阅读说明：本技术 维替泊芬在制备抗新型冠状病毒SARS-CoV-2药物中的用途 (Application of verteporfin in preparation of medicine for resisting novel coronavirus SARS-CoV-2 ) 是由 谢幼华 谷陈建 郭慧敏 吴旸 瞿涤 袁正宏 张�荣 王玉燕 朱园飞 徐巍 于 2020-11-04 设计创作，主要内容包括：本发明属于医药技术领域,涉及维替泊芬新的药用用途,具体涉及维替泊芬在制备抗新型冠状病毒SARS-CoV-2药物中的用途。本发明经实验证实,维替泊芬通过对体外细胞培养Vero-E6(非洲绿猴肾细胞)进行干预,能有效抑制新型冠状病毒的感染,所述的维替泊芬可进一步制备治疗新型冠状病毒引起的新型冠状病毒病,为治疗和控制新型冠状病毒病提供新的药物。(The invention belongs to the technical field of medicines, relates to a new medicinal application of verteporfin, and particularly relates to an application of verteporfin in preparation of a medicine for resisting novel coronavirus SARS-CoV-2. Experiments prove that the verteporfin can effectively inhibit the infection of the novel coronavirus by intervening in vitro cell culture Vero-E6 (African green monkey kidney cells), and the verteporfin can be further used for preparing a novel coronavirus disease caused by the novel coronavirus disease and provides a novel medicine for treating and controlling the novel coronavirus disease.)

1. The use of Verteporfin of formula 1 in the preparation of a medicament against a novel coronavirus SARS-CoV-2, wherein the title of Verteporfin is Verteporfin, the molecular formula is C82H84N8O16,

2. the use of claim 1, wherein verteporfin is resistant to the novel coronavirus by inhibiting the infection of the novel coronavirus SARS-CoV-2.

3. A medicament for the treatment of a novel coronavirus disease, characterized by: the drug takes verteporfin as a prodrug.

4. A pharmaceutical composition for the treatment of a novel coronavirus disease, characterized by: the pharmaceutical composition comprises verteporfin or a drug taking verteporfin as a prodrug and a pharmaceutically acceptable carrier.

5. The medicament or pharmaceutical composition according to claim 3 or 4, characterized in that said novel coronavirus Disease is a novel coronavirus Disease (Corona Virus Disease 2019, COVID-19), also known as novel coronavirus pneumonia.

Technical Field

The invention belongs to the technical field of medicines, relates to a new medicinal application of verteporfin, and particularly relates to an application of verteporfin in preparation of a medicine for resisting novel coronavirus SARS-CoV-2.

Background

A new type of coronavirus Disease (Corona Virus Disease 2019, COVID-19), also known as new type of coronavirus pneumonia, is a serious acute respiratory infectious Disease. The disease has strong infectivity and high lethality rate. The pathogen causing the novel coronavirus disease is a novel coronavirus (SARS-CoV-2). SARS-CoV-2 and the known severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) belong to the genus of the beta coronavirus belonging to the family Coronaviridae.

Clinical practice shows that the common symptoms of SARS-CoV-2 infection include respiratory tract symptoms, fever, cough, shortness of breath, dyspnea, etc. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. At present, there is no specific treatment method for SARS-CoV-2 caused diseases. Research shows that the Reidcisvir and the hydroxychloroquine have obvious inhibition effect on the replication of SARS-CoV-2 in vitro research. However, the clinical research shows that the hydroxychloroquine has no obvious treatment effect. While Reideciclovir has been reported to cure 1 case, in addition, the results of trials of Reideciclovir on 53 severe patients treated with isosexual medication show that 68% of severe patients have symptomatic relief after Reideciclovir but 13% of mortality, and 60% of patients report side effects. The above study of Reidesciclovir has few cases and there are significant limitations on data from isosexual medication.

Research shows that the novel coronavirus can be replicated in the Vero-E6 cell strain to cause obvious cytopathic effect (CPE) such as syncytium, cell shedding and the like, is a universal novel coronavirus cell infection model and is used for the separation, culture and pharmacodynamic determination of the novel coronavirus.

Verteporfin, the chinese alias: microtoporfin, english name: verteporfin. Verteporfin is a second generation porphyrin photosensitizer, can be activated by light (with the wavelength of 689nm) irradiation, and can be used for the treatment of macular degeneration through photodynamic therapy. Research shows that the medicine can selectively enter diseased blood vessels, and active oxygen is generated to occlude the diseased blood vessels through non-thermal laser irradiation, so that the leakage of the blood vessels is stopped, and the vision is preserved. Verteporfin is suitable for patients who are secondary to age-related ocular neovascularization, as well as pathological myopia or ocular histoplasmosis. To date, there has been no report of the use of verteporfin in inhibiting infection and replication of coronaviruses, particularly novel coronaviruses.

Disclosure of Invention

The invention aims to solve the problem that an effective anti-SARS-CoV-2 medicament is urgently needed at present based on the current situation of the prior art, provides a new medicinal application of verteporfin, and particularly relates to the application of verteporfin in preparing a medicament for resisting novel coronavirus SARS-CoV-2.

The invention evaluates the activity of the verteporfin against SARS-CoV-2 by an in vitro experiment; the in vitro experiment mainly comprises the determination of the inhibition of SARS-CoV-2 infection by the verteporfin, the determination of the cytotoxicity of the medicine and the like, provides the new application of the verteporfin in the medicine for resisting SARS-CoV-2, and the verteporfin can be used for preparing the medicine for resisting novel coronavirus SARS-CoV-2.

The purpose of the invention is realized by the following technical scheme:

the invention provides a new application of verteporfin in resisting SARS-CoV-2.

Further, the present invention provides a novel anti-coronavirus drug comprising verteporfin.

The invention carries out anti-SARS-CoV-2 drug screening, and the result shows that the known chemical substance of the verteporfin has obvious inhibition effect on the infection of SARS-CoV-2 on Vero-E6 cells, and further evaluates the anti-SARS-CoV-2 activity of the verteporfin through in vitro experiments; the in vitro experiment mainly comprises the steps of determination of inhibition of SARS-CoV-2 infection by the verteporfin, determination of cytotoxicity of the medicine and the like, and results show that the verteporfin can be used for preparing the anti-SARS-CoV-2 medicine and treating novel coronavirus diseases caused by SARS-CoV-2. A novel coronavirus Disease (Corona Virus Disease 2019, COVID-19), also known as novel coronavirus pneumonia.

The title of the invention is Verteporfin, molecular formula is C₈₂H₈₄N₈O₁₆The molecular structure is shown as formula 1.

One embodiment of the invention provides the application of verteporfin in preparing SARS-CoV-2 resisting medicine.

In another preferred embodiment of the invention, verteporfin is used in the preparation of anti-SARS-CoV-2 drugs and in the treatment of novel coronavirus drugs caused by anti-SARS-CoV-2.

The invention adopts the verteporfin to intervene in the in-vitro cell culture of Vero-E6 (Vero-Epimedium cell), and experimental results prove that the verteporfin can effectively inhibit the infection of the novel coronavirus, and the verteporfin can be used for preparing and treating the novel coronavirus diseases caused by the novel coronavirus.

Furthermore, the invention provides other new anti-new crown virus diseases medicines taking the verteporfin as a prodrug, a composition of the verteporfin and a pharmaceutically acceptable carrier and the like.

The invention has the following beneficial effects:

experiments show that the Vero-E6 (African green monkey kidney cell) cultured in vitro by the verteporfin can effectively inhibit the infection of SARS-CoV-2, provides a new application of the verteporfin as an anti-SARS-CoV-2 medicament, and provides a new medicament for treating and controlling novel coronavirus diseases.

Drawings

FIG. 1 is the results of an experiment in which the inhibition of infection by SARS-CoV-2 on Vero-E6 cells by Verteporfin at different concentrations was observed by cytopathic effect (CPE).

FIG. 2 is the experimental results of observing the inhibition of infection of SARS-CoV-2 on Vero-E6 cells by different concentrations of verteporfin through indirect immunofluorescence experiments.

FIG. 3 is a graph showing the inhibition effect of Vero-E6 on SARS-CoV-2 infection by Vero-E6 cell supernatant virus copy number by real-time quantitative PCR (Q-RT-PCR) method.

FIG. 4 is a graph of the toxic effect of verteporfin on Vero-E6 cells at various concentrations.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these specific embodiments.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology and the like, which are well known to those skilled in the art. These techniques are described in their entirety in the tool book, e.g., Bruce Alberts, cell molecular biology, 5 th edition (2002), or alternatively, according to the instructions provided by the manufacturer of the reagents.

Example 1

The inhibitory effect of different concentrations of verteporfin on the infection of SARS-CoV-2 on Vero-E6 cells was observed by cytopathic effect (CPE):

Vero-E6 cell suspension (4X 10) was seeded in 96-well plates⁴One/hole), placing the culture plate in an incubator for pre-culture for 12 hours, and enabling cells to grow in an adherent manner;

adding verteporfin with corresponding concentration for treating for 1 hour in advance, adding DMSO (dimethyl sulfoxide) into a solvent control, and adding no medicine into a negative control;

thereafter, 200PFU SARS-CoV-2 virus (GenBank: MT121215.1) was added to each well except for the negative control, and 12 hours after infection, PBS was washed twice, and a new culture medium containing verteporfin was added, and after culturing at 37 ℃ for 48 hours, cytopathic effect was observed microscopically.

The experimental results are shown in FIG. 1, and show that normal Vero-E6 cells (negative control) grow normally after 48 hours of culture, no cytopathic effect is observed, Vero-E6(DMSO solvent control) infected with SARS-CoV-2 shows obvious cytopathic effect after 48 hours of culture, obvious cell shedding appears, Vero-E6 cells after 0.31 mu M Verteporfin treatment have no obvious cytopathic effect after SARS-CoV-248 hours of infection, and Vero-E6 cells after 0.16 mu M Verteporfin treatment show cytopathic effect, which indicates that 0.31 mu M Verteporfin can effectively inhibit SARS-CoV-2 infection.

Example 2

The inhibitory effect of different concentrations of verteporfin on the infection of SARS-CoV-2 on Vero-E6 cells was observed by indirect immunofluorescence experiments:

cells were treated as in example 1, washed 2 times for 5 minutes each with PBS 48 hours after viral infection;

adding 4% paraformaldehyde, and fixing at room temperature for 15 minutes;

discarding the solution, adding PBS solution containing 0.5% Triton X-100, acting at room temperature for 10 min, washing with PBS for 2 times, each time for 5 min;

adding a freshly prepared PBS solution containing 5% BSA, blocking for 1 hour at room temperature, and washing for 5 minutes for 2 times with PBS;

adding mouse anti-N protein (SARS-CoV-2) polyclonal antibody diluted at a ratio of 1:1000, and acting at room temperature for 1 hour;

PBS wash 3 times, each for 5 minutes;

adding a FITC-goat anti-mouse IgG secondary antibody diluted by 1:10000, and acting for 1 hour at room temperature; PBS wash 3 times, each for 5 minutes;

adding DAPI solution to act for 20 seconds, washing with PBS for 2 times, each time for 5 minutes;

and (4) observing under a fluorescence microscope.

The results are shown in FIG. 2 and show that, consistent with the results of example 1, Vero-E6 cells were treated with 0.31. mu.M Verteporfin before infecting SARS-CoV-2, no N protein of SARS-CoV-2 could be detected in the cells, and two other groups (DMSO solvent control and 0.16. mu.M Verteporfin treatment) could detect a green positive signal.

Example 3

The real-time quantitative PCR (Q-RT-PCR) method detects the copy number of Vero-E6 cell supernatant virus to verify the inhibition effect of the verteporfin on the infection of SARS-CoV-2:

treating the cells as in example 1;

after 48 hours, cell supernatants were collected for RNA extraction.

RNA extraction:

adding 100 mu L of cell supernatant into 300 mu L of TRIzol lysate, and fully mixing and lysing;

adding 200 mu L chloroform, shaking vigorously, standing for 2-3 min, centrifuging at 4 deg.C and 12000g for 15 min;

sucking supernatant, adding 500 mu L of isopropanol, standing for 10 minutes, and centrifuging at 4 ℃ at 12000g for 10 minutes;

adding 1mL of 75% ethanol to wash the RNA precipitate; centrifuging at 7500g for 5 min at 4 ℃;

discarding 75% ethanol, drying, adding 20-50 μ L water to dissolve RNA precipitate;

reverse transcription of RNA:

RNA reverse transcription one-step cDNA was synthesized using the Tiangen FastKing method (Tiangen, KR 118). mu.L of 5 XFastKing-RT SuperMix, 20-2. mu.g of RNA, was added to 20. mu.L of the reaction system, reacted at 42 ℃ for 15 minutes, and then reacted at 95 ℃ for 3 minutes.

Real-time quantitative pcr (qrtpcr);

real-time quantitative PCR was performed using the Tiangen SYBR Green I chimeric fluorescence method (Tiangen, FP 205). mu.L of 2 XSuperReal PreMix Plus, 1. mu.L each of primers (10. mu.M), and 1. mu.L of template cDNA were added to 20. mu.L of the reaction mixture and then the mixture was made up with water. The reaction conditions were as follows: pre-denaturation: 15 minutes at 95 ℃; amplification was performed for 40 cycles: 94 ℃ for 10 seconds, 55 ℃ for 20 seconds; 20 seconds at 72 ℃; melting curve analysis was performed. The specific primer aiming at SARS-CoV-2N protein gene is adopted as the amplification primer: an upstream primer: 5'-GGGGAACTTCTCCTGCTAGAAT-3', respectively; a downstream primer: 5'-CAGACATTTTGCTCTCAAGCTG-3' are provided.

The results are shown in FIG. 3, and show that the viral RNA level in the supernatant of Vero-E6 cells infected with SARS-CoV-2 is obviously reduced along with the increase of the concentration of the Verteporfin drug, which indicates that the Verteporfin has obvious inhibition effect on the infection of SARS-CoV-2, and the half effective concentration (EC50) is 0.028 μ M.

Example 4

And (3) detecting the toxic effect of the verteporfin on Vero-E6 cells:

Vero-E6 cell suspension (1X 10) was seeded in 96-well plates⁴One/well), the plate was placed in an incubator for 12 hours;

the test drugs with different concentrations are added into the culture wells, and the control group is a solvent DMSO.

The effect of the drug on cell proliferation was examined after 48 hours. The Dojindo CCK-8 endpoint method kit (ck04) was used. Discarding the excess medium in the wells, and adding 100 μ L of serum-free medium containing 10% CCK8 solution into each well;

placing the culture plate in an incubator to incubate for 1-4 hours;

the absorbance at 450nm was then measured using a microplate reader.

As shown in FIG. 4, Vero-E6 cells were not cytotoxic at a concentration of 2.5. mu.M or less, and the median cytotoxic concentration (CC50) was 10.33. mu.M. The Selection Index (SI) was 369(SI ═ CC50/EC 50).

In conclusion, the research of the invention shows that the verteporfin can inhibit the infection of SARS-CoV-2 to Vero-E6 cells; after being treated by low-concentration verteporfin (nM level), SARS-CoV-2 can not infect Vero-E6 cell, thus providing new antiviral application for verteporfin application; therefore, the novel function of the verteporfin in preventing and treating SARS-CoV-2 infection is developed, and a novel thought and a novel method are provided for the research and development of novel medicaments for treating the novel coronavirus diseases.

It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.

The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.