CN114288306B - Use of composition in preparing medicine for inhibiting SARS-CoV-2 virus replication - Google Patents

Abstract

The present application relates to the field of medicines, in particular to the use of a composition in the preparation of anti-Bunyavirus or SARS‑CoV‑2 virus drugs. The composition provided by the application is effective against bunya virus and anti-SARS-CoV-2 virus.

Description

Application of composition in the preparation of medicines for inhibiting SARS-CoV-2 virus replication

This application was submitted on July 20, 2021, the application date was July 20, 2021, and the title of the invention is "Application of Toosendanin in the Preparation of Drugs for the Prevention and/or Treatment of Bunia Virus and New Coronavirus Infection Diseases" , the divisional application of the invention patent application with application number 202110821896.X.

technical field

The present application relates to the field of medicines, in particular to the use of a composition in the preparation of anti-Bunyavirus or SARS-CoV-2 virus medicines.

Background technique

Toosendanin (Toosendanin, TSN) is a tetracyclic triterpene compound in Melia toosendan Sieb.et Zucc., and it is the main medicinal component of Meliaceae.

The structural formula of toosendanin is as follows:

Toosendan has a variety of pharmacological activities, such as anti-botulinum neurotoxin, specifically acting on a variety of ion channels in nerve and cardiomyocytes and inhibiting insect feeding and larval development. In recent years, it has been reported that toosendanin can induce the death and apoptosis of tumor cells (such as liver cancer cells, neural tumor cells and lymphoma cells), which suggests that toosendanin has a good prospect in inhibiting tumor cell proliferation.

This application provides a new application of toosendanin.

Contents of the invention

The purpose of the embodiment of the present application is to provide a composition in the preparation of anti-bunya virus or SARS-CoV-2 virus medicine, which aims to provide a new application of toosendanin.

The application provides the use of a composition in the preparation of anti-bunyavirus medicine, the composition includes toosendanin or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments of the present application, the composition is used in the preparation of anti-fever with thrombocytopenia syndrome virus drugs.

In some embodiments of the present application, the composition is used in the preparation of a medicament for inhibiting the spread of fever with thrombocytopenia syndrome virus.

In some embodiments of the present application, the composition is used in the preparation of anti-Rift Valley fever virus drugs.

In some embodiments of the present application, the composition is used in the preparation of a medicament for inhibiting the spread of Rift Valley fever virus.

In some embodiments of the present application, the composition further includes pharmaceutically acceptable excipients.

The present application also provides the use of a composition in the preparation of anti-SARS-CoV-2 virus medicine, said composition comprising toosendanin or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments of the present application, the composition is used in the preparation of a drug for inhibiting SARS-CoV-2 virus replication.

In some embodiments of the present application, the composition further includes pharmaceutically acceptable excipients.

In some embodiments of the present application, the dosage form of the composition is that the dosage form of the drug is a dosage form administered through the gastrointestinal tract or an injection.

The application of the composition provided in the embodiments of the present application in the preparation of anti-Bunya virus or SARS-CoV-2 virus medicine has at least the following beneficial effects:

Toosendanin or its pharmaceutically acceptable salt, solvate or hydrate is effective against bunya virus and anti-SARS-CoV-2 virus; further, toosendanin or its pharmaceutically acceptable salt, The solvate or hydrate is effective against fever with thrombocytopenia syndrome virus and anti-Rift Valley fever virus.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the accompanying drawings that are required in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Fig. 1 is the cytotoxicity test result of Toosendanin in Experimental Example 1.

Fig. 2 is the detection result of antiviral activity of different concentrations of Toosendanin in Experimental Example 2.

Fig. 3 shows the effect of Toosendanin on SFTSV replication in Experimental Example 2.

Fig. 4 is the result of the effect of Toosendanin on RVFV replication in Experimental Example 3.

Figure 5 is the effect of Toosendanin on SFTSV infection of C57BL/6 mice in Experimental Example 4.

Figure 6 is the result of the influence of Toosendanin on the replication of SARS-CoV-2 in Experimental Example 5.

Wherein, the vehicle group in Fig. 2, Fig. 4 and Fig. 5 refers to the vehicle control group.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The application of the composition of the embodiment of the present application in the preparation of anti-Bunya virus or SARS-CoV-2 virus medicine will be specifically described below.

In the first aspect, the present application provides a use: the use of a composition in the preparation of an anti-bunyavirus drug, the composition comprising toosendanin or a pharmaceutically acceptable salt, solvate or Hydrate.

Bunyaviruses are spherical, enveloped, and segmented negative-sense RNA viruses of order 1.

For example, the use of the above composition in the preparation of anti-fever with thrombocytopenia syndrome virus drugs.

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) belongs to the order Bunyavirales, the family Phenuiviridae, and the genus Bandavirus. It is a new type of Bunyavirus.

In some embodiments of the present application, it is used in the preparation of inhibiting the transmission of fever with thrombocytopenia syndrome virus.

Ticks are known natural hosts of SFTSV, and tick bites are the main cause of human infection with SFTSV. Toosendanin or its pharmaceutically acceptable salt, solvate or hydrate can inhibit the transmission of fever with thrombocytopenia syndrome virus; therefore, it has preventive and therapeutic effects.

For example, the use of the above composition in the preparation of anti-Rift Valley fever virus drugs. Rift Valley fever virus (RVFV) belongs to the family Phenuiviridae and belongs to the genus Phlebovirus, and is a highly pathogenic bunyavirus. RVFV is transmitted by mosquitoes and can infect animals and humans.

Toosendanin or its pharmaceutically acceptable salts, solvates or hydrates can be used to prepare drugs for inhibiting the spread of Rift Valley fever virus.

In some embodiments, the above composition further includes pharmaceutically acceptable excipients.

In the second aspect, the present application also provides a use: the use of the composition in the preparation of anti-SARS-CoV-2 virus medicine, the composition includes toosendanin or its pharmaceutically acceptable salt, solvate or Hydrate.

The novel coronavirus (SARS-CoV-2) belongs to the family Coronaviridae and belongs to the genus Betacoronavirus.

In the embodiment of the present application, toosendanin or its pharmaceutically acceptable salt, solvate or hydrate has curative effect on novel coronavirus (SARS-CoV-2).

In some embodiments of the present application, toosendanin or its pharmaceutically acceptable salt, solvate or hydrate can inhibit the replication of SARS-CoV-2 virus; therefore, the composition inhibits SARS-CoV-2 Use in Viral Replication Medicines.

Further, the composition also includes pharmaceutically acceptable auxiliary materials.

It should be noted that, in the first aspect and the second aspect of use above, the dosage form of the drug may be tablet, capsule, granule, pill, powder, ointment, powder or oral liquid.

As an example, the above pharmaceutically acceptable excipients are water, ethanol, starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, inorganic salts, mannitol, starch slurry, carboxymethyl cellulose Sodium cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, CMS-Na, L-HPC, cross-linked PVP, CCNa, magnesium stearate, micronized silica gel, Talc, hydrogenated vegetable oil, PEG, magnesium lauryl sulfate, lactose, corn starch, calcium carbonate, calcium phosphate, sodium lauryl sulfate, Tween-80, boric acid, stearic acid, sodium chloride, sodium benzoate, acetic acid Sodium, Macrogol, Liquid Paraffin, Dicalcium Phosphate, Dicalcium Phosphate, Lactose, Pregelatinized Starch, Povidone, Citric Acid, Polysorbate 80, Paraffin, Hydrogenated Vegetable Oil, Glycine, CAP, AEA, One or more of acrylic resin, cyclodextrin, gum arabic, gelatin and sodium alginate.

The administration object of the medicament prepared by the composition of the present application is not limited to human beings, for example, other mammals may be used. Any method of administration can be, for example, oral or parenteral administration. The method of parenteral administration may adopt intravenous, arterial, bone marrow, dura mater, cardiac, transdermal, subcutaneous, cavity, nasal, enteral, topical, sublingual or rectal administration methods.

Further, pharmaceutically acceptable auxiliary materials can be selected according to the dosage form.

It should be further noted that, in addition to auxiliary materials, the composition of the present application may also include other pharmaceutically acceptable active ingredients.

Alternatively, the preparation method based on toosendanin may also include pharmaceutically acceptable impurities.

For example the composition may be an extract of Melia azedarach, Melia toosendan.

The above-mentioned "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not hinder the action of the active ingredient after human administration, and usually does not cause allergic reactions such as gastrointestinal disorders and dizziness, or similar reactions.

The above-mentioned pharmaceutically acceptable salts may preferably be pharmaceutically acceptable acid addition salts formed from free acids. Described free acid can all use organic acid and inorganic acid. And described organic acid besides this, can also use citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methylsulfonic acid acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, carbonic acid, and aspartic acid. In addition to the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid can also be used.

The characteristics and performance of the present application will be further described in detail below in conjunction with experimental examples and embodiments.

Experimental example 1

Cytotoxicity Detection of Toosendanin

In HUVECs cells, the cytotoxicity of Toosendanin was tested. HUVECs cells were seeded in a 96-well cell culture plate at 1×10 ⁴ cells/well, and cultured in a 5% CO ₂ incubator at 37°C for 12-16 h, respectively with 0.2 μM, 0.62 μM, 1.85 μM, 5.6 μM, 16.7 μM, 50 μM, 150 μM and 450 μM Toosendanin were treated, and each group had three replicate wells, and the control group was added with the same amount of dimethyl sulfoxide (DMSO). After 24 hours of drug action, 10 μl of CCK8 was added, incubated at 37°C for 1 hour, and the OD _450nm was detected to analyze the cell viability; the detection results are shown in Figure 1 .

Among them, CCK8 is a kit for the determination of cell viability, and its solution contains It is a water-soluble tetrazolium salt, which can be oxidized and reduced to orange-yellow water-soluble formazan (Formazan) by intracellular dehydrogenase, and the amount of formazan produced is directly proportional to the number of living cells.

After testing, as shown in Figure 1, the CC ₅₀ of Toosendanin is greater than 450 μM. In the subsequent examples, the concentration of Toosendanin is 1-40 μM, which is within the safe and non-toxic range.

Experimental example 2

Detection of Antiviral Activity of Toosendanin on SFTSV

Vero cells were seeded in a 24-well cell culture plate at 2×10 ⁵ cells/well, cultured in a 5% CO ₂ incubator at 37°C for 12-16 hours, and treated with Toosendanin at a concentration of 5 μM, 10 μM, 20 μM, and 40 μM , the negative control was added with the same volume of DMSO. After incubation for 1 hour, infect with SFTSV. After incubation at 37°C for 2 hours, replace the medium with 2% FBS DMEM medium. After 24 hours, detect the effect of Toosendanin on virus replication by titer. The results are shown in Figure 2. At different concentrations The compound Toosendanin had a significant inhibitory effect on the replication of SFTSV under all conditions. Further detect the IC ₅₀ of Toosendanin inhibiting SFTSV in Vero cells, and the inhibition curve of SFTSV is shown in Figure 3; the dots in Figure 3 represent the inhibition rate of drug inhibition of virus replication, and the triangles represent cytotoxicity. As can be seen from Figure 3, Toosendanin Significantly inhibits the activity of SFTSV, the calculated IC ₅₀ is 1.2 μM. The CC ₅₀ in Vero cells is greater than 250 μM, and the SI index is greater than 208.3.

It shows that toosendanin has the function of inhibiting the activity of SFTSV, and can prepare the medicine for resisting fever with thrombocytopenia syndrome virus.

Experimental example 3

Effect of Toosendanin on RVFV Replication

HUVECs cells were seeded in 24-well cell culture plates at 2×10 ⁵ cells/well, and cultured in a 5% CO ₂ incubator at 37°C for 12-16 hours, using concentrations of 0.2 μM, 0.62 μM, 1.85 μM, and 5.6 μM , 16.7 μM, 50 μM Toosendanin were treated, and the negative control was added with the same volume of DMSO. After incubation for 1 hour, they were infected with Rift Valley Fever Virus (RVFV) belonging to the genus Phlebovirus. After incubation at 37°C for 2 hours, the medium was replaced with DMEM medium with 2% FBS. After 24 hours, real-time fluorescent quantitative PCR (qPCR) method Detect virus gene copy number in the supernatant, as shown in Figure 4; As can be seen from Figure 4, under different concentration conditions, compound Toosendanin all has significant inhibition to the replication of Rift Valley fever virus (RVFV); It has the effect of inhibiting the replication of Rift Valley fever virus.

Experimental example 4

Effects of Toosendanin on C57BL/6 Mice Infected with SFTSV

C57BL/6 mice have a sound immune system and a clean genetic background, which can be used for drug protection evaluation experiments. The mice were raised in SPF environment throughout the whole process, and the mice were divided into two groups: (1) SFTSV+vehicle group (n=5), (2) SFTSV+Toosendanin group (n=5). Mice were infected by intraperitoneal injection, with an inoculation volume of 100 μL (10 ⁵ FFU/mouse), and the mock group was treated with an equal volume of DMEM medium. Intraperitoneal injection, the dose of Toosendanin was 1mg/kg/d, starting 3 days before infection, once a day, for a total of 6 days. The entire experimental procedure strictly complied with the guidelines of the National Institutes of Health as stipulated in the Instructions for the Care and Use of Animals. The daily monitoring indicators of mice include: arched back, piloerection, activity, stress response, body weight, etc. On the third day after SFTSV infection, the mice were dissected, and the spleen was taken to detect the viral load. The results are shown in Figure 5. Compared with the control group, the virus titer in the spleen of the mice in the administration group decreased significantly. Tip Toosendanin can significantly inhibit the replication of SFTSV in mice.

TSN in Figure 5 represents the SFTSV+Toosendanin group.

Experimental example 5

Effect of Toosendanin on the Replication of SARS-CoV-2

The IC ₅₀ and CC ₅₀ of Toosendanin inhibiting SARS-CoV-2 in Vero-E6 cells were detected, and the results are shown in the inhibition curve of SARS-CoV-2 in Figure 6. The squares in Figure 6 represent the inhibition rate of drug inhibition of virus replication, Dots indicate cytotoxicity, and Toosendanin significantly inhibits the activity of SARS-CoV-2, and its calculated _IC50 is 0.24 μM. The _CC50 in Vero-E6 cells is greater than 450 μM, and the SI index is greater than 1875.

It shows that toosendanin can inhibit the replication of SARS-CoV-2 virus, and can be used to prepare anti-SARS-CoV-2 virus medicine.

Based on the above-mentioned Experimental Example 1-Experimental Example 5, the embodiment of the present application provides a medicine containing toosendanin or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The drug can at least be used against Bunia virus, against fever with thrombocytopenia syndrome virus, against Rift Valley fever virus and against SARS-CoV-2 virus.

In summary, it can be seen that toosendanin or its pharmaceutically acceptable salts, solvates or hydrates are resistant to bunyavirus, fever with thrombocytopenia syndrome virus, Rift Valley fever virus and SARS-CoV -2 virus works.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, there may be various modifications and changes in the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included within the protection scope of this application.

Claims (3)

1. The application of a composition in preparing a medicine for inhibiting SARS-CoV-2 virus replication is characterized in that the active ingredient of the composition is toosendanin or pharmaceutically acceptable salts thereof.

2. The use of claim 1, the composition further comprising a pharmaceutically acceptable adjuvant.

3. The use according to claim 1, wherein the composition is in the form of a gastrointestinal tract administration or injection.