Application of verapamil hydrochloride in preparation of anti-PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) medicine

Abstract

The invention relates to the technical field of western medicine production, in particular to application of verapamil hydrochloride in preparation of anti-PRRSV (porcine reproductive and respiratory syndrome virus) drugs, including application of verapamil hydrochloride in preparation of drugs for inhibiting PRRSV (porcine reproductive and respiratory syndrome virus) infection. The invention further discloses application of the verapamil hydrochloride in preparation of drugs for preventing and treating symptoms caused by PRRSV infection and a pharmaceutical composition containing the verapamil hydrochloride. Through related pharmacological experiments and virus replication experiment researches of the verapamil hydrochloride, the invention reveals that the verapamil hydrochloride can inhibit the replication of a plurality of PRRSV-1 and PRRSV-2 strains in a broad spectrum, can be used as an effective component of a medicine for preventing and treating PRRSV infection, and provides valuable theoretical basis and reference for clinical medication of the PRRSV.

Description

Technical Field

[0001] The invention relates to the technical field of western medicine production, in particular to application of verapamil hydrochloride in the preparation of an anti-PRRSV drug. Background Art

[0002] Verapamil hydrochloride, English name: Verapamil Hydrochloride, CAS number: 152-11-4; molecular formula: C27H39ClN2O4; molecular weight: 491.06. The molecular structure is as follows Figure 1 As shown, it is an L-calcium channel blocker (calcium antagonist), which can selectively dilate coronary arteries, increase coronary flow, inhibit myocardial excitability and atrioventricular conduction, and is mainly used to treat paroxysmal supraventricular tachycardia; hypertension, angina pectoris, arrhythmia, cerebrovascular disease, finger vasospasm, abdominal pain, esophageal achalasia, migraine, pulmonary hypertension, etc. At present, there are no reports on the use of verapamil hydrochloride for the prevention and control of PRRSV at home and abroad.

[0003] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which seriously endangers global pig production. It is commonly known as "blue ear disease". This virus infection mainly leads to reproductive disorders in sows and respiratory diseases in growing pigs, as well as secondary infections in diseased pig herds, causing huge economic losses to pig farming companies.

[0004] PRRSV is a member of the order Alphavirales, family Arteriviridae, and genus Arterivirus. PRRSV includes two subtypes: PRRSV-1 and PRRSV-2. PRRSV-1 is prevalent in Europe and Russia, while PRRSV-2 is prevalent in North America and Asia. In recent years, the new NADC30-like strain has spread globally and gradually become one of the dominant strains, causing a huge impact on the pig farming industry. Given that the replication mechanism of PRRSV is not fully understood, there is currently no effective vaccine or drug for the prevention and control of PRRSV, and an economical and efficient anti-PRRSV drug is urgently needed. Summary of the invention

[0005] The purpose of the present invention is to provide an application of verapamil hydrochloride in the preparation of an anti-PRRSV drug, so as to solve the problem that there is no report on the use of verapamil hydrochloride for preventing and controlling PRRSV at home and abroad.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention discloses an application of verapamil hydrochloride in preparing an anti-PRRSV drug, including the application of verapamil hydrochloride in preparing a drug for inhibiting PRRSV infection, the application of verapamil hydrochloride in preparing a drug for preventing and treating symptoms caused by PRRSV infection, and a pharmaceutical composition containing verapamil hydrochloride.

[0008] Preferably, the maximum safe mass concentration of the verapamil hydrochloride in PRRSV susceptible cells is 80 μM (μmol / L), and the PRRSV susceptible cells include MARC-145 and PAM cells.

[0009] Preferably, the verapamil hydrochloride has an inhibitory effect on a variety of PRRSV strains, including PRRSV-1 strains and PRRSV-2 strains, the PRRSV-1 strains include GZ11-G1 and P073-3, and the PRRSV-2 strains include highly pathogenic strains SD16, JXA1, classic strains VR-2332, CH-1a and popular strains NADC30-like.

[0010] Preferably, the verapamil hydrochloride exerts a blocking effect at multiple stages of PRRSV replication, including the early infection stage, the viral gene transcription and protein synthesis stage, and the viral release stage.

[0011] Preferably, the verapamil hydrochloride is used to reduce viremia caused by PRRSV infection and reduce the number of viral copies in the blood of pigs.

[0012] Preferably, the verapamil hydrochloride is used to alleviate lung damage in weaned piglets caused by PRRSV infection.

[0013] Preferably, the verapamil hydrochloride is applied to pigs via oral or injection administration for the prevention or treatment of PRRSV infection, wherein the oral dosage is 8 mg / kg, which is started 1 day before virus infection and continuously administered on the 1st, 3rd, 5th and 8th days after infection.

[0014] Preferably, the symptoms caused by PRRSV infection include fever, anorexia, reproductive disorders in pregnant sows, and pneumonia commonly occurring in weaned piglets.

[0015] Preferably, the pharmaceutical composition is used to prepare a drug for preventing PRRSV infection, and the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier or excipient, the carrier or excipient being selected from one or more of a filler, a disintegrant or a solvent, the filler comprising microcrystalline cellulose, starch, lactose, and calcium hydrogen phosphate, the disintegrant comprising cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, and corn starch, and the solvent comprising water, ethanol, and propylene glycol.

[0016] Preferably, the pharmaceutical composition is formulated for oral administration, injection or topical application, including tablets, capsules, or injection solutions.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Broad-spectrum anti-PRRSV activity

[0019] Multi-strain inhibition: Verapamil hydrochloride has a significant inhibitory effect on a variety of PRRSV strains (including PRRSV-1 and PRRSV-2), indicating that it has a wide range of antiviral activity.

[0020] (2) Multi-stage inhibition

[0021] Comprehensive blocking: Verapamil hydrochloride can block multiple stages of PRRSV replication (including virus adsorption, entry, replication and release), effectively interfering with the virus life cycle.

[0022] (3) High security

[0023] Clinical safety: As a known cardiovascular drug, the safety of verapamil hydrochloride has been widely verified, reducing the risk and cost of new drug development.

[0024] Low cytotoxicity: Verapamil hydrochloride has no obvious toxicity to PRRSV-susceptible cells (such as MARC-145 and PAM) at the maximum safe mass concentration of 80 μM (μmol / L), ensuring its safety in animals.

[0025] (4) Significantly reduce viremia

[0026] Reduce viral load: Verapamil hydrochloride can significantly reduce the viremia caused by PRRSV infection and reduce the transmission and spread of the virus in the body.

[0027] (5) Reduce lung damage

[0028] Protect lung tissue: Verapamil hydrochloride significantly reduces lung damage in weaned piglets caused by PRRSV infection and improves lung pathology.

[0029] (6) Improve clinical symptoms

[0030] Relieve symptoms: Verapamil hydrochloride can effectively relieve clinical symptoms caused by PRRSV infection, such as fever, anorexia, reproductive disorders in pregnant sows and pneumonia commonly seen in weaned piglets, and improve the quality of life and production performance of animals.

[0031] (7) Easy to prepare and use

[0032] Multiple dosage forms: Verapamil hydrochloride can be formulated into multiple dosage forms, such as tablets, capsules, or injections, to facilitate use in different scenarios;

[0033] Good stability: Verapamil hydrochloride has good chemical stability and physical stability, and is easy to store and transport.

[0034] (8) Economical and efficient

[0035] Cost-effectiveness: As a known drug, verapamil hydrochloride has relatively low production and preparation costs and has high economic benefits;

[0036] Reduce losses: By effectively controlling PRRSV infection, reduce economic losses caused by virus transmission and improve the overall benefits of the pig farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they provide further detailed explanations but do not constitute a limitation of the present invention.

[0038] Figure 1 This is the molecular formula diagram of verapamil hydrochloride;

[0039] Figure 2 The toxicity test results of verapamil hydrochloride on PRRSV susceptible cells in the present invention;

[0040] Figure 3 The results of the different doses of verapamil hydrochloride in the present invention inhibiting the replication of PRRSV SD16;

[0041] Figure 4 The results show that the verapamil hydrochloride in the present invention has an inhibitory effect on multiple PRRSV-1 and PRRSV-2 strains;

[0042] Figure 5 It is a schematic diagram of the experimental setup of different treatment methods of verapamil hydrochloride and PRRSV infection in the present invention;

[0043] Figure 6 Verapamil hydrochloride in the present invention exerts an inhibitory effect at multiple stages of the PRRSV replication cycle;

[0044] Figure 7 Verapamil hydrochloride in the present invention cannot directly inactivate PRRSV;

[0045] Figure 8 Verapamil hydrochloride in the present invention reduces the number of viral copies in the blood of PRRSV-infected piglets;

[0046] Fig. 9The results show that the verapamil hydrochloride in the present invention significantly reduces the lung damage of piglets caused by PRRSV infection (HE staining). DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] (I) Toxicity test of verapamil hydrochloride on PRRSV susceptible cells

[0049] MARC-145, and PAM cells were seeded in 96-well plates (1×10 4 / well), when it grows to about 80%, add 100 μL of culture medium containing different concentrations of verapamil hydrochloride (0 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM) to each well. At the same time, set up DMSO negative control and blank control, with 4 replicate wells in each group. After incubating in a 37°C, 5% CO2 incubator for 24 hours, add 10 μL of CCK-8 solution to each well and continue to culture for 2 hours. Use an enzyme reader to read the absorbance value at OD450 nm, and use the following formula to calculate the effect of verapamil hydrochloride on the activity of MARC-145 and PAM cells, and evaluate the maximum safe mass concentration of verapamil hydrochloride.

[0050] Cell viability (%) = [A1-A2] / [A3-A2] × 100%

[0051] A1 test group: absorbance values ​​of wells with cells, CCK-8 solution and drug solution

[0052] A2 Blank control: absorbance value of the well with culture medium and CCK-8 solution but no cells

[0053] A3 Negative control: absorbance value of the well with cells, CCK-8 solution but no drug solution

[0054] The results are as follows Figure 2 As shown, the concentration of verapamil hydrochloride (less than 160 μM) has no significant effect on the viability of susceptible cells, and 80 μM is the maximum safe mass concentration of verapamil hydrochloride.

[0055] (II) Inhibitory effect of verapamil hydrochloride on PRRSV replication

[0056] Example 1. Verapamil hydrochloride inhibits the replication of PRRSV SD16 in multiple susceptible cell lines

[0057] MARC-145 and PAM cells were inoculated in 12-well cell plates, respectively. When the cell confluence reached 80%, different concentrations of verapamil hydrochloride (5μM, 10μM, 20μM, 40μM, and 80μM) were added and cultured for 2h. After 0.1MOI PRRSVSD161h, the virus solution was discarded and replaced with 3% FBSDMEM or RPMI 1640 containing the corresponding concentration of drugs. The DMSO-treated group and the normal cell untreated group were set up as the control group. The cells were collected 24h after infection, and the total cell RNA was extracted with the help of TRIzol reagent and reverse transcribed into cDNA. The changes in the transcription level of PRRSV ORF7 gene were determined by qPCR. At the same time, the cells were collected and the changes in the expression level of PRRSV N protein were analyzed by Western blot method. The results are shown in Figure 3 As shown, verapamil hydrochloride significantly reduced the expression of PRRSV ORF7 at the transcriptional level and protein level in a dose-dependent manner.

[0058] Example 2. Verapamil hydrochloride has a broad-spectrum anti-PRRSV infection effect

[0059] MARC-145 cells were inoculated into 12-well culture plates. When the cells were 80% confluent, they were pretreated with verapamil hydrochloride (80 μM / mL) for 2 h, and then infected with 0.1 MOI PRRSV-1 strains (GZ11-G1 and P073-3) or PRRSV-2 strains (SD16, JXA1, VR-2332, CH-1a, and NADC30-like) for 1 h. The liquid was discarded and replaced with 3% FBSDMEM containing 80 μM verapamil hydrochloride. The DMSO-treated group was used as a negative control. Cell supernatants and cells were collected 24 h after virus infection and TCID 50 The supernatant progeny virus titer and PRRSV N protein expression were detected by Western blot. The results showed that verapamil hydrochloride could significantly reduce the infection of multiple PRRSV-1 and PRRSV-2 strains in susceptible cells ( Figure 4 ).

[0060] Example 3. Experiment to determine the effect of verapamil hydrochloride on multiple stages of the PRRSV replication cycle

[0061] Refer to the instructions Figure 5 To further explore the replication stage of PRRSV at which verapamil hydrochloride exerts its antiviral effect, MARC-145 cells were inoculated in 12-well plates (1×10 5 / well), and when the cells grew to 70%, the control group, the verapamil hydrochloride pretreatment group (Pre-treatment), the verapamil hydrochloride and PRRSV co-treatment group (Co-treatment), the verapamil hydrochloride group incubated with PRRSV after infection (Post-treatment) and the verapamil hydrochloride full-stage treatment group (All stages-treatment) were set up respectively. The dose of PRRSV SD16 inoculated was 0.1MOI. After 24 hours of virus infection, the cells were collected and the replication of PRRSV was determined by qPCR and Western blot. The results are shown in Figure 6 As shown, the expression levels of viral ORF7 gene at the transcriptional level and protein level in the verapamil hydrochloride drug Pre, Co, and Post treatment groups were significantly lower than those in the DMSO treatment control group, suggesting that verapamil hydrochloride exerts an antiviral effect in multiple replication stages of PRRSV infection.

[0062] Example 4. Determination of whether verapamil hydrochloride has direct inactivation activity for PRRSV

[0063] In order to detect whether verapamil hydrochloride directly affects PRRSV activity, MARC-145 cells were seeded in 12-well plates (1×10 5 / well), after overnight culture. Verapamil hydrochloride (80μM) was incubated with different doses of PRRSV SD16 (0.1MOI, 0.5MOI and 1MOI) at 37℃ for 2h. 3% FBSDMEM was replaced 1h after inoculation of cells. Cell supernatants were collected 24h after virus infection, and the number of viral copies was detected by qPCR. The results showed that verapamil hydrochloride could not directly inactivate PRRSV ( Figure 7 ).

[0064] Example 5. Verapamil hydrochloride anti-PRRSV infection test in pigs

[0065] Nine blue ear disease double negative weaned piglets were purchased and randomly divided into three groups (n=3 / group), namely group NO.1 (Mock control group), group NO.2 (DMSO treatment group), and group NO.3 (verapamil hydrochloride treatment group). The pigs in groups NO.2 and NO.3 were challenged with PRRSV SD163×10 5 TCID 50 Verapamil hydrochloride (8 mg / kg) was taken orally 1 day before the virus infection. The drug was administered on days 1, 3, 5, and 8 after virus infection. Clinical symptoms were observed every day. Blood was collected from different groups of pigs 7 days after virus infection, and the number of virus copies was detected by qPCR ( Figure 8The pigs were euthanized on the 28th day after virus infection, and the lung tissues were fixed with formaldehyde and stained with HE to evaluate the protective effect of verapamil hydrochloride on lung damage induced by PRRSV infection in piglets ( Fig. 9 ).

[0066] In summary, the present invention is the first to systematically evaluate the inhibitory effect of verapamil hydrochloride on PRRSV infection in vitro and in vivo, and confirms that it has inhibitory effects on multiple PRRSV-1 and PRRSV-2 strains. In-depth analysis found that verapamil hydrochloride can affect multiple replication stages of PRRSV, thereby proving that verapamil hydrochloride can be used to prepare drugs for preventing and treating PRRSV infection.

[0067] The application of the verapamil hydrochloride of the present invention in the preparation of anti-PRRSV drugs has the following advantages:

[0068] (1) Broad-spectrum antiviral activity

[0069] Multi-strain inhibition: Verapamil hydrochloride has a significant inhibitory effect on a variety of PRRSV strains (including PRRSV-1 and PRRSV-2), indicating that it has a wide range of antiviral activity.

[0070] Dose dependence: The antiviral effect of verapamil hydrochloride increases with increasing dose, showing good dose dependence.

[0071] (2) Multi-stage inhibition

[0072] Comprehensive blocking: Verapamil hydrochloride can block multiple stages of PRRSV replication (including virus adsorption, entry, replication and release), effectively inhibiting the life cycle of the virus.

[0073] (3) High security

[0074] Low cytotoxicity: Verapamil hydrochloride has no obvious toxicity to PRRSV-susceptible cells (such as MARC-145 and PAM) at the maximum safe mass concentration of 80 μM (μmol / L), ensuring its safety in animals.

[0075] Clinical safety: As a known cardiovascular drug, the safety of verapamil hydrochloride has been widely verified, reducing the risk and cost of new drug development.

[0076] (4) Significantly reduce viremia

[0077] Reduce viral load: Verapamil hydrochloride can significantly reduce the viremia caused by PRRSV infection and reduce the transmission and spread of the virus in the body.

[0078] (5) Reduce lung damage

[0079] Protect lung tissue: Verapamil hydrochloride significantly reduces lung damage in weaned piglets caused by PRRSV infection, improves lung pathology, and increases the survival rate and health of animals.

[0080] (6) Improve clinical symptoms

[0081] Relieve symptoms: Verapamil hydrochloride can effectively relieve clinical symptoms caused by PRRSV infection, such as fever, anorexia, reproductive disorders in pregnant sows and pneumonia commonly seen in weaned piglets, and improve the quality of life and production performance of animals.

[0082] (7) Easy to prepare and use

[0083] Multiple dosage forms: Verapamil hydrochloride can be formulated into a variety of dosage forms, such as tablets, capsules, or injections, to facilitate use in different scenarios.

[0084] Good stability: Verapamil hydrochloride has good chemical stability and physical stability, and is easy to store and transport.

[0085] (8) Economical and efficient

[0086] Cost-effectiveness: As a known drug, verapamil hydrochloride has relatively low production and preparation costs and high economic benefits. By effectively controlling PRRSV infection, the economic losses caused by virus transmission can be reduced and the overall benefits of the pig farming industry can be improved.

[0087] Summarize:

[0088] The present invention systematically reveals for the first time the multiple beneficial effects of Verapamil hydrochloride in the fight against PRRSV infection, including broad-spectrum antiviral activity, multi-stage inhibitory effects, high safety, significant reduction of viremia, alleviation of lung damage, improvement of clinical symptoms, ease of preparation and use, and economic efficiency. Through the pharmacological experiments and virus replication experiments conducted on Verapamil hydrochloride, the present invention reveals that Verapamil hydrochloride can broadly inhibit the replication of multiple PRRSV-1 and PRRSV-2 strains, and can be used as an effective ingredient for the prevention and treatment of PRRSV infection, providing a valuable theoretical basis and reference for the clinical use of PRRSV.

[0089] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An application of verapamil hydrochloride in the preparation of an anti-PRRSV drug, characterized in that: The invention comprises the use of verapamil hydrochloride in preparing a medicine for inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection, the use of verapamil hydrochloride in preparing a medicine for preventing and treating symptoms caused by PRRSV infection, and a pharmaceutical composition containing verapamil hydrochloride.

2. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The maximum safe mass concentration of the verapamil hydrochloride in PRRSV susceptible cells is 80 μM (μmol / L), and the PRRSV susceptible cells include MARC-145 cells and PAM cells.

3. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The verapamil hydrochloride has an inhibitory effect on a variety of PRRSV strains, including PRRSV-1 strains and PRRSV-2 strains. The PRRSV-1 strains include GZ11-G1 and P073-3, and the PRRSV-2 strains include highly pathogenic strains SD16, JXA1, classic strains VR-2332, CH-1a and popular strains NADC30-like.

4. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The verapamil hydrochloride exerts a blocking effect at multiple stages of PRRSV replication, including the early infection stage, the viral gene transcription and protein synthesis stage, and the replication release stage.

5. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The verapamil hydrochloride is used for reducing viremia caused by PRRSV infection and reducing the number of virus copies in the blood of pigs.

6. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The verapamil hydrochloride is used for alleviating lung damage in weaned piglets caused by PRRSV infection.

7. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The verapamil hydrochloride is applied to pigs via oral or injection administration to prevent or treat PRRSV infection, wherein the oral dosage is 8 mg / kg, which is started one day before virus infection and is continuously administered on the 1st, 3rd, 5th and 8th days after infection.

8. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The symptoms caused by PRRSV infection include fever, anorexia, reproductive failure in pregnant sows, and pneumonia commonly occurring in weaned piglets.

9. The use of verapamil hydrochloride according to claim 1 in the preparation of anti-PRRSV drugs, characterized in that: The pharmaceutical composition is used to prepare a drug for preventing PRRSV infection. The pharmaceutical composition also includes at least one pharmaceutically acceptable carrier or excipient, which is selected from one or more of a filler, a disintegrant or a solvent. The filler includes microcrystalline cellulose, starch, lactose, and calcium hydrogen phosphate. The disintegrant includes cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, and corn starch. The solvent is water, ethanol, and propylene glycol.

10. The use of verapamil hydrochloride according to claim 9 in the preparation of anti-PRRSV drugs, characterized in that: The pharmaceutical composition is formulated for oral administration, injection or topical application, including tablets, capsules, or injection solutions.

Images