Use of thalicarpine in preparation of antiviral drug

Abstract

The present invention belongs to the technical field of antiviral drugs. Provided is the use of thalicarpine in the preparation of an antiviral drug. The present invention demonstrates at the cellular level that thalicarpine has an inhibitory effect on a variety of viruses causing common infectious diseases in domestic pigs, especially having a significant inhibitory effect on the 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. Further disclosed in the present invention are an antiviral composition composed of an active ingredient, i.e. thalicarpine, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient, and the use of the composition in the preparation of a viral infection inhibitor and / or in the preparation of a pharmaceutical preparation for preventing and / or treating a viral infectious disease, thereby providing a basis for the development of a new broad-spectrum antiviral drug.

Description

Application of thalidomide in preparation of antiviral drugs TECHNICAL FIELD

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

[0002] African swine fever (ASF) is an acute and severe infectious disease caused by African swine fever virus (ASFV) infection, with a mortality rate of 100%. ASF was first discovered in Kenya in 1921, and then gradually spread to sub-Saharan Africa, Europe, the Americas, Asia and more than 60 countries and regions, and the epidemic area is increasing, which is the top threat to the global pig industry. In August 2018, China first appeared ASF epidemic, and then spread rapidly throughout the country, which seriously damaged the economic interests of China's pig industry. At present, there is no vaccine and treatment drug to provide effective prevention and treatment for ASF, and 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 in the global pig industry caused by porcine reproductive and respiratory syndrome virus (PRRSV). The virus is transmitted through direct or indirect contact, and its characteristics are reproductive failure in sows and respiratory disease in pigs of all ages. PRRSV has characteristics such as antigen variation, macrophage tropism, persistent infection, antibody-dependent enhancement, secondary and mixed infection, and so far there is no effective prevention and treatment method. At present, the protective effect of PRRS vaccine on the disease is limited, therefore, it is of great significance to develop new, safe, efficient and broad-spectrum anti-PRRSV drugs.

[0004] Porcine circovirus (PCV) is a non-enveloped icosahedral virus containing a single-stranded negative sense circular DNA genome, belonging to the Circoviridae family, Circovirus genus, and is one of the smallest animal viruses ever discovered. There are currently four 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 populations worldwide. PCV2 mainly attacks 5-12 week old weaned piglets and is the main pathogen of postweaning multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, proliferative and necrotic pneumonia, reproductive disorders, peripartum myocarditis, congenital tremor in piglets and other diseases, which are collectively referred to as porcine circovirus-associated diseases, which seriously affect pork production.

[0005] Porcine epidemic diarrhea virus (PEDV) is the pathogen of porcine epidemic diarrhea, and PEDV belongs to the Coronaviridae family, Alphacoronavirus genus, and is a single-stranded positive-sense RNA virus with an envelope. PEDV has brought huge economic losses to the global pig industry and has become one of the most threatening viral diseases in pigs. The main means of prevention and control of PEDV is to rely on vaccines to produce mucosal immunity, however, the main victim group of PEDV is the mammalian piglets whose mucosal immune system has not yet developed completely, therefore, the current immune effect is not ideal.

[0006] The above viruses have high mutation frequency, fast pathogenic speed, high mortality, and unsatisfactory vaccine protection, and there is a lack of safe and effective specific drugs in clinical practice, therefore, 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 double benzyl isoquinoline alkaloid, Thalicarpine is derived from the rhizome of the Ranunculaceae Thalictrum foliolosum DC. plant, and has good biological activity, such as anti-tumor, anti-oxidation, anti-platelet aggregation, antihypertensive, antibacterial and the like, but there is no related report on antiviral research at present. SUMMARY

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

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

[0010] In one aspect of the present application, there is provided a use of thalidomide in the preparation of an antiviral drug.

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

[0012] Preferably, the virus includes African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV).

[0013] In another aspect of the present application, there is provided an antiviral drug composition comprising an active ingredient of thalidomide or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

[0014] Preferably, the active ingredient of thalidomide in the drug composition accounts for 0.1% to 99.9% of the total weight.

[0015] Preferably, the drug composition further comprises one or more other antiviral drugs.

[0016] Preferably, the other antiviral drugs are compounds and / or traditional Chinese medicine extracts with antiviral activity, and / or traditional Chinese medicine compositions with antiviral effects.

[0017] In yet another aspect of the present application, there is provided a use of the above-mentioned antiviral drug 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.

[0018] The administration of the pharmaceutical preparation is oral or injection or external use.

[0019] The pharmaceutical preparation includes, but is not limited to, tablet, capsule, granule, gel, dispersion, oral liquid, injection, spray, drop, ointment, etc. Advantages:

[0020] The present application proves from the cellular level that thalidomide can inhibit a variety of RNA viruses and DNA viruses, especially African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 virus, 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 DRAWINGS

[0021] Figure 1 is the cell survival rate of PAMs cells treated with thalidomide;

[0022] Figure 2 is the CC of thalidomide on PAMs cells 50 Fitting results;

[0023] Figure 3 is a Western Blot method for determining the expression of p72 protein of African swine fever virus inhibited by thalidomide on PAMs cells;

[0024] Figure 4 is a software analysis of the gray value of the Western Blot protein band, the inhibition rate of the corresponding concentration is calculated, and the IC 50 value is fitted; Figure 5 is a Western Blot method for determining the expression of PRRSV virulence protein M protein inhibited by thalidomide on Vero cells, and the ratio of M protein to internal reference protein β-actin is calculated according to the protein gray value;

[0025] Figure 6 is a qPCR method for determining the transcription level of PCV2 Cap gene inhibited by thalidomide and positive control ribavirin on PK15 cells;

[0026] Figure 7 is the cell activity inhibition rate of thalidomide on Vero cells. DETAILED DESCRIPTION

[0027] The above will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter to the following examples. Any technology realized based on the content of the present application is within the scope.

[0028] Example 1 In vitro inhibition of African swine fever virus (ASFV) by thalidomide

[0029] 1. Determination of the half cytotoxic concentration (CC 50 ) of thalidomide on PAMs

[0030] Recover porcine alveolar macrophage PAMs, inoculate 2×10 4 cells / well in a 96-well plate, and after 6h of cell adhesion, discard the cell culture solution and add different concentrations of thalidomide (1 μM, 3 μM, 10 μM, 30 μM, 60 μM, 100 μM) to each well, three replicate wells for each concentration, set up negative control and blank control, continue to incubate and culture for 24h, then add 10 μL of CCK-8 (cell counting kit-8) to each well, continue to incubate for 2h, and measure the OD value at 450nm with a microplate reader. Calculate the cell survival rate % according to the formula: cell survival rate % = (A sample - A blank) / (A negative - A blank) x 100%, and the results are shown in Figure 1. The CC 50 value of thalidomide is fitted, as shown in Figure 2. The CC 50The concentration of thalidomide was 32.89 μM, and it did not exhibit significant toxicity to PAM cells (cell viability > 90%) when the concentration of thalidomide was less than 10 μM. Therefore, there was no significant difference between the treated and untreated cell groups when the concentration of thalidomide was less than 10 μM.

[0031] 2. The inhibitory activity of thalidomide on African swine fever virus infection in PAMs cells.

[0032] PAMs cells at 2×10 6 Cells were seeded per well in 6-well plates. After complete cell adhesion, different concentration gradients of thalidomide (0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM) were added to the cells. After incubation for 2 h, 0.1 MOI of virus adsorption was added to each well for 1 h. The cells were washed twice with PBS, and different concentration gradients of thalidomide (0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM) were added. After culturing for 24 h, the cells were harvested, and the expression of p72 protein was determined by immunoblotting. The results are shown in Figure 3. Image J analysis was used to obtain the gray values ​​of p72 protein and the internal control band in each lane. The inhibition rate of thalidomide against ASFV at each concentration was calculated using the formula: 1 - [(gray value of target band in experimental group / gray value of internal control band in experimental group) / (gray value of target band in control group / gray value of internal control band in control group)]. The half-maximal inhibitory concentration (IC50) of thalidomide against ASFV was then fitted based on the inhibition rate. 50 As shown in Figure 4, the IC50 of thalidomide... 50 =3.509 μM. Selectivity index SI = CC 50 / IC 50 The higher the SI value, the larger the therapeutic window and safe concentration of the drug, and the stronger the inhibitory effect. The SI value of thalidomide is 9.37.

[0033] Example 2: In vitro inhibitory effect of thalidomide on porcine reproductive and respiratory syndrome virus (PRRSV)

[0034] Vero cells (African green monkey kidney cells) were seeded in 6-well plates and cultured for 6 to 8 hours. Vero cells were then infected with 0.1 MOI PRRSV. After 1 hour of adsorption, the washing solution was discarded. Different concentrations of thalidomide (0 μM, 2.5 μM, 5 μM, 10 μM) were added to the cells, with 0 μM serving as a negative control. Cells were collected 36 hours after viral infection, and the expression of viral virulence protein M was detected by immunoblotting (Figure 5). The results showed that thalidomide significantly inhibited the expression of PRRSV virulence protein M in a dose-dependent manner.

[0035] Example 3: In vitro inhibitory effect of thalidomide on porcine circovirus type 2 (PCV2)

[0036] PK15 cells (porcine kidney cells) were inoculated in 24-well plates, and when the cells grew to about 80%, the PK15 cells were infected with PCV2 at a MOI of 0.1. After 2 hours of adsorption, the infection solution was removed and washed, and different concentrations of thalidomide (0 μM, 2.5 μM, 5 μM, 10 μM) and the positive control ribavirin (10 μM) were added to the cells. After 48 hours of viral infection, the PCV2 cap gene copy number was detected by qPCR.

[0037] The PCV2 cap primer sequence is:

[0038] PCV2 cap-F: 5'-TACATTTCCAGCAGTTTG-3';

[0039] PCV2 cap-R: 5'-CTCCCGCCATACCATAA-3';

[0040] The results (Figure 6) show that the Cap gene copy number of each thalidomide group was significantly reduced in a dose-dependent manner compared with the infection group.

[0041] Example 4 In vitro inhibition of porcine epidemic diarrhea virus (PEDV) by thalidomide

[0042] Vero cells (African green monkey kidney cells) were inoculated in 96-well plates, and when the cells grew to about 80%, the Vero cells were infected with PEDV at a MOI of 0.1. After 2 hours, the infection solution was removed and washed, and different concentrations of thalidomide (0 μM, 2.5 μM, 5 μM, 10 μM) were added to the cells. After 24 hours of viral infection, the culture medium was removed, CCK-8 was added, and incubation was continued for 2 hours. The OD value was measured at 450 nm by a microplate reader, and the cell activity was calculated. The results (Figure 7) show that thalidomide can significantly inhibit the proliferation of PEDV in a dose-dependent manner.

Claims

1. Use of thalidomide in the preparation of an antiviral drug.

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

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

4. An antiviral drug composition, which is composed of active ingredient thalidomide or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable carriers and / or excipients.

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

6. The pharmaceutical composition according to claim 4, wherein The drug 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 traditional Chinese medicine extracts with antiviral activity, and / or traditional Chinese medicine compositions with antiviral effect.

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

9. The pharmaceutical composition according to claim 8, characterized by The drug preparation is administered orally or by injection or externally.

10. The pharmaceutical composition according to claim 9, characterized by The drug preparation includes tablets, capsules, granules, gels, dispersions, oral liquids, injections, sprays, drops, and ointments.

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