阅读说明：本技术 小分子化合物肌醇六磷酸酯钠水合物在制备抗SARS-CoV-2药物中的应用 (Application of small molecular compound inositol hexaphosphate sodium hydrate in preparation of anti-SARS-CoV-2 medicine ) 是由 丁翠玲 彭浩然 赵兰娟 赵平 唐万达 江亮亮 唐海琳 戚中田 于 2021-10-21 设计创作，主要内容包括：本发明公开了小分子化合物肌醇六磷酸酯钠水合物在制备抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)药物中的应用,所述抗SARS-CoV-2药物是以肌醇六磷酸酯钠水合物为唯一的活性成份,或包含肌醇六磷酸酯钠水合物的药物组合物,所述抗SARS-CoV-2药物是指预防或治疗SARS-CoV-2感染的药物。本发明利用SARS-CoV-2的易感细胞系,包括非洲绿猴肾细胞Vero E6以及人肺腺癌细胞Calu-3,检测肌醇六磷酸酯钠水合物的抗SARS-CoV-2活性。实验结果显示,肌醇六磷酸酯钠水合物能有效抑制SARS-CoV-2对上述易感细胞的感染,且细胞毒性较小,有希望作为有效抗SARS-CoV-2感染的药物,具有应用前景。(The invention discloses an application of small molecular compound sodium inositol hexaphosphate hydrate in preparing a medicine for resisting severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), wherein the medicine for resisting SARS-CoV-2 takes sodium inositol hexaphosphate hydrate as a unique active component or a medicine composition containing the sodium inositol hexaphosphate hydrate, and the medicine for resisting SARS-CoV-2 is a medicine for preventing or treating SARS-CoV-2 infection. The invention uses susceptible cell line of SARS-CoV-2, including Vero E6 of African green monkey kidney cell and Calu-3 of human lung adenocarcinoma cell, to detect the anti-SARS-CoV-2 activity of inositol hexaphosphate sodium hydrate. The experimental result shows that the sodium phytate hydrate can effectively inhibit the infection of SARS-CoV-2 to the susceptible cells, has small cytotoxicity, is hopeful to be used as a medicine for effectively resisting SARS-CoV-2 infection, and has application prospect.)

1. The application of small molecular compound inositol hexaphosphate sodium hydrate in preparing medicine for resisting SARS-CoV-2.

2. The use of sodium phytate hydrate according to claim 1 for the preparation of a medicament against SARS-CoV-2, characterized in that: the anti-SARS-CoV-2 medicine is medicine for preventing or treating SARS-CoV-2 infection.

3. The use of sodium phytate hydrate according to claim 2 for the preparation of a medicament against SARS-CoV-2 infection, characterized in that: the anti-SARS-CoV-2 medicine is a medicine composition with inositol hexaphosphate sodium hydrate as the only active component or with inositol hexaphosphate sodium hydrate.

4. The use of sodium phytate hydrate according to claim 3 for the preparation of a medicament against SARS-CoV-2 infection, characterized in that: the content of inositol hexaphosphate sodium hydrate in the anti-SARS-CoV-2 medicine is 0.1-99 wt%.

5. The use of sodium phytate hydrate according to claim 4 for the preparation of a medicament against SARS-CoV-2, characterized in that: the pharmaceutical formulation is at least one of a capsule, a suspension, a tablet, a powder, an emulsion, a solution, a syrup, or an injection.

6. The use of sodium phytate hydrate according to claim 5 for the preparation of a medicament against SARS-CoV-2, characterized in that: the administration route of the pharmaceutical preparation is oral administration, injection or respiratory inhalation.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a small molecular compound sodium phytate hydrate in preparation of anti-SARS-CoV-2 medicines.

Background

Sodium phytate hydrate (Sodium phytate hydrate) is a precursor of other inositol phospholipids and pyrophosphates, approved for clinical use by the U.S. FDA, and is used as a hypocalcemic agent and complexing agent to remove trace heavy metal ions. There is no literature reporting its role in anti-SARS-CoV-2.

The chemical structural formula of sodium phytate hydrate is as follows (CAS No. 14306-25-3):

severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is a newly-appeared pathogen of high-infection and high-pathogenicity infectious diseases, and is mainly transmitted through respiratory tract by means of droplet, close contact and the like. The disease caused by SARS-CoV-2 infection is called coronavirus 2019(COVID-19), which often includes lower respiratory tract infection, namely viral pneumonia, and is mainly manifested by fever, dry cough, hypodynamia and the like, and a few patients are accompanied by upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Severe cases often develop dyspnea after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and hemorrhagic coagulation dysfunction, multiple organ failure, and the like. Since the outbreak of 2019, the epidemic situation rapidly spreads all over the world, and poses great threat to human health and huge damage to the global social and economic development. Although many types of vaccines have been widely distributed globally for a short period of time, the vaccines have limited ability to prevent infection and new variants continue to emerge and rapidly replace previously circulating strains. Thus, humans are at long-term risk for SARS-CoV-2 infection and disease. Reidesciclovir (Remdesivir) is an adenosine analog, a broad-spectrum antiviral agent, and has inhibitory activity against a variety of RNA viruses, including Ebola and coronavirus. Although Reidesciclovir has received the U.S. food and drug administration's immediate use authorization for COVID-19, its clinical efficacy is relatively limited. In view of this, the search and development of drugs effective against SARS-CoV-2 infection are important tasks in the world of biological medicine, and are urgent needs for protecting human health.

Disclosure of Invention

The invention aims to provide the application of sodium inositol hexaphosphate hydrate in preparing anti-SARS-CoV-2 medicaments.

The chemical structural formula of the sodium phytate hydrate is as follows:

the invention provides an application of small molecular compound inositol hexaphosphate sodium hydrate in preparing anti-SARS-CoV-2 medicine.

The application of the sodium phytate hydrate in the preparation of the anti-SARS-CoV-2 medicine is characterized in that: the anti-SARS-CoV-2 medicine is medicine for preventing or treating SARS-CoV-2 infection.

The application of the sodium phytate hydrate in the preparation of the anti-SARS-CoV-2 infection medicine is characterized in that: the anti-SARS-CoV-2 medicine is a medicine composition with inositol hexaphosphate sodium hydrate as the only active component or with inositol hexaphosphate sodium hydrate.

The application of the sodium phytate hydrate in the preparation of the anti-SARS-CoV-2 infection medicine is characterized in that: the content of inositol hexaphosphate sodium hydrate in the anti-SARS-CoV-2 medicine is 0.1-99 wt%.

The application of the sodium phytate hydrate in the preparation of the anti-SARS-CoV-2 medicine is characterized in that: the pharmaceutical formulation is at least one of a capsule, a suspension, a tablet, a powder, an emulsion, a solution, a syrup, or an injection.

The application of the sodium phytate hydrate in the preparation of the anti-SARS-CoV-2 medicine is characterized in that: the administration route of the pharmaceutical preparation is oral administration, injection or respiratory inhalation.

The invention utilizes an experimental operation system of SARS-CoV-2 infected susceptible cells to screen candidate micromolecule medicaments capable of inhibiting SARS-CoV-2 infection from a clinically approved medicament micromolecule library, screens out inositol hexaphosphate sodium hydrate, can effectively inhibit the infection of SARS-CoV-2 to Vero E6 of African green monkey kidney cells and Calu-3 of human lung adenocarcinoma cells, has small cytotoxicity, can be used as a potential anti-SARS-CoV-2 medicament, and has application prospect.

Drawings

FIG. 1. inhibitory effect of sodium phytate hydrate (Sodiumphytate hydrate) on SARS-CoV-2 infection at different concentrations.

Wherein, A: in Vero E6, analyzing the dose effect of Ruidexiwei and inositol hexaphosphate sodium hydrate on the inhibition effect of SARS-CoV-2; b: in human lung adenocarcinoma cell Calu-3, the dose effect analysis of the inhibitory effect of Reidesciclovir and sodium inositol hexaphosphate hydrate on SARS-CoV-2 was performed. After 18 hours of treatment, infection with SARS-CoV-2 was detected in each well by immunofluorescence, positive cells in each well were counted by a BioTek staining 5Imaging Reader, and then the inhibition rate (%) of the drug against SARS-CoV-2 infection was calculated as 1- (number of positive cells in drug-treated well/number of positive cells in DMSO-treated well) × 100%, and an EC50 value was calculated from the inhibition rate at each concentration. Each value is expressed as the mean ± standard deviation of 3 biological replicates. Black: the number of cells; gray: the rate of viral infection; EC50(concentration for 50% of maximum effect): half maximal effect concentration.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

The sodium phytate hydrate used in the examples of the present invention can be obtained commercially.

Example 1

First, experimental medicine, reagent and material

1. A compound: the small molecule drug library (product number: L1300-Z349373) approved for clinical use by the U.S. FDA contains 2580 small molecule compounds, purchased from Selleck, USA, all dissolved in DMSO at a concentration of 10. mu.M. Reidesciclovir (Remdesivir) from Selleck, USA, dissolved in DMSO at a concentration of 10. mu.M.

2. Vero E6 of African green monkey kidney cells and Calu-3 of human lung adenocarcinoma cells were purchased from Shanghai cell institute of Chinese academy of sciences and preserved by the biomedical protective research laboratory of naval medical department of the university of naval medical university of the people's liberation army.

DMEM complete cell culture solution containing 10% fetal bovine serum, 0.03% glutamine, non-essential amino acids, ampicillin and streptomycin 100U/mL, adjusted to pH 7.4.

4. Cell digest, containing 0.25% trypsin, was prepared in phosphate buffered saline.

SARS-CoV-2 virus was isolated and cultured from samples of nasopharyngeal swabs from COVID-19 patients at the department of the third Biosafety level (P3) laboratory of the university of military medical science, the gene sequence of which is shown in GenBank Accession No. MT622319, and all experimental procedures involving SARS-CoV-2 infection were carried out at the department of the university of military medical science P3 laboratory.

6. Rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody was purchased from Beijing Yiqian Shenzhou science and technology, Inc. (Sino Biological # 40143-T62).

Alexa Fluor 488-labeled anti-rabbit IgG is a product of Thermo Fisher, USA.

II, an experimental method:

screening anti-SARS-CoV-2 compound from approved small molecule medicine bank

Vero E6 of Vero cells of Vero, which were cultured in a T25 flask, were inoculated into a 96-well plate at 10000 cells per well in 100. mu.L of medium, and after 12 hours, 50. mu.L of SARS-CoV-2 diluted with the culture medium and 50. mu.L of the small molecule compound diluted with the culture medium were added per well. SARS-CoV-2 virus dose is 1X 10³Ffu (focus forming units) (i.e. the amount of virus infecting 1000 Vero E6 cells), final concentration of small molecule compound 5 μ M, repeated 3 wells using 5 μ M of rendesivir (Remdesivir) as positive control and DMSO as negative control, which contained an equal volume of solvent. After 18 hours, the infection of SARS-CoV-2 in each well of cells was detected by immunofluorescence, as follows: removing the culture medium from the culture plate by suction, adding 0.1mL of methanol to each well, placing the culture plate in a refrigerator at-20 ℃, after 20 minutes, taking out the culture plate, removing the methanol by suction, washing the wells with Phosphate Buffer Solution (PBS) once per well, then adding 100 μ L of PBS containing 3% Bovine Serum Albumin (BSA) (hereinafter referred to as 3% BSA-PBS), placing the plate on a horizontal shaking table, slowly shaking at room temperature for 1 hour, removing 3% BSA-PBS from the culture plate, adding 0.1mL of 1% BSA-PBS containing rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (antibody 500 times dilution) to each well, slowly shaking at room temperature for 1 hour, removing the rabbit polyclonal antibody working solution from the culture plate, washing each well with PBS for 3 times, then adding 0.1mL of 1% BSA-PBS containing fluorescein AlexaFluor 488-labeled anti-rabbit IgG (fluorescein lucifugal antibody 1500 times dilution), slowly shaking at room temperature for 1 hour, removing fluorescein antibody working solution in the culture plate by suction, adding 0.1mL of DAPI cell nucleus staining solution into each well, slowly shaking for 10 minutes at room temperature in a dark place, removing the DAPI cell nucleus staining solution in the culture plate by suction, washing each well for 3 times by PBS (phosphate buffer solution), counting green fluorescence positive cells of each well cell by using a cell Imaging and analyzing system (BioTek staining 5Imaging Reader), and then calculating the inhibition rate (%) of the drug to SARS-CoV-2 infection to be 1- (number of drug-treated well positive cells/number of DMSO-treated well positive cells) to be 100%.

And (3) taking the small molecular drug with the inhibition rate of more than 95% and the difference between the cell nucleus count and the DMSO-treated pore diameter of not more than 5% as a candidate antiviral drug, and further determining the cytotoxicity and antiviral activity of the small molecular drug. The screening results show that: the inhibition rate of the Ruidexiwei as a positive control on SARS-CoV-2 infection is more than 99 percent; 98 compounds of 2580 compounds reach the standard of candidate antiviral drugs, wherein the inhibition rate of sodium phytate hydrate on SARS-CoV-2 infection is more than 99%. The published research papers and patent of invention are referred to, and reports on the activity of sodium phytate hydrate against SARS-CoV-2 are not found.

Cytotoxicity assay for sodium (di) phytate hydrate

Respectively inoculating Vero E6 cultured Vero cells of African green monkey kidney cells and Calu-3 cultured human lung adenocarcinoma cells into a 96-well plate, respectively inoculating 10000 cells in each well and 100 mu L of DMEM culture solution, sucking out the original culture solution after 12 hours, adding 100 mu L of DMEM culture solution containing inositol hexaphosphate sodium hydrate diluted in concentration gradient into each well, wherein the final concentrations of the inositol hexaphosphate sodium hydrate are respectively 0, 0.064, 0.32, 1.6, 8, 40 and 100 mu M, and taking the DMEM culture solution containing DMSO as a control. Three duplicate wells were set for each drug concentration and corresponding volume of DMSO, placed at 37 deg.C and 5% CO₂Culturing in an incubator. After 48 hours, 100. mu.L of CellTiter-Glo luminescence cell activity detection reagent was added to each well, and after incubation at room temperature for 20min, the chemiluminescence value of each well cell was measured on a microplate reader (chemiluminescence value represents cell activity). And calculating the ratio of the average chemiluminescence value of the drug-treated well cells to the chemiluminescence value of the cells containing the DMSO solvent with corresponding volume, wherein the closer the ratio is to 1, the smaller the influence of the drug on the cell growth is, and the general ratio of more than 0.9 is considered to be unobvious in cytotoxicity.

The results are shown in Table 1, where sodium phytate hydrate is not significantly toxic to both cell lines at concentrations less than or equal to 40. mu.M, i.e.at these concentrations cell proliferation is not significantly affected by sodium phytate hydrate. When the concentration of sodium phytate hydrate is equal to or higher than 100. mu.M, there is a certain inhibitory effect on the growth of both cells, and the results show that the cytotoxicity of sodium phytate hydrate is lower.

TABLE 1 toxicity (chemiluminescence ratio) of sodium phytate hydrates at various concentrations on Vero E6 and Calu-3 cell lines

Activity detection of (tri) inositol hexaphosphate sodium hydrate for resisting SARS-CoV-2 infection

Vero E6 of Vero cells of Vero and Calu-3 of human lung adenocarcinoma cells cultured in T25 cell culture plates were inoculated into 96-well plates, 10000 cells per well, 100. mu.L of medium were added, and after 12 hours, SARS-CoV-2 (1X 10. mu.L of culture medium) diluted with 50. mu.L of LDMEM was added per well³FFU), and 50 μ L of sodium phytate hydrate (diluted with DMEM medium) serially diluted two-fold starting from a maximum concentration of 20 μ M to a minimum concentration of 0.0625 μ M, with remidesivir (Remdesivir) as a positive control and DMSO containing an equal volume of solvent as a negative control, each concentration being repeated in 3 wells. After 18 hours, the cells in each well were tested for infection with SARS-CoV-2 by immunofluorescence as described above. The green fluorescence positive cells per well were counted using a cell Imaging and analysis system (BioTek staining 5Imaging Reader), and then the inhibition rate (%) of the drug against SARS-CoV-2 infection was calculated as 1- (number of drug-treated well positive cells/number of DMSO-treated well positive cells) × 100%. EC50 values were calculated from the inhibition ratios at each concentration. As shown in FIG. 1, the EC50 of Reidesciclovir for inhibiting SARS-CoV-2 in Vero E6 cell is 0.7741. mu.M, and the EC50 for inhibiting SARS-CoV-2 in Calu-3 cell is 0.6403. mu.M; the EC50 of sodium phytate hydrate for inhibiting SARS-CoV-2 in Vero E6 cell was 0.8365. mu.M, and EC50 for inhibiting SARS-CoV-2 in Calu-3 cell was 0.8944. mu.M. The results show that sodium phytate hydrate can effectively inhibit SARS-CoV-2 from infecting Vero E6 and Calu-3 cells.

(IV) identification of Effect stage of inositol hexaphosphate sodium hydrate inhibiting SARS-CoV-2 infection

Inoculating Vero E6 Vero and Calu-3 to 96-well plate with 10000 cells per well and 100 μ L culture medium, adding 80 μ LSARS-CoV-2(2 × 10) per well after 12 hr³FFU), the culture supernatant was aspirated after 1 hour, and the wells were washed three times with PBS buffer. Respectively after infection0. mu.L of sodium phytate hydrate with a concentration of 50. mu.M was added at 1, 2, 4, 6 and 8h (hpi) (i.e.the final concentration of sodium phytate hydrate was 10. mu.M, and 0 h of the virus was added while adding sodium phytate hydrate), and the infection of SARS-CoV-2 in the cells of each well was detected by immunofluorescence at 10 h after infection, as described above. The green fluorescent positive cells per well were counted using a cell Imaging and analysis system (BioTek staining 5Imaging Reader), and then the inhibition rate (%) of the drug against SARS-CoV-2 infection was calculated as 1- (number of positive cells in drug-treated wells/number of positive cells in DMSO-treated wells) 100%.

The results are shown in Table 2, and the addition of sodium phytate hydrate 4 hours after SARS-CoV-2 infection of Vero E6 and Calu-3 cells still can significantly inhibit virus infection, indicating that the sodium phytate hydrate may act on the replication stage of the virus.

TABLE 2 Effect of sodium phytate hydrate treatment on different stages of SARS-CoV-2 infection

The above experimental results show that the sodium phytate hydrate can effectively inhibit SARS-CoV-2 infection, and mainly acts on virus replication stage.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.