Use of thalidomide in the preparation of an antiviral drug

Abstract

The application provides application of thalidomide in preparation of antiviral drugs and belongs to the technical field of antiviral drugs. The application proves that thalidomide has inhibiting effect on viruses of various common infectious diseases of domestic pigs at the cellular level, especially has significant inhibiting 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 application also discloses an antiviral composition composed of active ingredient thalidomide or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers and / or excipients, and discloses application of the composition in preparation of viral infection inhibitors and / or in preparation of pharmaceutical preparations for preventing and / or treating viral infectious diseases, and provides a basis for development of broad-spectrum antiviral new drugs.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug technology, specifically relating to the application of thalidomide in the preparation of antiviral drugs. Background Technology

[0002] African swine fever (ASF) is an acute and highly contagious disease caused by the African swine fever virus (ASFV), with a mortality rate as high as 100%. ASF was first discovered in Kenya in 1921 and subsequently spread to more than 60 countries and regions in sub-Saharan Africa, Europe, the Americas, and Asia, with the affected area continuously increasing, making it the number one threat to the global pig industry. In August 2018, my country reported its first ASF outbreak, which quickly spread throughout the country, severely damaging the economic interests of my country's pig farming industry. Currently, there are no vaccines or treatments for ASF, providing effective prevention and treatment methods. Safe and effective drugs can improve the survival rate of infected pigs, reduce economic losses, and stop the spread of the virus.

[0003] Porcine reproductive and respiratory syndrome (PRRS) is one of the most prevalent infectious diseases affecting the pig industry worldwide, caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). This virus is transmitted through direct or indirect contact and is characterized by reproductive failure in sows and respiratory disease in pigs of all ages. Due to its antigenic variation, macrophage tropism, persistent infection, antibody-dependent enhancement, secondary and mixed infections, there are currently no effective prevention and control methods for PRRSV. At present, PRRS vaccines offer limited protection against the disease; therefore, the development of novel, safe, effective, and broad-spectrum anti-PRRSV drugs is of great significance.

[0004] Porcine circovirus (PCV) is a non-enveloped icosahedral virus containing a single-stranded negative-sense circular DNA genome. It belongs to the family Circoviridae and the genus Circovirus, and is one of the smallest animal viruses discovered to date. Currently, four main PCV serotypes are known: 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 primarily affects weaned piglets aged 5-12 weeks and is a major pathogen causing various diseases, including weaned piglet multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, proliferative necrotizing pneumonia, reproductive disorders, peripartum myocarditis, and congenital tremors in piglets. These diseases and syndromes are collectively referred to as porcine circovirus-related diseases, which seriously impact pork production.

[0005] Porcine epidemic diarrhea virus (PEDV) is the pathogen of porcine epidemic diarrhea. PEDV belongs to the family Coronaviridae, genus Alphacoronavirus, and is an enveloped, single-stranded, positive-sense RNA virus. PEDV has caused enormous economic losses to the global pig industry and has become one of the most threatening viral diseases in pigs. The main control method for PEDV relies on vaccines to induce mucosal immunity; however, the primary victim group of PEDV is suckling piglets whose mucosal immune systems are not yet fully developed, therefore, current immunization efforts are not ideal.

[0006] Due to the high mutation rate, rapid onset of disease, and high mortality rate of the aforementioned viruses, the protective effect of vaccines is not ideal, 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 dibenzylisoquinoline alkaloid. Thalictrine is derived from the rhizome of Thalictrum foliolosum DC., a plant belonging to the genus Thalictrum in the Ranunculaceae family. It has good biological activities, such as antitumor, antioxidant, antiplatelet aggregation, hypotensive and antibacterial effects, but there are currently no reports on its antiviral properties. Summary of the Invention

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

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

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

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

[0012]

[0013] 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).

[0014] In another aspect, the present invention provides an antiviral pharmaceutical composition comprising the 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 further contains one or more other antiviral drugs.

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

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

[0019] The drug preparation can be administered orally, by injection, or topically.

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

[0021] Beneficial effects:

[0022] This invention demonstrates at the cellular level that thalidomide can inhibit a variety of RNA and DNA viruses, especially 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 various viral infectious diseases. Attached Figure Description

[0023] Figure 1 The cell survival rate of PAMs cells after the action of thalidomide;

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

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

[0026] Figure 4 The software analyzes the grayscale values ​​of protein bands in Western blotting, calculates the inhibition rate at corresponding concentrations, and fits the IC50. 50 Numerical value; Figure 5 The Western Blot method was used to determine the inhibition of PRRSV virulence protein M protein expression by thalidomide in Vero cells, and the ratio of M protein to internal reference protein β-actin was calculated based on the protein gray value.

[0027] Figure 6 This study used qPCR to determine the level of PCV2 Cap gene transcription inhibition by thalidomide and the positive control ribavirin in PK15 cells.

[0028] Figure 7 It is the inhibition rate of thalidomide on the cell activity of Vero cells. Detailed Implementation

[0029] The above content will be further described in detail below through specific embodiments. However, this should not be construed as limiting the scope of the above subject matter to the following embodiments. All technologies implemented based on the content of this invention fall within the scope.

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

[0031] 1. Half-maximal cytotoxic concentration (CMC) of thalidomide against PAMs 50 ) Measurement

[0032] Resuscitate porcine alveolar macrophages (PAMs) at 2 × 10⁻⁶ 4 Cells were seeded per well in 96-well plates. After 6 hours of complete cell adhesion, the cell culture medium was aspirated, and different concentrations of thalidomide (1 μM, 3 μM, 10 μM, 30 μM, 60 μM, 100 μM) were added to each well, with three replicates for each concentration. A negative control and a blank control were also included. After incubation for 24 hours, 10 μL of CCK-8 (Cell Counting Kit-8) was added to each well, and incubation continued for 2 hours. The OD value was measured at 450 nm using a microplate reader. The cell viability percentage (%) was calculated using the formula: (Sample A - Blank A) / (Negative A - Blank A) × 100%. The results are shown below. Figure 1Fitting the CC of thalidomide 50 Value, such as Figure 2 As shown, the CC effect of thalidomide on PAMs 50 The 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.

[0033] 2. The inhibitory activity of thalidomide on African swine fever virus infection in PAMs cells.

[0034] 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 hours, 0.1 MOI of virus adsorption solution was added to each well for 1 hour. Cells were washed twice with PBS, and then different concentration gradients of thalidomide (0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM) were added. After culturing for 24 hours, cells were harvested, and p72 protein expression was determined by immunoblotting. The results are shown below. Figure 3 As shown. Image J analysis of protein band gray values ​​yielded the p72 protein gray value / internal control band gray value for each lane. The inhibition rate of thalidomide against ASFV at various concentrations was calculated using the formula: 1 - [(target band gray value in the experimental group / internal control band gray value in the experimental group) / (target band gray value in the control group / internal control band gray value in the control group)]. Based on the inhibition rate, the half-maximal inhibitory concentration (IC50) of thalidomide against ASFV was fitted. 50 .Depend on Figure 4 It was learned that 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.

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

[0036] Vero cells (African green monkey kidney cells) were seeded in 6-well plates and cultured for 6 to 8 hours. The cells were then infected with 0.1 MOI PRRSV. After 1 hour of adsorption, the washings were 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 infection, and the expression of the viral virulence protein M was detected by immunoblotting. Figure 5The results showed that thalidomide significantly inhibited the expression of PRRSV virulence protein M in a dose-dependent manner.

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

[0038] PK15 cells (pig kidney cells) were seeded in 24-well plates. When the cells grew to about 80%, they were infected with PCV2 at 0.1 MOI. After 2 hours of adsorption, the washing solution was discarded. 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. The copy number of the PCV2 cap gene was detected by qPCR 48 hours after viral 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 The results showed that, compared with the infection group, the copy number of the Cap gene in each group of thalidomide 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 seeded in 96-well plates. When the cells reached approximately 80% cell growth, they were infected with 0.1 MOI PEDV. After 2 hours, the washings were discarded, and different concentrations of thalidomide (0 μM, 2.5 μM, 5 μM, 10 μM) were added to the cells. 24 hours after infection, the culture medium was discarded, and CCK-8 was added. Incubation continued for 2 hours. OD values ​​were measured at 450 nm using a microplate reader, and cell viability was calculated. Results ( Figure 7 This indicates that thalidomide can significantly inhibit PEDV proliferation in a dose-dependent manner.

Claims

1. The application of thalidomide as the sole active ingredient in the preparation of antiviral drugs, wherein the chemical structural formula of thalidomide is shown in Formula I: Formula I; the virus is African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, or porcine epidemic diarrhea virus.

2. The application according to claim 1, characterized in that, The antiviral drug is composed of the active ingredient thalidomide or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

3. The application according to claim 2, characterized in that, The mass percentage of thalidomide or its pharmaceutically acceptable salt in the antiviral drug is 0.1%-99.9%.

4. The application according to claim 2, characterized in that, The antiviral drug also contains one or more other antiviral drugs.

5. The application according to claim 4, characterized in that, The other antiviral drugs are compounds with antiviral activity and / or extracts of traditional Chinese medicine, and / or compositions of traditional Chinese medicine with antiviral effects.

6. The use of thalidomide as the sole active ingredient in the preparation of viral infection inhibitors and / or in the preparation of pharmaceutical formulations for the prevention and / or treatment of viral infectious diseases, characterized in that, The virus in question is African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, or porcine epidemic diarrhea virus.

7. The application according to claim 6, characterized in that, The drug preparation is administered orally or by injection.

8. The application according to claim 7, characterized in that, The pharmaceutical preparations include tablets, capsules, granules, gels, dispersants, oral liquids, injections, sprays, and drops.

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