WO2022001079A1 - Use of azacytidine in preparation of antiviral drugs - Google Patents

Abstract

Provided is the use of Azacytidine in the preparation of antiviral drugs. Viruses are those with a short course of disease. Azacytidine can effectively inhibit the replication of viruses and reduce injury to human bodies caused by a cytokine storm, and furthermore allows human bodies to have enough time to generate antibodies, thereby improving the cure rate of patients.

Description

Application of a kind of Azacytidine in the preparation of antiviral drugs technical field

The invention relates to the field of antiviral drugs, and more particularly to the application of Azacytidine in the preparation of antiviral drugs.

Background technique

Virus is a kind of non-cellular organism that is small in size, simple in structure, contains only one nucleic acid (DNA or RNA), and must be parasitic in living cells and reproduce by means of replication. Virus is a non-cellular life form. It consists of a long nucleic acid chain and a protein shell. Viruses do not have their own metabolic mechanisms and do not have an enzyme system. Therefore, the virus leaves the host cell and becomes a chemical substance without any life activity and unable to reproduce independently. Its replication, transcription, and translation capabilities are all carried out in the host cell. When it enters the host cell, it can use the substances and energy in the cell to complete life activities and generate it according to the genetic information contained in its own nucleic acid. A new generation virus like it.

Due to the small size of the virus, some can cause gastroenteritis in humans and pigs; some are parasitic in the human tonsils and lack reproductive capacity, but another helper virus, adenovirus, invades the tonsils. With help, pathogenic bacteria can also multiply. Among the microRNA viruses, there are polioviruses that can cause polio, many viruses that can cause myocarditis, meningitis, epidemic chest myalgia, laryngotracheitis and gastroenteritis, and rhinoviruses that can cause colds and flu , Foot-and-mouth disease virus can cause oral inflammation and hoof ulceration in cattle, pigs, sheep and other livestock, and often lead to large-scale deaths.

When the virus enters the human body, it is easy to replicate in the human respiratory tract and follow the oral secretions for direct transmission, thereby forming a large-scale infection.

Most of the diseases caused by viruses are the main infectious diseases of human beings. Viruses can invade different tissues and organs and cause diseases by infecting cells. Common diseases caused by viruses are:

① Epidemic diseases: influenza, common cold, measles, mumps, polio, infectious hepatitis, polio;

② Chronic susceptibility: Hepatitis B, AIDS (AIDS)

③ Latent infection: herpetic keratitis, venereal herpes virus.

According to the mechanism of action of antiviral drugs, current antiviral drugs can be divided into the following categories: [1]

Penetration and uncoating inhibitors, DNA polymerase inhibitors, reverse transcriptase inhibitors, nucleosides, non-nucleosides, protein inhibitors, neuraminidase inhibitors, broad-spectrum antivirals: ribavirin, interfering White.

technical problem

Due to the diversity of viruses, many viruses do not have specific treatment drugs, such as the new coronary pneumonia virus that occurred at the end of 2019 and SARS that occurred in 2003.

technical solutions

The purpose of the present invention is to provide the application of a known drug in antiviral.

According to the above purpose, the application of Azacytidine (also known as 5-Azacytidine) in the preparation of antiviral drugs is firstly provided. The virus is a virus with a short course of disease. The virus with a short course of disease here includes a Viruses that cause people to die quickly also include viruses that experience a certain incubation period without symptoms, and can cause people to die quickly after the onset of the disease.

More specifically, the antiviral drug is a drug that inhibits virus replication.

More specifically, the antiviral drug is a drug for reducing cytokine storm.

More specifically, reducing the toxic side effects of antiviral drugs

More specifically, the virus is a virus that relies on RNA as a gene carrier, or RNA as an intermediate metabolite.

More specifically, the virus is a coronavirus.

More specifically, the coronavirus is a novel coronavirus.

More specifically, the virus is SARS virus, MERS virus or nCoV-2019 virus.

Also provided is an application of Azacytidine in the preparation of a medicine for treating atypical pneumonia, where the atypical pneumonia is pneumonia caused by a virus.

More specifically, the antiviral drug is a drug that slows down the progression of acute viral disease.

More specifically, the dosage of the antiviral drug for human use is 1-10000 mg/m ² .

beneficial effect

Virus infection, damages cells after virus infection. Some articles show that the SARS nsp1 protein can shear the RNA of cells, resulting in the degradation of cellular RNA and damage to cells; virus infection leads to an increase in IgG in the blood, and immune cells in the blood overreact to cause an immune storm , and immune storms are often the most deadly. This mechanism is similar to Ebola; the virus progresses quickly, the body has no time to produce the corresponding antibodies, and the body has respiratory failure, so the rescue method is often based on a ventilator.

AIDS drugs can inhibit the replication of SARS in the early stage, but because AIDS is a targeted drug, how effective it is on non-targeted SARS may still need to be studied and verified. Current research is that the vast majority of HIV and flu drugs are ineffective against the new crown. Glead released remdesivir as a nucleoside analog to stop SARS virus RNA synthesis, but the current results are also ineffective. The reasons are mainly divided into two points. The first RNA polymerase has fidelity, so it has a relatively strong recognition effect on nucleoside analogs, resulting in the RNA that will not be integrated into the virus, such as ribavirin. Secondly, most protease inhibitors are also targeted drugs, so the specificity of the protein structure is also very high, so it is difficult for the new crown to work.

And the current treatment of cytokine storm, also known as immune storm, mainly uses glucocorticoids, and excessive use of glucocorticoids can cause serious sequelae, such as femoral head necrosis, pulmonary fibrosis, and other sequelae, in 2003 SARS Especially during a pandemic.

The mechanism of Azacytidine (hereinafter referred to as Aza) in the treatment of new crown

1. Aza is a highly similar nucleoside analog, and all current polymerases in eukaryotic cells cannot specifically reject the integration of Aza.

2. As a nucleoside analog, Aza can be highly efficiently integrated into cellular RNA.

3. Integrating into the RNA of the virus can effectively disrupt the high-speed translation mechanism of the virus, thereby slowing down the virus infection

4. At the same time, the RNA integrated into the cell itself may also play a role in preventing the virus from cutting the host RNA, thereby protecting the cell from being destroyed.

5. At the same time, this drug can also slow down the response of immune cells by integrating Aza into immune cell RNA, thereby weakening the immune storm.

To sum up, through research, it was found that Azacytidine can effectively inhibit the replication of the virus and reduce the damage caused by immune storms to the human body, so that the human body has enough time to produce antibodies, thereby improving the cure rate of patients.

Description of drawings

FIG. 1 is the survival curve of the mice of Example 1 under immune storm.

FIG. 2 is the expression level of the cytokine TNF-α in Example 1. FIG.

FIG. 3 shows the number of virus infections in Example 2. FIG.

FIG. 4 is the control regression curve of Example 2. FIG.

Figure 5 shows the expression levels of cytokines (tumor necrosis factor, interleukin-6) under different drug concentrations in Example 3.

BEST MODE FOR CARRYING OUT THE INVENTION

The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted here that the description of these embodiments is used to help the understanding of the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

Example 1 Azacytidine inhibits immune storm

BALB/cj mice were injected with PDL1 to create an immune storm model, so that the mice developed a severe immune storm.

Then BALB/cj mice were injected with PDL1 and Azacytidine at the same time. According to the description of the experimenter, the immune storm state of the mice was significantly relieved. Finally, the number of mice that died due to immune storm also decreased significantly, as shown in Figure 1, the survival curve of mice under immune storm. The mice in the PD-L1-treated group died rapidly due to the immune storm. After the injection of azacytidine, the immune storm of the mice in the PD-L1 treatment group was significantly relieved, and the survival curve was significantly prolonged.

The immune storm indicator factor TNF-a in the lung tissue was measured. As shown in Figure 2, it was found that the concentration of the immune storm indicator factor TNF-a in the lungs of mice injected with Azacytidine was significantly reduced.

Therefore, it is believed that Azacytidine has the effect of suppressing the immune storm, which can relieve the immune storm caused by the late SARS, and will not produce sequelae such as glucocorticoids.

Embodiments of the present invention

Example 2 Azacytidine inhibits virus replication

Experimental steps:

BHK21-hACE2 cells are plated, 2X104 cells per well of 96-well plate, so that the density can reach 70%-80% after 12 hours;

Dilution of Small Molecule Drugs:

Aza (2mM) was diluted as follows: 10ul Aza (2mM) + 90ul DMEM to make the final concentration 0.2mM, take 0.2mM sample 50ul + 150ul DMEM as gradient 1, take 50ul gradient 1 + 100ul DMEM as gradient 2, in turn 3 fold dilution, a total of 8 gradients;

Chloroquine-NIDVD (20mM) was diluted as follows: 10ul Chloroquine-NIDVD (20mM)+990ulDMEM to make the final concentration 0.2mM, the post-dilution method was the same as that of Aza (2mM), a total of 8 gradients;

Take 80ul+20ul rVSV-SARS-2 (3X10e5pfu/ml) of each graded dilution of the drug, mix and incubate at 37° for 1h;

Aspirate and discard the supernatant of BHK21-hACE2 cells and add 100ul of the sample after incubation in step 3: Drug 80ul+20ul rVSV-SARS-2;

After 12h, take pictures to observe and record the number of positive cells;

Calculation.

As shown in Figure 3, it can be seen from the data that under the condition of low drug concentration, many viruses can infect this cell, so most cells will express fluorescent proteins, and when the drug concentration increases, the drug inhibits the virus Infection or virus expression and replication, so that in the case of high concentrations of drugs, it can be seen that the infection of the virus is significantly reduced, and the number of fluorescence is significantly reduced. Therefore, it can be clearly seen that the drugs Aza and chloroquine have the same effect on the inhibition of the new coronavirus.

The percentage reduction in fluorescence number was plotted against drug concentration as inhibition rate as an index of viral infection. Through research, it was found that the effect and concentration of this drug and chloroquine in inhibiting the new coronavirus are similar and similar, as shown in Figure 4.

Mock is the blank control group (DMSO).

Inhibition rate=(the number of blank control fluorescence - the number of fluorescence at drug concentration)/the number of blank control fluorescence (y-axis)

Drug concentration (x-axis)

As shown in Figure 4, according to the regression curve made above. It was found that Aza and chloroquine were similar in inhibiting virus infection efficiency. The cytotoxicity of the compound was analyzed by Cell Counting Kit to analyze the cell viability and state, and plotted the cell viability and drug concentration. It was found that as the drug concentration increased, the cell viability decreased, which proved that the drug toxicity increased with the concentration.

The cytotoxicity of Aza is slightly higher than that of chloroquine between 2.5 and 5, but the cytotoxicity of Aza is lower than that of chloroquine when the concentration is greater than 5, which proves that the cell viability of Aza is better than that of chloroquine and the toxicity is lower at high concentrations.

 

Example 3 Immune storm detection

293T cells were transiently transfected with S protein (S protein granules), and 24 hours later, they were digested and plated in a 24-well plate, with 2*105 cells per well. After 24h, S-CART and different concentrations of Aza drug (1 uM, 5 uM, 10 uM) were added. S protein can stimulate the corresponding S-cart to produce immune storm, and immune storm-related factors are secreted into the supernatant. The supernatant was collected after 48h. Cytokines (tumor necrosis factor, interleukin-6) in the supernatant were detected by ELISA to determine the severity of the immune storm.

As shown in Figure 5, the horizontal axis represents the drug concentration, and the vertical axis represents the signal intensity (indicated concentration) of detected cytokines. It can be clearly seen that the cytokines IL6 and TNFa in the supernatant are significantly reduced at high concentrations above 5uM, indicating that Aza can obviously inhibit the immune storm.

 

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and alterations to these embodiments still fall within the protection scope of the present invention.

Industrial Applicability

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Claims (9)

An application of Azacytidine in the preparation of antiviral drugs, characterized in that the virus is a virus with a short course of disease. The application according to claim 1, wherein the antiviral drug is a drug that inhibits virus replication. The application according to claim 2, wherein the antiviral drug is a drug for reducing cytokine storm. The application according to claim 1, wherein the virus is a coronavirus. The application according to claim 4, wherein the coronavirus is a novel coronavirus. The application according to claim 1, wherein the virus is SARS virus, Ebola virus, MERS virus or nCoV-2019 virus. An application of Azacytidine in the preparation of a medicine for treating atypical pneumonia, characterized in that the atypical pneumonia is pneumonia caused by a virus. The application according to claim 1, wherein the antiviral drug is a drug for slowing down the development of an acute viral disease course. The application according to claim 1, characterized in that, the dosage of the antiviral drug for human use is 1-10000 mg/m2.