Use of scopolamine as an african swine fever virus inhibitor

Abstract

The application belongs to the technical field of veterinary drugs and antiviral technology, and particularly relates to application of hyoscyamine as an African swine fever virus inhibitor. Through in-vitro cytotoxicity test, fluorescent quantitative PCR, half red blood cell adsorption test and other methods, it is confirmed that hyoscyamine has a significant inhibitory effect on African swine fever virus (ASFV) in a concentration range of 0.625 μM to 10 μM, and the effect is in a dose-dependent manner; the half effective concentration (IC 50 ) of hyoscyamine is 0.5222 μM, the half cytotoxicity concentration (CC 50 ) is 197.3 μM, the selectivity index is high, and the safety is good. Mechanism research shows that hyoscyamine mainly inhibits the nucleic acid replication and protein synthesis stages of viruses, rather than directly killing viruses. The application has the advantages of easy availability of raw materials, low cost, strong antiviral activity, low cytotoxicity and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug and antiviral technology, specifically relating to the application of hyoscyamine as an inhibitor of African swine fever virus. Background Technology

[0002] African swine fever (ASF) is a contagious disease in pigs caused by the African swine fever virus (ASFV). It is characterized by acute, febrile, hemorrhagic, high morbidity and mortality rates. It is a notifiable disease by the World Organisation for Animal Health (OIE), and is classified as a Class A animal pathogen in my country. Currently, there is a lack of widely available and safe vaccines; therefore, strict biosecurity measures and culling are the primary methods of control. Since its introduction to my country in 2018, ASF has caused significant and ongoing economic losses to the pig farming industry. Therefore, the development of anti-ASFV drugs is of great importance.

[0003] Hyoscyamine is an AChR inhibitor. In vitro activity studies: In steady-state kinetic measurements, L-hyoscyamine increased the turnover number of TPase activity from 0.19 min⁻¹ to 2.11 min⁻¹. R-(+)-hyoscyamine caused the [3H]NMS shift to bind to muscarinic acetylcholine receptor subtypes (m1-m5), with pKi values ​​of 8.67, 8.51, 7.46, 8.56, and 8.53, respectively. In CHO cells, hyoscyamine prevented stimulant-induced cAMP production, with an EC50 of 7.8 nM. S-(-)-hyoscyamine enhanced the synthesis of forskolin-stimulated cyclic AMP in the membranes of the mouse heart (atrium and ventricle) by 24%. In vivo activity studies: In conscious rats, L-hyoscyamine (20 mg / kg) prolonged the cycle time of migrating electromyographic complex (MMC) from 17.6 minutes to 29.0 minutes. Hyoscyamine molecular formula: C 17 H 23 NO3, relative molecular mass: 289.37. CAS Registry Number 101-31-5, chemical structure as follows:

[0004] Currently, there is no publicly available research or technology regarding the effects of hyoscyamine on African swine fever virus. Summary of the Invention

[0005] The purpose of this invention is to provide the application of hyoscyamine as an inhibitor of African swine fever virus.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The use of hyoscyamine or its pharmaceutically acceptable salts in the preparation of drugs against African swine fever virus.

[0008] Furthermore, the drug is used to inhibit the replication and / or protein synthesis of African swine fever virus in host cells.

[0009] Furthermore, the drug is a formulation made with hyoscyamine or a pharmaceutically acceptable salt thereof as the active ingredient, and supplemented with veterinary pharmaceutically acceptable excipients or carriers.

[0010] Furthermore, the excipient or carrier is selected from at least one of diluents, adhesives, lubricants, disintegrants, surfactants, preservatives, and antioxidants.

[0011] Furthermore, the formulation is an injection, powder, granule, or tablet.

[0012] Furthermore, the African swine fever virus is genotype II African swine fever virus.

[0013] Furthermore, the host cell is a porcine alveolar macrophage.

[0014] Terminology Explanation

[0015] 1. Pharmaceutically acceptable salts

[0016] The term "pharmaceuticalally acceptable salt" refers to an acid addition salt formed by hyoscyamine with an inorganic or organic acid that is veterinary or pharmaceutically acceptable. This salt retains the biological activity of hyoscyamine and is non-toxic or minimally toxic to the host animal at the administered dose.

[0017] Preferably, the salt includes, but is not limited to:

[0018] Inorganic acid salts: hydrochloride, hydrobromide, sulfate, phosphate, nitrate;

[0019] Organic acid salts: acetate, oxalate, citrate, tartrate, succinate, maleate, fumarate, benzoate, salicylate, lactate, malate;

[0020] Sulfonates: methanesulfonate, benzenesulfonate, p-toluenesulfonate.

[0021] 2. Host cell

[0022] The term "host cell" refers to cells that can infect and support the replication of African swine fever virus (ASFV) in vivo or in vitro. In this invention, host cells specifically refer to porcine macrophages, particularly porcine alveolar macrophages (PAMs). ASFV exhibits strict macrophage tropism, and porcine alveolar macrophages are the primary natural target cells of ASFV in pigs; therefore, PAMs were selected as the model cells in the in vitro efficacy evaluation of this invention.

[0023] 3. Porcine alveolar macrophages (PAM)

[0024] The porcine alveolar macrophages were obtained from bronchoalveolar lavage fluid of healthy pigs and purified through in vitro adhesion. In this embodiment of the invention, PAM cells were cultured in RPMI-1640 medium containing 9% fetal bovine serum in a 37°C, 5% CO2 incubator.

[0025] 4. Veterinary pharmaceutically acceptable excipients or carriers

[0026] The term "veterinary pharmaceutically acceptable excipients or carriers" refers to inactive ingredients routinely used in veterinary drug formulations that are non-toxic or low-toxic to the target animal (pig) and do not affect the antiviral activity of hyoscyamine. Specifically, this includes, but is not limited to:

[0027] Diluents: starch, lactose, microcrystalline cellulose, mannitol, dextrin;

[0028] Adhesives: Hydroxypropyl methylcellulose, polyvinylpyrrolidone, sodium carboxymethyl cellulose;

[0029] Lubricants: magnesium stearate, talc, polyethylene glycol;

[0030] Disintegrants: Sodium croscarmellose, croscarmellose, sodium carboxymethyl starch;

[0031] Surfactants: Tween-80, Span-80, polyoxyethylene castor oil;

[0032] Preservatives: benzyl alcohol, parabens, potassium sorbate;

[0033] Antioxidants: Vitamin C, Vitamin E, Butylated hydroxyanisole (BHA).

[0034] 5. Genotype II African swine fever virus

[0035] The "genotype II African swine fever virus" refers to an ASFV strain classified as genotype II based on the ASFV B646L gene (encoding the p72 protein) sequence. The ASFV used in this embodiment is a genotype II strain, but this does not limit the scope of protection of this invention. This invention is also applicable to antiviral applications of other genotypes (such as genotype I) of ASFV strains.

[0036] 6. Regarding "Dosage Forms"

[0037] The term "injectable" includes injection solutions and lyophilized powder for injection, administered via intravenous, intramuscular, or subcutaneous injection.

[0038] The term "powder" includes mixing powder or drinking water additive powder.

[0039] The term "granules" includes soluble granules or suspended granules.

[0040] The term "tablets" includes regular tablets, dispersible tablets, effervescent tablets, or chewable tablets.

[0041] The preparation methods for the above dosage forms are all conventional pharmaceutical processes known to those skilled in the art.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention reveals for the first time a novel use of hyoscyamine as an inhibitor of African swine fever virus. It has comprehensive advantages such as strong antiviral activity, clear mechanism of action, low cytotoxicity, readily available raw materials, and mature formulation. Hyoscyamine raw materials are widely available, inexpensive, and safe and reliable, and have great potential value in the development of new drugs for the prevention and treatment of African swine fever. It provides a practical and feasible new solution for the clinical prevention and control of African swine fever and has good application prospects. Attached Figure Description

[0044] Figure 1 This is a CCK-8 assay to detect the cytotoxicity of different concentrations of hyoscyamine on PAM cells;

[0045] Figure 2 The effect of hyoscyamine on ASFV proliferation in PAM cells was determined using qPCR.

[0046] Figure 3 Using HAD 50 To determine the effect of hyoscyamine on ASFV titer in PAM cells;

[0047] Figure 4 The effect of hyoscyamine on different stages of ASFV proliferation in PAM cells was determined using qPCR. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these embodiments are commercially available.

[0049] All of the following experiments have obtained biosafety clearance and African swine fever laboratory activity clearance:

[0050] The Biosafety Level 3 Laboratory (ABSL-3) of the China Institute of Veterinary Drug Control has obtained permission from the Ministry of Agriculture and Rural Affairs for research on the African swine fever pathogen and has been registered with the Ministry of Agriculture and Rural Affairs, meeting the requirements of the national biosafety level.

[0051] Unless otherwise specified, all reagents used in the experiments are common commercially available reagents; and all procedures used in the experiments are known in the art unless otherwise specified. The ASFV used in the following examples is genotype II ASFV.

[0052] The following examples first utilize the CCK-8 assay (Cell Counting Kit-8) to detect the toxicity of hyoscyamine to porcine alveolar macrophages (PAM) and determine its safe working concentration. Subsequently, an established ASFV infection model was used to evaluate the protective effect of hyoscyamine against ASFV-infected PAM cells. The evaluation methods primarily included quantitative real-time PCR (qPCR) and half-maximal adsorption saturation (HAD) assays. 50 Experiments, experiments at different stages of action, etc.

[0053] This invention, through in vitro cytotoxicity assays, found that hyoscyamine has low cytotoxicity. The median cytotoxic dose (CC) of hyoscyamine in the in vitro cytotoxicity assay was [data missing]. 50 The value was 197.3 μM.

[0054] This invention demonstrates that hyoscyamine significantly inhibits ASFV through in vitro cell experiments, with the inhibitory effect increasing with increasing drug concentration. The median effective dose (IC50) of hyoscyamine in in vitro cell experiments against ASFV is... 50 The value is 0.5222 μM.

[0055] Example 1: CCK-8 assay for detecting the cytotoxicity of different concentrations of hyoscyamine on PAM cells

[0056] 1.25×10 5PAM cells were seeded per well in 96-well plates and incubated at 37°C for at least 2 hours to allow the cells to adhere. Hyoscyamine stock solution (10000 μM) was serially diluted with 1640 containing 9% serum. The dilutions were 2000 μM, 1000 μM, 500 μM, 250 μM, and 125 μM. Each dilution was repeated in triplicate, with 20 μL per well, plus 80 μL of 1640 containing 9% serum to a total volume of 200 μL. The final concentrations of hyoscyamine were 200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM. A control group (cells only, no hyoscyamine) and a blank group (no cells, no hyoscyamine) were also included. Cells were incubated at 37°C for 72 hours. Discard the supernatant, add 10 μL of CCK-8 (protected from light) and 90 μL of serum-free 1640 to each well, ensuring no air bubbles are present. Incubate at 37°C for 2 hours. Use a microplate reader to measure the OD value. 450 Read the OD value of each well. Calculate the results: Cell viability = (Experimental group - Blank group) / (Control group - Blank group) × 100%.

[0057] from Figure 1 It can be seen that at a concentration of 50 μM, hyoscyamine resulted in a PAM cell survival rate of over 90% and did not affect PAM cell proliferation.

[0058] Example 2: qPCR determination of the effect of hyoscyamine on ASFV proliferation in PAM cells

[0059] 1.25×10 5 PAM cells were seeded per well in a 96-well plate and allowed to adhere for at least 2 hours. Hyoscyamine was serially diluted with 1640 solution containing 9% serum to obtain dilutions of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 0 μM. 10 μL of PAM cell supernatant was discarded. Each dilution was repeated three times, with 20 μL of hyoscyamine added to each well, followed by 90 μL of ASFV (MOI=0.16) to a total volume of 200 μL. The final drug concentrations were 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, and 0 μM. The cells were incubated at 37°C for 72 hours, followed by three freeze-thaw cycles at -80°C. ASFV was collected and nucleic acids were extracted. The copy number of ASFV was detected by qPCR using p72 primers and probes (as shown in Table 1), and the inhibition rate of hyoscyamine against ASFV was calculated.

[0060] Table 1. p72 primer and probe sequences

[0061] The test results are as follows Figure 2 As shown, the hyoscyamine of the present invention has a significant inhibitory effect on ASFV at concentrations ranging from 0.625 μM to 10 μM, and exhibits a dose-dependent effect.

[0062] Example 3: HAD 50 Determining the effect of hyoscyamine on ASFV titers in PAM cells

[0063] 1.25×10 5 PAM cells were seeded per well in a 96-well plate and allowed to adhere for at least 2 hours. Hyoscyamine was serially diluted with 1640 solution containing 9% serum to obtain dilutions of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 0 μM. 10 μL of PAM cell supernatant was discarded. Each dilution was repeated three times, with 20 μL of hyoscyamine added to each well, followed by 90 μL of ASFV (MOI=0.16) to a total volume of 200 μL. The final drug concentrations were 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0 μM. The cells were incubated at 37°C for 72 hours, followed by three freeze-thaw cycles at -80°C to collect ASFV. 1.25 × 10⁻⁶ cells / well 5 PAM was laid per well in a 96-well plate and allowed to adhere for at least 2 hours. ASFV treated with various hyoscyamine concentrations was diluted 10-fold to 10⁻⁶. -8 100 μL of the mixture was added to each well, along with 20 μL of 1% red blood cells. The cells were incubated at 37°C. Red blood cell agglutination was observed daily for 7 consecutive days. The half-maximal adsorbed dose (HAD) of ASFV was calculated. 50 The method used was the Reed-Muench method.

[0064] The test results are as follows Figure 3 As shown, the hyoscyamine of the present invention has a significant inhibitory effect on the viral titer of ASFV infected PAM cells at concentrations of 0.625 μM to 10 μM, and exhibits a clear dose-dependent effect.

[0065] Example 4: qPCR determination of the effect of hyoscyamine on different stages of ASFV proliferation in PAM cells

[0066] 1.25×10 5PAM cells were seeded per well in a 96-well plate and allowed to adhere for at least 2 hours. Hyoscyamine was serially diluted with 1640 M serum containing 9% serum to 50 μM, 25 μM, and 0 μM. ASFV (MOI = 0.16) was used. Each dilution was performed in triplicate. ① Direct killing: To investigate the direct killing effect of the drug, 20 μL of 50 μM, 25 μM, and 0 μM hyoscyamine were added to 90 μL of ASFV and incubated at 37°C for 1 hour. The mixture was then diluted 20-fold to a concentration where hyoscyamine had no inhibitory effect. 10 μL of PAM cell supernatant was discarded, and 110 μL of the dilution mixture was added to the PAM cells, for a total volume of 200 μL. The cells were incubated for 72 hours. ② Pre: Used to investigate the blocking effect of the drug or its influence on host factors. Add 20 μL of 50 μM, 25 μM, and 0 μM hyoscyamine to PAM cells, then bring the volume to 200 μL with 1640 containing 9% serum. Incubate at 4°C for 2 hours. Wash three times with 1640. Add 90 μL of ASFV and 110 μL of 1640 containing 9% serum to PAM cells, and incubate at 4°C for 2 hours. Wash three times with 1640. Add 200 μL of 1640 containing 9% serum to PAM cells, and incubate at 37°C for 72 hours. ③During: Discard 10 μL of PAM cell supernatant, add 20 μL of 50 μM, 25 μM, and 0 μM hyoscyamine and 90 μL of ASFV to PAM cells, and incubate at 37°C for 2 hours. Wash three times with 1640. Add 200 μL of 1640 containing 9% serum to PAM cells, and incubate at 37°C for 72 hours. ④Post-1: Investigating the inhibitory effect of the drug on viral nucleic acid replication and protein synthesis. 90 μL of ASFV was added to PAM cells, then the volume was increased to 200 μL with 1640 diluted with 9% serum. The cells were incubated at 4°C for 2 hours, followed by three washes with 1640. 20 μL of 50 μM, 25 μM, and 0 μM hyoscyamine were added to PAM cells, then the volume was increased to 200 μL with 1640 diluted with 9% serum. The cells were incubated in a cell culture incubator for 72 hours. ⑤Post-2: Investigating the inhibitory effect of the drug on viral nucleic acid replication and protein synthesis. 90 μL of ASFV was added to PAM cells, then the volume was increased to 200 μL with 1640 diluted with 9% serum. The cells were incubated in a cell culture incubator at 37°C for 2 hours, followed by three washes with 1640. 20 μL of 50 μM, 25 μM, and 0 μM hyoscyamine were added to PAM cells, and then 200 μL of 1640 solution with 9% serum was added to make up the volume. Cells were incubated for 72 h. After 72 h, cells incubated at different stages were subjected to three freeze-thaw cycles at -80°C to collect ASFV and extract nucleic acids. The copy number of ASFV was detected using qPCR with p72 primers and probes, and the differences in ASFV copy number at different stages were compared.

[0067] The test results are as follows Figure 4 As shown, the hyoscyamine of the present invention inhibits the nucleic acid replication and protein synthesis stages of the virus, but does not directly kill ASFV.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of hyoscyamine or its pharmaceutically acceptable salts in the preparation of drugs against African swine fever virus.

2. The application according to claim 1, characterized in that, The drug is used to inhibit the replication and / or protein synthesis of African swine fever virus in host cells.

3. The application according to claim 1 or 2, characterized in that, The drug is a formulation made with hyoscyamine or its pharmaceutically acceptable salt as the active ingredient, and supplemented with veterinary pharmaceutically acceptable excipients or carriers.

4. The application according to claim 3, characterized in that, The excipient or carrier is selected from at least one of diluents, adhesives, lubricants, disintegrants, surfactants, preservatives, and antioxidants.

5. The application according to claim 3, characterized in that, The preparation is an injection, powder, granule or tablet.

6. The application according to claim 1, characterized in that, The African swine fever virus mentioned is genotype II African swine fever virus.

7. The application according to claim 1, characterized in that, The host cells are porcine alveolar macrophages.

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