阅读说明：本技术 苯甲酸雌二醇或其药物可接受盐在制备抗冠状病毒的药物中的应用 (Application of estradiol benzoate or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines ) 是由 徐伟 刘叔文 杨婵 牛晓阁 潘晓彦 肖庚富 孙静 赵金存 于 2020-07-21 设计创作，主要内容包括：本发明公开了一种苯甲酸雌二醇或其药物可接受盐在制备抗冠状病毒的药物中的应用。本发明首次提出苯甲酸雌二醇或其药物可接受盐在制备抗冠状病毒的药物中的应用,该应用扩大了苯甲酸雌二醇的使用范围。特别是在全球新冠肺炎流行的情况下,为抑制SARS-CoV-2导致的新冠肺炎提供新的药物。(The invention discloses an application of estradiol benzoate or pharmaceutically acceptable salts thereof in preparing anti-coronavirus medicines. The invention provides the application of the estradiol benzoate or the pharmaceutically acceptable salt thereof in preparing the anti-coronavirus medicines for the first time, and the application expands the application range of the estradiol benzoate. Particularly provides a new medicine for inhibiting the new coronary pneumonia caused by SARS-CoV-2 under the condition of the global new coronary pneumonia epidemic.)

1. Application of estradiol benzoate or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines, wherein the molecular formula of estradiol benzoate is C₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

2. Use of estradiol benzoate of formula C or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting the entry of a coronavirus into a target cell₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

3. The application of the estradiol benzoate analogue or the pharmaceutically acceptable salt thereof in preparing the anti-coronavirus medicament.

4. Use of estradiol benzoate analogues or pharmaceutically acceptable salts thereof in the manufacture of a medicament for inhibiting entry of a coronavirus into a target cell.

5. An anti-coronavirus pharmaceutical composition comprising as an active substance estradiol benzoate having the formula C or a pharmaceutically acceptable salt thereof₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

6. A pharmaceutical composition for inhibiting the entry of coronaviruses into target cells, characterized in that it contains as an active substance estradiol benzoate or a pharmacologically acceptable salt thereofReceiving a salt of estradiol benzoate having the formula C₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

7. An anti-coronavirus pharmaceutical composition comprising an estradiol benzoate analogue or a pharmaceutically acceptable salt thereof as an active substance.

8. A pharmaceutical composition for inhibiting entry of a coronavirus into a target cell, which comprises an estradiol benzoate analog or a pharmaceutically acceptable salt thereof as an active substance.

9. The use according to any one of claims 3 to 4 or the pharmaceutical composition according to any one of claims 7 to 8, wherein the estradiol benzoate analogue comprises 17 α -estradiol, 17 β -estradiol, estradiol cypionate, estradiol dipropionate, estradiol valerate.

10. The use of any one of claims 1 to 4 or the pharmaceutical composition of any one of claims 5 to 8, wherein the coronavirus comprises HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV-2 and MERS-CoV; preferably, the coronavirus is SARS-CoV-2.

Technical Field

The invention relates to the technical field of medicines, in particular to application of estradiol benzoate or pharmaceutically acceptable salts thereof in preparation of anti-coronavirus medicines.

Background

The previously known human-infecting coronavirus are 6 kinds, namely human coronavirus HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, and severe acute respiratory syndrome coronavirus SARS-CoV and middle east respiratory syndrome coronavirus MERS-CoV. SARS-CoV-2 is the 7 th coronavirus which is known to infect human, is a single-stranded RNA positive strand enveloped beta coronavirus, and has a genome total length of about 26-32 kb. SARS-CoV-2 entering into host cell is mediated by transmembrane Spike S glycoprotein (S), S protein can be split into S1 and S2 subunit by protease on host cell membrane surface, wherein S2 is coronavirus evolution conservative domain, has Heptad repeat sequence 1 (HR 1) and Heptad repeat sequence 2 (HR 2), is responsible for mediating fusion of virus and cell membrane, and S1 can bind with ACE2 receptor on cell membrane surface, so S protein has both receptor binding activity and membrane fusion activity. Therefore, inhibiting the activity of S protein can prevent virus from entering host cells, and S1 and S2 subunits can be used as targets for screening antiviral drugs. At present, no specific medicine aiming at SARS-CoV-2 exists, and no approved medicine is available on the market.

Estradiol Benzoate (EB) is an estrogen class of drug. Has similar action to estradiol, can promote endometrial hyperplasia, enhance uterine smooth muscle contraction, and promote mammary gland development and hyperplasia: the large dose inhibits prolactin release, acts against antiandrogens, and increases calcium deposition in bone. The traditional Chinese medicine composition is mainly used for supplementing estrogen deficiency in clinic, such as atrophic vaginitis, vaginal dryness, female gonad dysfunction, menopausal vasomotor symptoms, primary ovarian failure and the like. No applications of estradiol benzoate and estradiol benzoate analogues in the aspect of resisting coronavirus exist.

Disclosure of Invention

The invention aims to provide a novel anti-coronavirus drug.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the invention provides an application of estradiol benzoate or pharmaceutically acceptable salt thereof in preparing an anti-coronavirus drug, wherein the molecular formula of the estradiol benzoate is C₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

In another aspect, the invention provides an application of estradiol benzoate or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting coronavirus from entering target cells, wherein the molecular formula of the estradiol benzoate is C₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

The invention further provides an application of the estradiol benzoate analogue or the pharmaceutically acceptable salt thereof in preparing the anti-coronavirus medicament.

In another aspect, the invention provides an application of estradiol benzoate analogue or pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting coronavirus from entering target cells.

In a further aspect of the present invention, there is provided an anti-coronavirus pharmaceutical composition comprising as an active substance estradiol benzoate having the formula C or a pharmaceutically acceptable salt thereof₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

In a further aspect of the present invention, there is provided a pharmaceutical composition for inhibiting the entry of coronavirus into a target cell, comprising as an active substance estradiol benzoate having the formula C or a pharmaceutically acceptable salt thereof₂₅H₂₈O₃Molecular weight of 376.49, and structural formula

In a further aspect of the present invention, there is provided an anti-coronavirus pharmaceutical composition comprising an estradiol benzoate analogue or a pharmaceutically acceptable salt thereof as an active substance.

In a further aspect of the present invention, there is provided a pharmaceutical composition for inhibiting the entry of coronavirus into a target cell, which comprises as an active substance an estradiol benzoate analogue or a pharmaceutically acceptable salt thereof.

Further, the estradiol benzoate analogs include 17 α -estradiol, 17 β -estradiol, estradiol cypionate, estradiol dipropionate, estradiol valerate.

Further, the coronavirus includes HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Severe acute respiratory syndrome coronavirus SARS-CoV, Severe acute respiratory syndrome coronavirus 2SARS-CoV-2 and eastern respiratory syndrome coronavirus MERS-CoV; preferably, the coronavirus is SARS-CoV-2.

Further, the pharmaceutical composition is an injection, a gel, a tablet and a patch.

The invention has the following beneficial effects:

the invention discovers the application of the estradiol benzoate and the estradiol benzoate analogue in the preparation of the anti-coronavirus medicine for the first time, and the application expands the application range of the estradiol benzoate and the estradiol benzoate analogue. The estradiol benzoate is used as antiviral medicine, and the experiment shows that the estradiol benzoate can effectively inhibit SARS-CoV-2 on in-vitro cultured cell Vero-E6, and half of the estradiol benzoate is effectiveConcentration EC₅₀6.72 μ M; estradiol benzoate is used for inhibiting SARS-CoV-2 entry stage, inhibiting SARS-CoV-2S pseudovirus infection 293T/ACE2 cell, and inhibiting half inhibitory concentration IC₅₀0.27. mu.M; estradiol benzoate has no obvious cytotoxicity in the effective concentration range. Therefore, can be used for preparing anti-SARS-CoV-2 medicaments.

Drawings

FIG. 1 is a graph showing the inhibition rate of Estradiol Benzoate (EB) against SARS-CoV-2 at different concentrations in example 1 of the present invention, in which the abscissa represents the concentration of estradiol benzoate and the ordinate represents the inhibition rate of estradiol benzoate against SARS-CoV-2 with respect to the solvent group as a control, and the half effective concentration EC of estradiol benzoate against SARS-CoV-2 was calculated from the inhibition rate₅₀The value is obtained.

FIG. 2 is a graph showing the inhibition rate of Estradiol Benzoate (EB) to inhibit SARS-CoV-2S pseudovirus entry into target cells at different concentrations in example 2 of the present invention, wherein the abscissa represents the concentration of estradiol benzoate, and the ordinate represents the inhibition rate of estradiol benzoate to inhibit SARS-CoV-2S pseudovirus entry using solvent group as control, and the half inhibition concentration IC50 value of estradiol benzoate to inhibit SARS-CoV-2S pseudovirus entry is determined.

FIG. 3 is a graph of the viability of Estradiol Benzoate (EB) versus Vero-E6 cells in example 3 of the invention, wherein the abscissa represents the concentration of estradiol benzoate and the ordinate represents the percentage of cell survival of Vero-E6 cells after administration of varying concentrations of estradiol benzoate, relative to the solvent group.

FIG. 4 is a graph showing the cell activity inhibition rate of Estradiol Benzoate (EB) on 293T/ACE2 cells in example 3, wherein the abscissa represents the concentration of estradiol benzoate, the ordinate represents the percentage inhibition of the activity of estradiol benzoate on 293T/ACE2 cells at different concentrations with a solvent group as a control, and the half lethal dose CC of estradiol benzoate on 293T/ACE2 cells is determined₅₀The value is obtained. .

Detailed Description

For a better understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings, and the scope of the invention is not limited to the following examples.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The following examples mainly establish SARS-CoV-2 live virus and SARS-CoV-2S pseudovirus in vitro cell infection model to evaluate the anti-SARS-CoV-2 activity of estradiol benzoate, and confirm that estradiol benzoate has the anti-SARS-CoV-2 infection ability, and simultaneously has the effect of inhibiting SARS-CoV-2 from entering target cells, and provides an application of estradiol benzoate in preparing anti-novel coronavirus medicines.

Vero-E6 and 293T cells adopted by the invention are purchased from American ATCC, and 293T cells stably over-expressing human SARS-CoV-2 receptor protein ACE2 are constructed and stored by the unit.

The cell growth culture solution adopted in the embodiment of the invention comprises the following components: DMEM basal medium, wherein fetal bovine serum with a total volume of 10% and ampicillin/streptomycin with a total volume of 1% are added, and the culture solution is stored at 4 ℃ and preheated in a water bath at 37 ℃ before use.

The estradiol benzoate adopted in the embodiment of the invention is purchased from Shanghai ceramic Biotechnology limited company, and the purity is more than 99%.

SARS-CoV-2 used in the examples of the present invention was isolated from infected persons who were studied for the Wuhan virus and amplified for storage.

Pseudovirus packaging plasmids and sources thereof in the examples of the invention: pseudovirus packaging skeleton plasmid pNL4-3.Luc. R-E-for southern medical university identification and preservation, the disclosed optimized full-length SARS-CoV-2S protein core plasmid pcDNA3.1-SARS-CoV-2-Spike was offered by professor Lu of Shanghai double-den university.

The single luciferase assay kit used in the examples of the present invention was purchased from PROMEGA, usa and includes cell lysate and luciferase reaction substrate.

Pharmacological experiment part

Example 1 measurement of inhibitory Activity of estradiol benzoate against SARS-CoV-2 in vitro

1. Drug inhibition activity assay:

1) taking Vero-E6 cells in logarithmic growth phase, 3 x 10^5 cells/well, inoculating in 48-well plate, 37 deg.C, 5% CO₂The culture was carried out overnight.

2) Pre-hatching with medicaments: the drug was diluted in DMEM medium containing 2% by volume fetal bovine serum. Initial drug concentration was set at 200 μ M (solvent in DMSO), drug was diluted three-fold, 3 multiple wells per concentration, for a total of 6 drug gradients (200, 66.67, 22.22, 7.41, 2.47, 0.82 μ M); solvent dimethyl sulfoxide (DMSO) is set as a control group, the control group is diluted by DMEM medium containing 2% fetal bovine serum in total volume, and the dimethyl sulfoxide with the same volume is given to the drug. After removing cell supernatant 1), 100. mu.l of diluted drug was added to each well of the experimental group in 48-well plate, 100. mu.l of diluted DMSO was added to the control group, and incubation was performed at 37 ℃ for 1 h.

3) Viral infection: mu.l of SARS-CoV-2 virus dilution (MOI 0.05) was added to each well of the 48-well plate, and incubation was continued at 37 ℃ for 1 h. The infected supernatant was removed well and the cells were washed once with 200. mu.l PBS. 200 mul of culture medium containing the drug at the corresponding concentration is added into the wells again, the culture is continued for 24h, and 150 mul of cell culture supernatant is collected for testing. Viral copy number was determined using qRT-PCR.

4) For the specific operation of Viral RNA Extraction, Takara MiniBEST Viral RNA/DNA Extraction Kit (Code No. 9766):

a. splitting the virus: mu.l of PBS (pH7.4) was added to 150. mu.l of the cell culture supernatant to make up to 200. mu.l. Add 200. mu.l VGB buffer, 20. mu.l Proteinase K and 1.0. mu.l Carrier RNA, mix well and lyse well in a 56 ℃ water bath for 10 minutes. Add 200. mu.l absolute ethanol to the lysate, suck well and mix well.

b. Column passing: the Spin Column was mounted on a Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12,000rpm for 2 minutes, and the filtrate was discarded.

c. Washing 1: mu.l of RWA buffer was added to Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.

d. And (3) washing 2: mu.l of RWB buffer was added to Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded. (RWB buffer to which a specified volume of 100% ethanol had been added). RWB buffer was added around the wall of the Spin Column.

e. And d, repeating the operation step.

f. Spin Column was mounted on the Collection Tube and centrifuged at 12,000rpm for 2 minutes.

g. And (3) elution: the Spin Column was mounted on a new 1.5ml RNase free collection tube, and 30. mu.l of RNase free dH was added to the center of the Spin Column membrane₂And O, standing for 5 minutes at room temperature. The RNA was eluted by centrifugation at 12,000rpm for 2 minutes.

5) Specific procedures for reverse transcription of viral RNA were described in Takara PrimeScriptTM RT reagent Kit with gDNA Eraser (Code No. RR047A):

a. removing the genomic DNA in the eluate: the reaction system is prepared on ice according to the following components

Reagent	Volume (μ l)
		5*gDNA Eraser Buffer	2.0
gDNA Eraser	1.0
		Total RNA	3.0
RNase Free dH2O	4.0
		Total volume	10.0

The sample was left to react at 42 ℃ for 2 min.

b. Reverse transcription reaction:

the samples were incubated at 37 ℃ for 15min and then heated at 85 ℃ for 5 sec.

6) qPCR assay virus copy number: reference is made to Takara TBPremix Ex Taq^TMII (TliRNaseH Plus, Code, No. RR820A) (Standard Curve method: RBD plasmid of known copy number as standard, specific primer targeting RBD). The reaction system was prepared on ice as follows:

reagent	Volume (μ l)
		TB Green Premix Ex Taq II(Tli RNaseH Plus)(2X)	10
Forward Primer(10μM)	1
		Reverse Primer(10μM)	1
ROX Reference Dye(50X)	0.4
		cDNA template	1
Sterilized water	6.6
		Total volume	20

The primer sequences are as follows:

RBD upstream Primer (Forward Primer): CAATGGTTTAACAGGCACAGG (SEQ ID NO: 1)

RBD downstream Primer (Reverse Primer): CTCAAGTGTCTGTGGATCACG (SEQ ID NO: 2)

And (3) computer detection: ABI7500 quantitative PCR instrument

Pre-denaturation: 95 ℃, 30 seconds, 1 cycle; and (3) PCR amplification: at 95 ℃, 5 seconds, 40 cycles; annealing: 30-34 seconds at 60 ℃; and (6) recording.

2. As a result: as shown in fig. 1;

the copy number of each sample was calculated from the standard curve. The drug-treated group inhibition rate was calculated with DMSO group copy number as a reference. Fitting a drug inhibition rate curve by using prism8.0 software according to the inhibition rates of drug treatment groups with different concentrations, and calculating the half effective concentration EC of Estradiol Benzoate (EB) acting on SARS-CoV-2 activity₅₀It was 6.27. mu.M.

EXAMPLE 2 detection of inhibitory Activity of estradiol benzoate against SARS-CoV-2S pseudovirus entry

1. The method comprises the following steps:

1) SARS-CoV-2S pseudovirus packaging:

HEK-293T cells in logarithmic growth phase 4 x 10^ 5/ml, 2ml per well were evenly seeded in 6-well plates。37℃、5％CO₂The cells were cultured in a cell incubator for 24 hours. Replacing fresh culture medium half an hour before transfection, respectively preparing plasmid diluent and transfection reagent (PolyJet) diluent by adopting 100 mul of blank DMEM culture medium, wherein the preparation proportion of each well is as follows (plasmid DNA needs to be extracted by adopting an extraction kit for removing endotoxin):

pNL4-3.Luc.R-E- 1000ng

pcDNA3.1-SARS-CoV-2-S 500ng

PolyJet 6μl

the preparation method comprises the following steps: the pNL4-3.Luc. R-E-plasmid and pcDNA3.1-SARS-CoV-2-Spike plasmid were added to 100. mu.l of blank DMEM medium simultaneously and mixed, and Polyjet was diluted with 100. mu.l of blank DMEM medium and mixed. The PolyJet dilutions were added to the plasmid dilutions and mixed well, incubated for 10 min at room temperature, and added well to HEK-293T cells. Culturing at 37 deg.C for 48 hr, collecting supernatant, centrifuging at 4000rpm for 10 min, and filtering with 0.45 μm sterile filter head to obtain SARS-CoV-2 pseudovirus.

2) Pseudovirus inhibition experiments:

293T cells (293T/ACE2) overexpressing the SARS-CoV-2 receptor ACE2 in logarithmic growth phase were plated evenly in 96 well plates at 1 × 10^ 4/well. Cultured in a cell culture chamber at 37 ℃ for 24 hours.

The initial concentration of the drug is set to 20 μ M, and 8 concentration gradients are diluted 2 times by volume in DMEM medium containing 2% fetal calf serum of total volume and are respectively 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 μ M. The DMSO solvent control was set up at 60 μ l per well volume, 3 replicates per concentration. 60 μ l of pseudovirus solution was added to the diluted drug, allowed to act at room temperature for 30 minutes, and 100 μ l/well was added to ACE2/293T cells and cultured at 37 ℃ for 48 hours. The medium was removed and the cells were washed once with 100. mu.l/well sterile PBS (pH7.4), 50. mu.l of 1X cell lysate was added to each well and lysed with shaking at room temperature for 15 minutes. Transferring 40 mul/hole cracking supernatant to 96-hole white enzyme label plate, adding isovolumic diluted luciferase substrate according to the specification of single luciferase detection kit, and immediately detecting fluorescence value and root by enzyme labelAnd judging the activity of the estradiol benzoate for inhibiting the virus adsorption according to the fluorescence value. Calculating the inhibition rate according to the corresponding relation between the fluorescence value and the drug concentration, drawing a curve, and calculating the half inhibition concentration IC of the estradiol benzoate₅₀。

2. As a result: as shown in fig. 2;

and (5) calculating the inhibition rate of the drug treatment group according to the fluorescence value by taking the DMSO solvent group as a control. Fitting a drug inhibition rate curve by using prism8.0 software according to the inhibition rates of drug treatment groups with different concentrations, and calculating the half inhibition concentration IC of Estradiol Benzoate (EB) for inhibiting SARS-CoV-2S protein pseudovirus from entering target cells₅₀It was 0.27. mu.M.

Example 3 cytotoxicity assay of estradiol benzoate

1. The method comprises the following steps:

1) cell inoculation:

Vero-E6, 293T-ACE2 cells in logarithmic growth phase were adjusted to 1 × 10^4 cells/well, seeded in 96-well plates at 100 μ L/well, and cultured overnight.

2) Designing the concentration of the medicine:

Vero-E6 cells: before administration, 6 concentration gradients are diluted by 3 times with DMEM medium containing 2% fetal calf serum in total volume, and the initial concentration of the drug is set to 200 μ M (200, 66.66, 22.22, 7.41, 2.47, 0.82 μ M); 100. mu.L of the diluted drug per well was added to Vero-E6 cells in 96-well plates in 1) to a final volume of 200. mu.L per well. 3 multiple wells were set for each drug concentration. The DMSO solvent treated group served as blank control.

293T-ACE2 cells: before administration, 8 concentration gradients were diluted 2-fold in DMEM medium containing 2% fetal bovine serum in total volume, the initial concentration of the drug was set at 100. mu.M (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78. mu.M), 100. mu.L of the diluted drug was added to 293T-ACE2 cells in 96-well plates 1) and the final volume of each well was 200. mu.L. 3 multiple wells were set for each drug concentration. The DMSO solvent treated group served as blank control.

3) Detecting the absorbance:

after 48h of incubation in the incubator, 10. mu.L of CCK-8 working solution was added to each well and the incubator was incubated for 3 hours. And (5) measuring the absorbance at 450nm by using a microplate reader.

4) According to the measured OD values, the survival rate of Vero-E6 cells and the inhibition rate of 293T-ACE2 cells by the drug at each concentration of the drug compared with the control group are respectively calculated.

2. As a result: as shown in fig. 3 and 4;

estradiol Benzoate (EB) had no significant toxic effect on Vero-E6 cells (FIG. 3) at 200. mu.M and in the effective concentration range.

The half lethal dose CC50 of Estradiol Benzoate (EB) to 293T/ACE2 cells is 39.80 mu M (figure 4), and the estradiol benzoate has no obvious toxic effect on 293T/ACE2 cells within an effective concentration range for inhibiting the entry of SARS-CoV-2 pseudovirus.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.

SEQUENCE LISTING

<110> southern medical university

Wuhan Institute of Virology, Chinese Academy of Sciences

GUANGZHOU INSTITUTE OF RESPIRATORY HEALTH

Application of estradiol benzoate or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines

<130> CP120010399C

<160> 2

<170> PatentIn version 3.3

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