Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), is a newly discovered virus in the genus Phlebovirus of the order Bunyavirales. The novel bunyavirus is mainly transmitted through tick bites, and its clinical manifestations include fever, thrombocytopenia, leukopenia, and in severe cases, it can lead to multiple organ failure, with a fatality rate of about 5 - 10%. Its genomic structure is similar to that of bunyaviruses, but the nucleotide sequence and encoded proteins are specific, and it has a low homology with other members of the genus Phlebovirus (such as Rift Valley fever virus).

[0003] Currently, there are no specific vaccines and antiviral drugs against this virus, and clinical treatment mainly relies on supportive therapies. Although the broad-spectrum antiviral drug Ribavirin shows certain anti-SFTSV activity in in vitro experiments, its clinical efficacy for patients with high viral loads is limited, and it may also cause adverse consequences such as anemia and hyperamylasemia. Favipiravir, one of the antiviral candidate drugs, shows better efficacy than Ribavirin in in vitro and animal models, but its efficacy in treating SFTS patients has not been verified. Summary of the Invention

[0004] Based on the above problems existing in the prior art, the present invention provides the application of the drug small molecule Simeprevir in the preparation of drugs against novel bunyavirus. The CAS number of Simeprevir is 923604 - 59 - 5. As a small molecule compound, Simeprevir was initially developed for the treatment of hepatitis C virus (HCV), and it exerts antiviral effects by inhibiting viral protease activity. The present invention discovers that Simeprevir has a significant inhibitory effect on the replication of SFTSV. Therefore, using Simeprevir to prepare drugs against novel bunyavirus has certain application value.

[0005] Among them, the dosage forms of the drugs against novel bunyavirus include powders, tablets, granules, capsules, solutions, emulsions, and suspensions.

[0006] Among them, the Simeprevir is the only effective component of the anti-Neobunyavirus drug.

[0007] The present invention also provides an oral tablet for anti-Neobunyavirus, which contains the drug small molecule Simeprevir.

[0008] Among them, the oral tablet for anti-Neobunyavirus is made of the following components and amounts: Simeprevir 100 mg / tablet, microcrystalline cellulose 120 mg / tablet, crospovidone 20 mg / tablet, magnesium stearate 5 mg / tablet, hydroxypropyl methylcellulose 10 mg / tablet, polyethylene glycol-4000 15 mg / tablet, and silicon dioxide 5 mg / tablet.

[0009] The present invention also provides an intravenous injection for anti-Neobunyavirus, which contains the drug small molecule Simeprevir.

[0010] Among them, the intravenous injection for anti-Neobunyavirus is made of the following components and amounts: Simeprevir 100 mg / vial, polysorbate-80 50 mg / vial, hydroxypropyl-β-cyclodextrin 400 mg / vial, and sodium chloride 45 mg / vial. The final volume of each vial of intravenous injection is 5 ml, which is made up to 5 ml with water for injection, and the pH of the injection is adjusted with sodium dihydrogen phosphate, and the range of the pH value is 6.5 - 7.0.

[0011] The beneficial effects of the present invention: Applying the drug small molecule Simeprevir to the preparation of anti-Neobunyavirus drugs provides a new application direction for Simeprevir, and provides a new direction for the preparation of anti-Neobunyavirus drugs, providing a new idea for the prevention and treatment of Neobunyavirus. Description of the Drawings

[0012] Figure 1 It is a curve graph of Simeprevir inhibiting virus replication.

[0013] Figure 2 It is an indirect immunofluorescence result graph of Simeprevir inhibiting the expression of Neobunyavirus natural virus NP protein. The colors in the attached drawings are part of the experimental results, so color drawings are used. Detailed Embodiments

[0014] The following describes the present invention in detail with reference to the drawings and specific embodiments.

[0015] Example 1: Application of the drug small molecule Simeprevir in the preparation of anti-Neobunyavirus drugs.

[0016] The CAS number of the small molecule simeprevir is 923604-59-5, and one Chinese translation is Ximipivei. Its structural formula is as follows: The above-mentioned small molecule drug simeprevir can be used in the preparation of drugs against novel bunyavirus. The dosage forms of the drugs against novel bunyavirus include pharmaceutically acceptable carrier dosage forms such as powders, tablets, granules, capsules, solutions, emulsions, and suspensions, etc. The simeprevir is the only active ingredient of the drugs against novel bunyavirus.

[0017] The small molecule drug simeprevir in this example has a significant inhibitory effect on the replication of novel bunyavirus, providing a new idea for the prevention and treatment of novel bunyavirus.

[0018] Example 2: An oral tablet against novel bunyavirus.

[0019] This example provides an oral tablet against novel bunyavirus, which contains the small molecule drug simeprevir and is specifically made from the following components and amounts: The amounts of each component in the above table are the amounts for one tablet dose, and each tablet contains 100 mg of simeprevir.

[0020] The preparation method of the oral tablet against novel bunyavirus provided in this example is as follows: Step S1 Raw material treatment: Pass the simeprevir raw material through an 80-mesh sieve, and pass the microcrystalline cellulose and crospovidone through a 60-mesh sieve to ensure the uniformity of the particles.

[0021] Step S2 Mixing: Place simeprevir, microcrystalline cellulose, crospovidone, and silicon dioxide in a high-efficiency mixer and mix at 200 rpm for 15 minutes to obtain a uniform mixture.

[0022] Step S3 Granulation: Dissolve hydroxypropyl methylcellulose in an appropriate amount of purified water to prepare a 5% (w / v) binder solution. Slowly add the binder solution to the mixture and granulate using a wet granulator (10-mesh sieve). After granulation, dry at 50 °C in a forced-air oven for 12 hours, and control the moisture content to be less than 2%.

[0023] Step S4 Screening and lubrication of granules: Screen the dried granules through a 20-mesh sieve, add magnesium stearate and polyethylene glycol 4000, and mix for 5 minutes.

[0024] Step S5 Tabletting: Use a rotary tabletting machine (punch die diameter 8 mm), adjust the pressure to a tablet weight of about 275 mg, and control the hardness at 60 - 80 N.

[0025] Step S6 Coating (optional): To improve stability, a film coating solution containing polyvinyl alcohol can be used for film coating, with a coating weight gain of approximately 3%.

[0026] Step S7 Quality control: Detect the content uniformity of the tablets (the content of simeprevir should be 95% - 105% of the labeled amount), the disintegration time limit (≤15 minutes), and the dissolution rate (the dissolution rate within 30 minutes should be ≥85%, using 0.1 M HCl as the medium).

[0027] The formulation provided in this example improves the solubility of simeprevir by adding polyethylene glycol 4000. Microcrystalline cellulose and cross-linked polyvinylpyrrolidone ensure the mechanical strength and rapid disintegration of the tablets, are suitable for oral administration, and meet the treatment needs of patients with acute SFTSV infection.

[0028] Example 3: An intravenous injection for anti - novel bunyavirus.

[0029] This example provides an intravenous injection for anti - novel bunyavirus, which contains the drug small molecule Simeprevir and is specifically prepared from the following components and amounts: The amounts of each component in the above table are for a 5 - ml intravenous injection. Each vial contains 100 mg of simeprevir, which is made up to 5 ml with injection water, and its pH is adjusted with sodium dihydrogen phosphate. The pH value ranges from 6.5 to 7.0.

[0030] The preparation method of the anti - novel bunyavirus intravenous injection provided in this example is as follows: Step S1 Preparation of inclusion complex: Dissolve hydroxypropyl - β - cyclodextrin in 2 mL of injection water, heat to 40°C, slowly add simeprevir, and stir for 30 minutes until completely dissolved to form an inclusion complex.

[0031] Step S2 Solubilization and mixing: Add polysorbate 80 to the inclusion complex solution, continue stirring for 10 minutes, and add sodium chloride until completely dissolved.

[0032] Step S3 pH adjustment: Adjust the pH of the solution to 6.5 - 7.0 with 0.1 M sodium dihydrogen phosphate solution, and add injection water to make up to 5 mL.

[0033] Step S4 Filtration and filling: Filter the solution through a 0.22 - μm microporous membrane to remove particles and bacteria, fill it into a 5 - mL sterile vial, and seal it under nitrogen protection.

[0034] Step S5 Sterilization: Sterilize with high - pressure steam at 121°C for 15 minutes to ensure the sterility of the preparation.

[0035] Step S6 Quality Control: Detect the clarity (no visible particles), the content of simeprevir (98%-102% of the labeled amount), the pH value (6.5-7.0), and the osmotic pressure (280-310 mOsm / kg) of the preparation. The sterility test and the pyrogen test comply with the requirements of the Chinese Pharmacopoeia.

[0036] Example 4: Testing the anti-SFTSV effect of the small molecule drug simeprevir.

[0037] Cell viability assay: Vero cells were seeded in 96-well plates at a density of 5000 cells per well. After 12 hours, the cells were treated with different concentrations of simeprevir. After incubation for 48 hours, the cytotoxicity of simeprevir was detected using a Cell Counting Kit-8 (CCK-8) (C0038, Beyotime) according to the manufacturer's instructions. 10 μL of CCK-8 reagent was added to each well of the 96-well plate, and the cells were incubated at 37 °C for 1 hour. The absorbance was read at a wavelength of 450 nm using a microplate reader (Bio-Rad). The absorbance of untreated cells (OD control) was used as a control. This assay was performed at least independently three times.

[0038] SFTSV infection and drug treatment: Vero cells were seeded in 24-well plates (105 cells / well). The next day, the cells were infected with medium containing SFTSV (MOI = 0.05) for 2 hours. After 2 hours, the medium was replaced with medium containing different concentrations of simeprevir (concentration gradient: 0, 0.05, 0.1, 0.52, 1.5625, 6.25, 12.5, 25, 50, 100 μM, two replicate wells for each concentration), and incubation was continued for 48 hours. After 48 hours, RT-qPCR experiments were performed to evaluate the viral replication in the cells. The RT-qPCR experiment used a two-step method: an equal amount of quantitative RNA was reverse transcribed into cDNA and then used for qPCR. The experimental data were analyzed using the 2-ΔΔCt relative quantification method.

[0039] RT-qPCR analysis: Total RNA was isolated using the RNeasy Mini Kit (74104, Qiagen) according to the manufacturer's instructions. cDNA was synthesized using the HiScript III RT SuperMix for qPCR kit (R323, Vazyme Biotech), and qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech) according to the manufacturer's instructions, with GAPDH as the internal control. The primers used were as follows: SFTSV forward: 5'-AAGAGTGCGTTCATTATTG-3'; SFTSV reverse: 5'-ATTGCCTRAGGACATTGGTGAG-3'; GAPDH forward: 5'-AGCCTTCCCAGTGGGTGAAGAC-3'; GAPDH reverse: 5'-CGGAGTCACACGGATTTGGTCG-3'.

[0040] The PCR amplification procedure was as follows: 30 seconds at 95°C, followed by 40 cycles (10 seconds at 95°C and 30 seconds at 60°C). The dose-response curve of viral RNA copies versus drug concentration was plotted using GraphPad Prism 8 software, and the results are as Figure 1 shown.

[0041] Indirect immunofluorescence assay: Vero cells were seeded in 12-well plates. After reaching 80% confluence, the SFTSV virus (MOI = 0.05) was added to the cells for 2 hours of infection. Then, the virus-containing supernatant was replaced with medium containing different concentrations of Simeprevir until the end of the experiment. All samples were washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde (P0099, Beyotime) for 15 minutes, and permeabilized with 0.1% Triton-X for 10 minutes. After blocking with 2% bovine serum albumin (BSA, A95115G, Sigma) for 1 hour at room temperature, the anti-NP primary antibody (SFTSV) was incubated at 37°C for 1 hour. After washing with 2% BAS, the goat anti-mouse IgG h&L secondary antibody (AlexaFluor 488) (1:200; ab150113, Abcam) (1:2000; ab150113 Abcam) was incubated at 37°C for one hour. The cells were stained with mounting medium containing DAPI (ab104139 Abcam). The samples were examined, and images were captured using a laser scanning confocal microscope system (Leica DMi8), as Figure 2 shown.

[0042] From Figure 1 and Figure 2 It can be seen from the results that simeprevir has an obvious inhibitory effect on the novel bunyavirus, can be applied to drugs against the novel bunyavirus, and provides a new idea for the prevention and treatment of the novel bunyavirus.

[0043] The above-described embodiments merely represent one implementation mode of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. Use of the small molecule drug Simeprevir in the preparation of a drug against novel Bunyavirus, wherein the CAS number of Simeprevir is 923604-59-5.

2. Use of the small molecule drug Simeprevir according to claim 1 in the preparation of a drug against the new Bunyavirus, characterized in that, The dosage forms of the drug against novel Bunyavirus include powder, tablets, granules, capsules, solutions, emulsions and suspensions.

3. Use of the small molecule drug Simeprevir according to claim 1 in the preparation of a drug against novel bunyavirus, characterized in that, Simeprevir is the only active ingredient of the drug against novel Bunyavirus.

4. An oral tablet against novel bunyavirus, characterized in that, It contains the small molecule drug Simeprevir.

5. An oral tablet against novel bunyavirus according to claim 4, characterized in that, It is made from the following components and amounts: Simeprevir 100 mg / tablet, microcrystalline cellulose 120 mg / tablet, crospovidone 20 mg / tablet, magnesium stearate 5 mg / tablet, hydroxypropyl methylcellulose 10 mg / tablet, polyethylene glycol-4000 15 mg / tablet and silicon dioxide 5 mg / tablet.

6. An intravenous injection against the new bunyavirus, characterized in that, It contains the small molecule drug Simeprevir.

7. An intravenous injection for anti-new bunyavirus according to claim 6, characterized in that, It is made from the following components and amounts: Simeprevir 100 mg / vial, polysorbate-80 50 mg / vial, hydroxypropyl-β-cyclodextrin 400 mg / vial and sodium chloride 45 mg / vial, and the final volume of each vial is 5 ml.

8. An intravenous injection for anti-new bunyavirus according to claim 7, characterized in that, It is made up to 5 ml with water for injection, and the pH of the injection solution is adjusted with sodium dihydrogen phosphate, and the pH value ranges from 6.5 to 7.0.

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