Application of thalicamine in preparation of antiviral drugs

Abstract

The invention provides application of thalictriine in preparation of antiviral drugs, and belongs to the technical field of antiviral drugs. The invention proves that the thalictriine has an inhibition effect on various common infectious disease viruses of domestic pigs from a cellular level, and particularly has a remarkable inhibition effect on proliferation of African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and porcine epidemic diarrhea virus (PEDV) in vitro. The invention also discloses an antiviral composition consisting of the active ingredient thalictriine or the pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient, and discloses an application of the composition in preparation of a virus infection inhibitor and / or a pharmaceutical preparation for preventing and / or treating virus infectious diseases. And a basis is provided for developing novel broad-spectrum antiviral drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of antiviral drugs, and particularly relates to the application of thalidomide in the preparation of antiviral drugs. Background Art

[0002] African swine fever (ASF) is an acute, highly contagious disease caused by infection with the African swine fever virus (ASFV), with a mortality rate of up to 100%. ASF was first discovered in Kenya in 1921, and then gradually spread to more than 60 countries and regions in sub-Saharan Africa, Europe, America, Asia, and other regions. The epidemic area continues to expand, and it is the number one threat to the global pig industry. In August 2018, the first ASF outbreak occurred in my country, and then quickly spread throughout the country, seriously damaging the economic interests of my country's pig industry. At present, there is no vaccine or therapeutic drug to provide effective prevention and treatment for ASF. Safe and effective drugs can improve the survival rate of infected pigs, reduce economic losses, and prevent the spread of the virus.

[0003] Porcine reproductive and respiratory syndrome (PRRS) is one of the most common infectious diseases affecting the pig industry worldwide, caused by the porcine reproductive and respiratory syndrome virus (PRRSV). The virus is transmitted through direct or indirect contact and is characterized by reproductive failure in sows and respiratory diseases in pigs of all ages. PRRSV has no effective prevention and treatment methods to date due to its antigenic variation, macrophage tropism, persistent infection, antibody-dependent enhancement, secondary and mixed infection. Currently, the PRRS vaccine has limited protective effect on the disease. Therefore, it is of great significance to develop new, safe, efficient and broad-spectrum anti-PRRSV drugs.

[0004] Porcine circovirus (PCV) Porcine circovirus (PCV) is a non-enveloped icosahedral virus containing a single-stranded negative-sense circular DNA genome. It belongs to the Circoviridae family and the Circovirus genus. It is one of the smallest animal viruses discovered so far. There are four main known PCV serotypes: PCV1, PCV2, PCV3 and PCV4. Since its discovery in 1998, PCV2 has been recognized as one of the most important pathogens in pig herds worldwide. PCV2 mainly attacks weaned piglets aged 5-12 weeks and is the main pathogen of many diseases such as multisystemic wasting syndrome in weaned piglets, porcine dermatitis and nephrotic syndrome, hyperplastic necrotizing pneumonia, reproductive disorders, peripartum myocarditis, and congenital tremor in piglets. These diseases and syndromes are collectively referred to as porcine circovirus-related diseases, which seriously affect pork production.

[0005] Porcine epidemic diarrhea virus (PEDV) is the pathogen of porcine epidemic diarrhea. PEDV belongs to the Coronaviridae family and the Alphacoronavirus genus. It is an enveloped single-stranded positive-sense RNA virus. PEDV has caused huge economic losses to the global pig industry and has become one of the most threatening viral diseases of pigs. The prevention and control of PEDV mainly relies on vaccines to produce mucosal immunity. However, the main victim group of PEDV is suckling piglets whose mucosal immune system has not yet fully developed. Therefore, the current immune effect is not ideal.

[0006] Because the above-mentioned viruses have high mutation frequencies, rapid pathogenicity, high mortality rates, unsatisfactory vaccine protection, and a lack of safe and effective specific drugs in clinical practice, it is of great significance to develop new, safe, efficient, and broad-spectrum antiviral drugs.

[0007] Thalicarpine, molecular formula C 41 H 48 N2O8 is a dibenzylisoquinoline alkaloid. Thalictrum alkaloids are derived from the rhizomes of Thalictrumfoliolosum DC., a plant of the genus Thalictrum in the Ranunculaceae family. It has good biological activities, such as anti-tumor, antioxidant, antiplatelet aggregation, antihypertensive, and antibacterial. However, there are currently no reports on antiviral research. Summary of the invention

[0008] The present invention aims to solve the technical problems in the prior art and provides the application of thalidomide in the preparation of antiviral drugs.

[0009] To achieve the above purpose, the specific technical solution of the present invention is as follows:

[0010] In one aspect, the present invention provides the use of thalidomide in the preparation of antiviral drugs.

[0011] The chemical structural formula of thalidomide is shown in Formula I:

[0012]

[0013] Preferably, the viruses include African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV).

[0014] Another aspect of the present invention provides an antiviral pharmaceutical composition, which comprises an active ingredient thalidomide or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

[0015] Preferably, the active ingredient thalidomide in the pharmaceutical composition accounts for 0.1%-99.9% of the total weight.

[0016] Preferably, the pharmaceutical composition also contains one or more other antiviral drugs.

[0017] Preferably, the other antiviral drugs are compounds with antiviral activity and / or Chinese medicine extracts, and / or Chinese medicine compositions with antiviral effects.

[0018] In another aspect, the present invention provides use of the above-mentioned antiviral pharmaceutical composition in the preparation of a viral infection inhibitor and / or in the preparation of a pharmaceutical preparation for preventing and / or treating viral infectious diseases.

[0019] The pharmaceutical preparation can be administered orally, by injection or externally.

[0020] The pharmaceutical preparations include, but are not limited to, tablets, capsules, granules, gels, dispersants, oral liquids, injections, sprays, drops, ointments and other dosage forms.

[0021] Beneficial effects:

[0022] The present invention proves at the cellular level that thalidomide can inhibit a variety of RNA viruses and DNA viruses, and is particularly effective against African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and porcine epidemic diarrhea virus, providing a potential broad-spectrum antiviral drug for the prevention and treatment of a variety of viral infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the cell survival rate of PAMs cells acted by thalassoside;

[0024] Figure 2 is the CC of thalassine on PAMs cells 50 Fitting results;

[0025] Figure 3 The Western Blot method was used to determine the inhibition of the expression of African swine fever virus p72 protein by thalidomide in PAMs cells;

[0026] Figure 4 The software analyzes the grayscale value of Western Blot protein bands, calculates the inhibition rate of the corresponding concentration, and fits the IC 50 Numeric value; Figure 5 The Western Blot method was used to determine the inhibition of the expression of PRRSV virulence protein M protein by thalidomide in Vero cells, and the ratio of M protein to the internal reference protein β-actin was calculated based on the protein gray value;

[0027] Figure 6 is the qPCR method to determine the inhibition of PCV2 Cap gene transcription level in PK15 cells by thalidomide and positive control ribavirin;

[0028] Figure 7 is the cell activity inhibition rate of thalidomide on Vero cells. DETAILED DESCRIPTION

[0029] The above contents will be further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter being limited to the following embodiments. All technologies realized based on the contents of the present invention are within the scope.

[0030] Example 1 In vitro inhibitory effect of thalidomide on African swine fever virus (ASFV)

[0031] 1. The median cytotoxic concentration (CC) of thalassine against PAMs 50 ) determination

[0032] Resuscitated porcine alveolar macrophages (PAMs) were used at 2 × 10 4 100 μL of CCK-8 (Cell counting kit-8, cell counting reagent) were added to each well, and the cells were incubated for 24 hours. The OD value was measured at 450 nm using an enzyme reader. The cell survival rate was calculated according to the formula % = (A sample - A blank) / (A negative - A blank) × 100%, and the results are as follows: Figure 1Fitting CC of thalidomide 50 Values, such as Figure 2 As shown, the CC of PAMs by thalassine 50 The concentration of thalidomide was 32.89 μM, and when the concentration of thalidomide was less than 10 μM, it had no obvious toxicity to PAMs cells (cell activity>90%). Therefore, when the concentration of thalidomide was less than 10 μM, there was no significant difference between the treated cell group and the untreated group.

[0033] 2. Thalictrine inhibits African swine fever virus infection in PAMs cells

[0034] PAMs cells were 2×10 6 Each well was inoculated with 100 μg / well of 6-well plates. After the cells were completely attached to the wall, different concentrations of thalidomide (0.5 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM) were added to the cells. After incubation for 2 h, 0.1 MOI of virus was added to each well for adsorption for 1 h. The cells were washed twice with PBS and different concentrations of thalidomide (0.5 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM) were added. After culturing for 24 h, the cells were collected and the expression of p72 protein was determined by immunoblotting. The results are shown in the figure. Figure 3 Image J was used to analyze the grayscale value of protein bands, and the grayscale value of p72 protein in each lane was obtained / the grayscale value of the internal reference band was obtained. The inhibition rate of ASFV at each concentration of thalidomide was calculated according to the formula = 1-[(grayscale value of the target band in the experimental group / grayscale value of the internal reference band in the experimental group) / (grayscale value of the target band in the control group / grayscale value of the internal reference band in the control group)], and the half inhibition concentration IC of thalidomide on ASFV was fitted according to the inhibition rate. 50 .Depend on Figure 4 It is known that the IC of thalassine 50 =3.509 μM. Selectivity index SI = CC 50 / IC 50 The larger the SI value, the larger the drug therapeutic window and safe concentration, and the stronger the inhibitory effect. The SI value of thalidomide is 9.37.

[0035] Example 2 In vitro inhibitory effect of thalassoline on porcine reproductive and respiratory syndrome virus (PRRSV)

[0036] Vero cells (African green monkey kidney cells) were inoculated in 6-well plates and cultured for 6 to 8 hours. Vero cells were infected with 0.1 MOI of PRRSV. After adsorption for 1 hour, the infection solution was discarded and washed. Different concentrations of thalidomide (0 μM, 2.5 μM, 5 μM, and 10 μM) were added to the cells, with 0 μM as a negative control. Cells were collected 36 hours after virus infection, and the expression of viral virulence protein M protein was detected by immunoblotting ( Figure 5), the results showed that thalidomide significantly inhibited the expression of PRRSV virulence protein M protein in a dose-dependent manner.

[0037] Example 3 In vitro inhibitory effect of thalassine on porcine circovirus type 2 (PCV2)

[0038] PK15 cells (pig kidney cells) were inoculated in a 24-well plate. When the cells grew to about 80%, PK15 cells were infected with 0.1 MOI of PCV2. After 2 hours of adsorption, the infection solution was discarded and washed. Different concentrations of thalidomide (0 μM, 2.5 μM, 5 μM, 10 μM) and positive control ribavirin (10 μM) were added to the cells. The PCV2 cap gene copy number was detected by qPCR method 48 hours after virus infection.

[0039] The PCV2 cap primer sequence is:

[0040] PCV2 cap-F: 5'-TACATTTTCCAGCAGTTTG-3';

[0041] PCV2 cap-R: 5'-CTCCCGCCATACCATAA-3';

[0042] result( Figure 6 ) showed that compared with the infected group, the Cap gene copy number in each thalidomide group was significantly reduced in a dose-dependent manner.

[0043] Example 4 In vitro inhibitory effect of thalidomide on porcine epidemic diarrhea virus (PEDV)

[0044] Vero cells (African green monkey kidney cells) were inoculated in 96-well plates. When the cells grew to about 80%, 0.1 MOI of PEDV was used to infect the Vero cells. After 2 hours, the infection solution was discarded and different concentrations of thalidomide (0 μM, 2.5 μM, 5 μM, and 10 μM) were added to the cells. After 24 hours of virus infection, the culture medium was discarded and CCK-8 was added. The cells were incubated for another 2 hours. The OD value was measured at 450 nm using an enzyme reader and the cell activity was calculated. The results were ( Figure 7 ) showed that thalidomide could significantly inhibit the proliferation of PEDV in a dose-dependent manner.

Claims

1. Application of thalidomide in the preparation of antiviral drugs.

2. The use according to claim 1, characterized in that: The chemical structural formula of thalidomide is shown in Formula I:

3. The use according to claim 1, characterized in that: The viruses include African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and porcine epidemic diarrhea virus.

4. An antiviral pharmaceutical composition, which is composed of an active ingredient, thalidomide or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

5. The pharmaceutical composition according to claim 4, characterized in that The mass percentage of thalidomide or its pharmaceutically acceptable salt in the pharmaceutical composition is 0.1%-99.9%.

6. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition also contains one or more other antiviral drugs.

7. The pharmaceutical composition according to claim 6, characterized in that The other antiviral drugs are compounds and / or Chinese medicine extracts with antiviral activity, and / or Chinese medicine compositions with antiviral effects.

8. Use of an antiviral pharmaceutical composition as claimed in any one of claims 4 to 7 in the preparation of a viral infection inhibitor and / or in the preparation of a pharmaceutical preparation for preventing and / or treating viral infectious diseases.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical preparation can be administered orally, by injection or externally.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical preparations include tablets, capsules, granules, gels, dispersants, oral liquids, injections, sprays, drops, and ointments.

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