Application of dibenzoylmethane to inhibition of coronavirus and treatment of related diseases caused by coronavirus

Abstract

The invention relates to application of dibenzoylmethane to inhibition of coronavirus and treatment of related diseases caused by the coronavirus. In the invention, an in-vitro cell experiment shows that the dibenzoylmethane can inhibit the proliferation of SARS-CoV-2, HCoV-229E and HCoV-OC43 in cells, and a very good inhibition effect can be achieved at a low dosage; in-vivo mouse experiments show that dibenzoylmethane can effectively inhibit the load of HCoV-OC43 in tissues and organs such as brain, spine, lung, kidney and the like, and has a good treatment effect on nerve and respiratory system diseases caused by HCoV-OC43 infection. The dibenzoylmethane has the advantages of good curative effect, high safety and the like, so that the dibenzoylmethane can be developed into a novel coronavirus inhibitor for treating diseases caused by coronavirus infection.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy, and particularly relates to use of dibenzoylmethane in inhibiting coronavirus or in preparing medicines for treating or preventing related diseases caused by coronavirus. Background Art

[0002] Coronavirus is a class of enveloped, single-stranded, positive-sense RNA viruses belonging to the Coronaviridae family. The Coronaviridae family is divided into four genera, including alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. Among them, alphacoronavirus and betacoronavirus mainly infect mammals, while gammacoronavirus and deltacoronavirus mainly infect birds. Coronavirus has a high degree of genetic diversity and variability, which makes it extremely capable of cross-species transmission and adaptation to new hosts.

[0004] Currently, effective treatments for coronavirus infections are still limited, especially in the face of the virus's easy mutation characteristics. The effectiveness of existing antiviral drugs and vaccines may weaken or even fail as the virus mutates. For example, multiple mutations of SARS-CoV-2 have produced a variety of variants that show significant differences in transmissibility, pathogenicity, and resistance to vaccines. Although current vaccines and treatments are effective to a certain extent, it is still a huge challenge to deal with possible new coronavirus mutations in the future.

[0005] In this context, the development of broad-spectrum antiviral drugs is particularly important. Broad-spectrum antiviral drugs can not only inhibit a variety of known coronaviruses, but also have the potential to fight against coronavirus variants that may appear in the future. The main advantage of broad-spectrum antiviral drugs is that they do not rely on the specific gene sequence or protein structure of the virus, but instead inhibit a variety of coronaviruses by interfering with the key biological processes of the virus or targeting the relevant mechanisms of the host cell. This broad-spectrum characteristic makes this type of drug more adaptable and effective in dealing with coronavirus mutations and new outbreaks.

[0006] The development of broad-spectrum anti-coronavirus drugs is not only of great significance for the current and future prevention and control of coronaviruses, but also provides a scientific basis and technical reserves for responding to other potential viral pandemics. In the context of globalization, cross-border virus transmission has become the norm. The development and reserve of broad-spectrum antiviral drugs will provide a solid guarantee for global public health security. At the same time, progress in this field will also promote the overall level of antiviral drug research and development, and provide more possibilities for humans to fight against various emerging and re-emerging viral diseases.

[0007] In summary, as the threat of coronavirus to human health becomes increasingly serious, the development of new antiviral compounds with broad inhibitory effects to meet the challenges of multiple coronaviruses and their variants has become a key direction of current drug research and development. Summary of the invention

[0008] The inventors of the present invention unexpectedly discovered that dibenzoylmethane has antiviral activity against a variety of coronaviruses and can be used for the treatment or prevention of related diseases caused by a variety of coronavirus infections, thereby completing the present invention.

[0009] The present invention provides use of dibenzoylmethane, a solvate or hydrate thereof, or a pharmaceutical composition comprising any of the foregoing in preparing a drug, wherein the drug is selected from:

[0010] i. Drugs used to prevent or treat diseases caused by coronavirus;

[0011] ii. Drugs used to inhibit the replication or reproduction of coronaviruses in mammalian cells, tissues or organs.

[0012] In a specific embodiment, the coronavirus is selected from:

[0013] (1) Coronaviruses that infect humans: severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus MERS-CoV (MERS-CoV), human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1);

[0014] (2) Coronaviruses that infect animals: porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIFV).

[0015] In a specific embodiment, the coronavirus is HCoV-OC43, SARS-CoV-2 Omicron variant BA.5 or HCoV-229E.

[0016] In a specific embodiment, the coronavirus is HCoV-OC43.

[0017] In a specific embodiment, the coronavirus-related disease is selected from respiratory diseases, encephalitis, encephalomyelitis, sepsis, septic shock, arrhythmia, myocarditis, liver function damage and renal function damage caused by coronavirus; porcine epidemic diarrhea caused by porcine epidemic diarrhea virus; and feline infectious peritonitis caused by feline infectious peritonitis virus.

[0018] In a specific embodiment, the respiratory disease caused by the coronavirus is selected from simple infection, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome caused by coronavirus.

[0019] In a specific embodiment, the simple infection caused by the coronavirus includes fever, cough and sore throat.

[0020] In a specific embodiment, in ii, the mammal includes bovines, equines, ovines, porcines, canines, felines, rodents, primates, wherein the preferred mammal is a human, a cat, a dog or a pig.

[0021] In a specific embodiment, in ii., inhibiting the coronavirus comprises reducing the nucleic acid load of the coronavirus in a cell culture, tissue or organ of a mammalian cell.

[0022] In a specific embodiment, the pharmaceutical composition comprises a therapeutically effective amount of dibenzoylmethane, a solvate or a hydrate thereof, and a pharmaceutically acceptable excipient.

[0023] In a specific embodiment, the dosage form of the pharmaceutical composition includes: tablets, capsules, aqueous solutions or suspensions or micronized suspensions or solutions, suppositories, ointments, sprays, lotions or creams, rectal suppository preparations or enemas, local transdermal patches, sterile injections or oil suspensions, but are not limited thereto.

[0024] In the present application, the pharmaceutical composition can be prepared into a desired dosage form according to a suitable administration method of the drug by a method known in the art.

[0025] Beneficial Effects

[0026] The in vitro cell experiments of the present invention show that dibenzoylmethane can inhibit the proliferation of SARS-CoV-2, HCoV-229E and HCoV-OC43 in cells, and can achieve good inhibitory effects at low doses.

[0027] The in vivo mouse experiments of the present invention show that dibenzoylmethane can effectively inhibit the load of HCoV-OC43 in tissues and organs such as the brain, spine, lungs and kidneys, and has a good therapeutic effect on neurological and respiratory diseases caused by HCoV-OC43 infection.

[0028] Therefore, dibenzoylmethane can be used as a coronavirus inhibitor for treating diseases caused by coronavirus infection, and it has the advantages of good efficacy and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The results of the in vitro inhibitory activity test of dibenzoylmethane at different concentrations against HCoV-OC43 in Example 1 are shown.

[0030] Figure 2 The results of the in vitro inhibitory activity test of dibenzoylmethane at different concentrations against HCoV-229E in Example 2 are shown.

[0031] Figure 3 The results of the in vitro inhibitory activity test of dibenzoylmethane at different concentrations against the SARS-CoV-2 Omicron variant (BA.5) in Example 3 are shown.

[0032] Figure 4 The results of the in vivo inhibitory activity test of dibenzoylmethane at different concentrations against HCoV-OC43 in the HCoV-OC43 infected mouse model in Example 4 are shown.

[0033] Figure 5 The results show the efficacy of different concentrations of dibenzoylmethane in Example 4 on organ damage caused by HCoV-OC43 in the HCoV-OC43 infected mouse model.

[0034] Figure 6 The cytotoxicity test results of dibenzoylmethane at different concentrations in Example 5 on five different cell lines including Huh-7 are shown. DETAILED DESCRIPTION

[0035] the term:

[0036] In the present invention, dibenzoylmethane (DBM for short) has the structure shown below:

[0037]

[0038] In the present invention, unless otherwise explicitly stated, the terms used in the present invention have the meanings defined below. Terms not explicitly defined in the present invention have the general meanings generally understood by those skilled in the art.

[0039] As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, the like, and combinations thereof, which are well known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, it may be considered for use in the therapeutic or pharmaceutical composition.

[0040] As used herein, "inhibit" refers to a reduction or suppression of a particular condition, symptom or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0041] As used herein, the term "treating" any disease or condition refers in one embodiment to ameliorating the disease or condition (i.e., arresting or slowing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" refers to improving at least one physical parameter, which may not be perceived by the patient. In another embodiment, "treating" refers to modulating the disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both.

[0042] As used herein, "prevention" refers to the administration of one or more pharmaceutical substances, particularly compounds of the present invention and / or pharmaceutically acceptable salts thereof, to an individual with a predisposition to the disease in question, in order to prevent the individual from developing the disease.

[0043] As used herein, "subject" refers to mammals, such as primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a preferred embodiment, the subject is a human, pig, or cat. In a preferred embodiment, the subject is a human.

[0044] Pharmaceutical composition of the present invention

[0045] According to the present invention, the pharmaceutical composition can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or administration via an explanted reservoir. Among them, oral, intraperitoneal or intravenous administration is preferred.

[0046] When administered orally, dibenzoylmethane, its solvate and / or its hydrate can be made into any oral acceptable preparation form, including but not limited to tablets, capsules, aqueous solutions or aqueous suspensions. Among them, the carriers generally used in tablets include lactose and corn starch, and lubricants such as magnesium stearate can also be added. The diluents generally used in capsule preparations include lactose and dry corn starch. Aqueous suspension preparations are usually mixed with active ingredients and suitable emulsifiers and suspending agents. If necessary, some sweeteners, aromatics or coloring agents can also be added to the above oral preparation forms.

[0047] When administered rectally, dibenzoylmethane, its solvates and / or its hydrates are generally prepared in the form of suppositories, which are prepared by mixing the drug with a suitable non-irritating excipient. The excipient is solid at room temperature and melts at rectal temperature to release the drug. Such excipients include cocoa butter, beeswax and polyethylene glycol.

[0048] When used topically, especially for treating affected areas or organs that are easily accessible by topical application, such as eyes, skin or lower intestinal neurological diseases, dibenzoylmethane, its solvate and / or its hydrate can be prepared into different topical preparations according to different affected areas or organs, as described below:

[0049] When applied topically to the eye, dibenzoylmethane, its solvate and / or its hydrate can be formulated into a micronized suspension or solution, the carrier used being isotonic sterile saline of a certain pH, wherein a preservative such as benzyl alkoxide chloride may or may not be added. In addition, for ophthalmic use, the compound can also be formulated into an ointment such as vaseline.

[0050] When applied topically to the skin, dibenzoylmethane, its solvate and / or its hydrate can be made into suitable ointment, lotion or cream preparation forms, wherein the active ingredient is suspended or dissolved in one or more carriers. The carriers that can be used for ointment here include but are not limited to: mineral oil, liquid vaseline, white vaseline, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; the carriers that can be used for lotion or cream include but are not limited to: mineral oil, sorbitan monostearate, Tween 60, hexadecyl ester wax, hexadecene aromatic alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0051] When applied topically to the lower intestinal tract, dibenzoylmethane, its solvate and / or its hydrate may be formulated as a rectal suppository or in a suitable enema formulation as described above. Alternatively, a topical transdermal patch may be used.

[0052] Dibenzoylmethane, its solvate and / or its hydrate can also be used in the form of sterile injection preparations, including sterile injection water or oil suspension, or sterile injection solution. Among them, the carriers and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.

[0053] The drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0054] In the present invention, the term "therapeutically effective amount" or "prophylactically effective amount" refers to an amount that is sufficient to treat or prevent a patient's disease but low enough to avoid serious side effects (at a reasonable benefit / risk ratio) within the scope of reasonable medical judgment. The therapeutically effective amount of a compound will vary depending on the specific compound selected (e.g., considering the potency, effectiveness and half-life of the compound), the selected route of administration, the disease to be treated, the severity of the disease to be treated, the age, size, weight and physical illness of the patient to be treated, the medical history of the patient to be treated, the duration of treatment, the nature of concurrent therapy, the desired therapeutic effect, and other factors, but can still be routinely determined by those skilled in the art.

[0055] It should also be noted that the specific dosage and method of use of dibenzoylmethane, its solvates and / or its hydrates for different patients are determined by many factors, including the patient's age, weight, gender, natural health, nutritional status, drug activity, time of administration, metabolic rate, severity of the disease, and the subjective judgment of the treating physician. The preferred dosage is 0.001-1000 mg / kg body weight / day.

[0056] After extensive and in-depth research and experiments, the inventor unexpectedly discovered that dibenzoylmethane has antiviral activity against a variety of coronaviruses and can be used to treat related diseases caused by their infection.

[0057] Experiments have shown that dibenzoylmethane has significant anti-coronavirus activity, and it is expected to have obvious advantages in the treatment of various coronavirus infections such as SARS-CoV-2 and human coronavirus OC43. The present invention was completed on this basis.

[0058] Specifically, the present invention discloses the use of dibenzoylmethane in anti-coronavirus, which can be used to prepare a drug for treating a disease, condition or indication caused by coronavirus infection. The virus is severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus MERS-CoV (Middle east respiratory syndrome coronavirus, MERS-CoV), human coronavirus OC43 (Human coronavirus OC43), human coronavirus 229E (Human coronavirus 229E), human coronavirus NL63 (Human coronavirus NL63), human coronavirus HKU1 (Human coronavirus HKU1), porcine epidemic diarrhea virus (PEDV) or feline infectious peritonitis virus (FIFV).

[0059] Specifically, the present invention provides the use of dibenzoylmethane in the preparation of coronavirus inhibitors, for example, in the preparation of drugs for treating and / or preventing and alleviating diseases caused by severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus MERS-CoV (Middle east respiratory syndrome coronavirus, MERS-CoV), human coronavirus OC43 (Human coronavirus OC43), human coronavirus 229E (Human coronavirus229E), human coronavirus NL63 (Human coronavirus NL63), human coronavirus HKU1 (Human coronavirus HKU1), porcine epidemic diarrhea virus (PEDV) and / or feline infectious peritonitis virus (FIFV). The dibenzoylmethane described herein can significantly inhibit the proliferation of coronaviruses such as SARS-CoV-2 and HCoV-OC43 in cells both in vitro and in vivo, and a low dose can achieve a good inhibitory effect, and has good clinical application prospects.

[0060] The present invention is described in detail below in conjunction with the examples. It should be noted that the examples of the present invention are limited to the description of the present invention and have no limiting effect. The relevant test methods and other various experimental operations involved in the examples are all routine techniques in the art. For the parts not particularly described in the text, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literature or related specifications, manuals, etc. before the application date of the present invention for implementation.

[0061] Materials and reagents

[0062] Dibenzoylmethane was purchased from MedChemExpress with a purity greater than 98%; VV116 was purchased from MedChemExpress with a purity greater than 98%; Solutol HS-15 was purchased from MedChemExpress; PEG400 was purchased from MedChemExpress; DMEM was purchased from Thermo Fisher Scientific; FBS was purchased from Thermo Fisher Scientific; and CellCounting Kit-8 (CCK-8) reagent was purchased from Glpbio.

[0063] Human hepatoma cell line (Huh-7), human rhabdomyosarcoma cells (RD), human diploid fibroblasts (MRC-5) and African monkey kidney cells (Vero E6) were purchased from the American Type Culture Collection (ATCC); the ACE2 and TMPRSS2 stable transfected lines of human embryonic kidney cells 293 expressing SV40 large T antigen (HEK293T-ACE2-TMPRSS2, HEK293T-AT) were donated by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0064] The in vivo and in vitro activity tests against the new coronavirus were carried out in a biosafety level 3 laboratory, and the SARS-CoV-2 Omicron variant strain (BA.5) of the new coronavirus was obtained from the National Virus Resource Bank.

[0065] The in vivo and in vitro activity tests against HCoV-OC43 and HCoV-229E were carried out in a biosafety level 2 laboratory, and the HCoV-OC43 and HCoV-229E viruses were obtained from the National Virus Resource Bank.

[0066] Embodiment 1:

[0067] Test method:

[0068] 1) Human rhabdomyosarcoma cells (RD) were evenly plated in a 48-well plate, with 50,000 to 60,000 cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a cell culture incubator at 37°C for 12 hours;

[0069] 2) Discard the cell supernatant, replace the medium with DMEM containing 2% (V / V) FBS containing dibenzoylmethane, and incubate in a 37°C cell culture incubator for 1 hour. Drug concentration design plan: Design 7 dibenzoylmethane concentrations, which are 5.00μM, 1.67μM, 0.56μM, 0.19μM, 0.06μM, 0.02μM, and 0.00μM respectively;

[0070] 3) Add coronavirus HCoV-OC43, MOI = 0.1 per well, tap to mix, and incubate in a 37°C cell culture incubator for 24 hours;

[0071] 4) Collect the cell supernatant, extract the viral RNA in the supernatant, and use qRT-PCR to detect the viral copy number.

[0072] Test results:

[0073] Results Figure 1 Quantitative evaluation of the virus inhibition rate by measuring the virus copy number through real-time quantitative polymerase chain reaction (qRT-PCR) can reflect the inhibitory effect of dibenzoylmethane on HCoV-OC43. Figure 1 It can be seen that dibenzoylmethane has a significant inhibitory effect on HCoV-OC43 in a dose-dependent manner, and can achieve significant therapeutic effects at a concentration as low as 0.56 μM.

[0074] Embodiment 2:

[0075] Test method:

[0076] 1) Human diploid fibroblasts (MRC-5) were evenly plated in a 48-well plate, with 50,000 to 60,000 cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a cell culture incubator at 37°C for 12 hours;

[0077] 2) Discard the cell supernatant, replace the medium with DMEM containing 2% (V / V) FBS containing dibenzoylmethane, and incubate in a 37°C cell culture incubator for 1 hour. Drug concentration design plan: Design 7 dibenzoylmethane concentrations, which are 10.00μM, 3.33μM, 1.11μM, 0.37μM, 0.12μM, 0.04μM, and 0.00μM respectively;

[0078] 3) Add coronavirus HCoV-229E, MOI = 0.1 per well, tap to mix, and incubate in a 37°C cell culture incubator for 48 hours;

[0079] 4) Collect the cell supernatant, extract the viral RNA in the supernatant, and use qRT-PCR to detect the viral copy number.

[0080] Test results:

[0081] Results Figure 2 Quantitative evaluation of the virus inhibition rate by measuring the virus copy number through real-time quantitative polymerase chain reaction (qRT-PCR) can reflect the inhibitory effect of dibenzoylmethane on HCoV-229E. Figure 2 It can be seen that dibenzoylmethane has a significant inhibitory effect on HCoV-229E in a dose-dependent manner, and can achieve significant therapeutic effects at a concentration as low as 1.11 μM.

[0082] Embodiment 3:

[0083] Test method:

[0084] 1) African monkey kidney cells (Vero E6) were evenly plated in a 48-well plate, with 50,000 to 60,000 cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a cell culture incubator at 37°C for 12 hours;

[0085] 2) Discard the cell supernatant, replace the medium with DMEM containing 2% (V / V) FBS containing dibenzoylmethane, and incubate in a 37°C cell culture incubator for 1 hour. Drug concentration design plan: Design 7 dibenzoylmethane concentrations, which are 20.00μM, 6.67μM, 2.22μM, 0.74μM, 0.24μM, 0.08μM, and 0.00μM respectively;

[0086] 3) Add the novel coronavirus SARS-CoV-2 Omicron variant (BA.5) at an MOI of 0.01 per well, tap to mix, and incubate in a cell culture incubator at 37°C for 24 hours;

[0087] 4) Collect the cell supernatant, extract the viral RNA in the supernatant, and use qRT-PCR to detect the viral copy number.

[0088] Test results:

[0089] Results Figure 3 . The quantitative evaluation of the virus inhibition rate by measuring the virus copy number through real-time quantitative polymerase chain reaction (qRT-PCR) can reflect the inhibitory effect of dibenzoylmethane on the SARS-CoV-2 Omicron variant (BA.5). Figure 3 It can be seen that dibenzoylmethane has a significant inhibitory effect on the SARS-CoV-2 Omicron variant (BA.5) and is dose-dependent, and can achieve significant therapeutic effects at concentrations as low as 6.67 μM.

[0090] Embodiment 4:

[0091] Test method:

[0092] 5-6 day old Balb / c mice were divided into five groups, 4-6 mice in each group, and infected with HCoV-OC43 virus (10000 TCID 50 / mouse), and then gavage was performed 1 hour later (day 0). The dosing scheme was that each group of Balb / c suckling mice was given dibenzoylmethane 500 mpk (mg per kg), dibenzoylmethane 200 mpk, and dibenzoylmethane 100 mpk, respectively. At the same time, a solvent control group (Vehicle group, the solvent was 40% (V / V) PEG400 + 10% (V / V) Solutol HS-15 + 50% (V / V) ultrapure water) and a positive control group (administered VV116 50 mpk) were set up. After that, each group of animals was gavage-administered once a day, and the behavioral changes and body weight changes of the mice were observed. On the 5th day, the animals were dissected, and the brain, spinal cord, lungs and kidneys of the mice were taken for qPCR detection of the number of viral copies in the tissues (viral load, viral copies), and the brain and lung tissue sections of the mice were stained with hematoxylin-eosin (HE) for observation.

[0093] Test results:

[0094] The results of qPCR detection of viral copy number in tissues are shown in Figure 4 In the HCoV-OC43 infected mouse model, quantitative evaluation of the virus inhibition rate by measuring the number of viral copies in the brain, spinal cord, lungs and kidneys of suckling mice by real-time quantitative polymerase chain reaction (qRT-PCR) can reflect the inhibitory effect of dibenzoylmethane on HCoV-OC43. Figure 4 It can be seen that compared with the solvent control group, administration of 500 mpk of dibenzoylmethane can reduce the viral load in the brain, spinal cord, lungs and kidneys of suckling mice to approximately 1 / 1400, 1 / 40, 1 / 20 and 1 / 60 of that of the solvent control group, respectively; the above results indicate that dibenzoylmethane has a significant inhibitory effect on HCoV-OC43 in mice.

[0095] The results of hematoxylin-eosin (HE) staining are shown in Figure 5 . In the mouse model of HCoV-OC43 infection, oral administration of DBM can significantly reduce the damage caused by viral infection in organs such as the brain and lungs. Compared with the control group, DBM treatment reduced the accumulation of immune effector cells and neuronal cell death in the brain, and reduced pulmonary fibrosis, pulmonary edema, and pulmonary sputum formation in the lungs. Therefore, it can be considered that DBM has a good therapeutic effect on neurological and respiratory diseases caused by HCoV-OC43 infection.

[0096] Embodiment 5:

[0097] Test method:

[0098] 1) Human liver cancer cell line (Huh-7) was evenly plated in a 96-well plate, with 20,000 to 30,000 cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a cell culture incubator at 37°C for 12 hours;

[0099] 2) Discard the cell supernatant, replace the medium with DMEM containing 2% (V / V) FBS containing dibenzoylmethane, and incubate in a 37°C cell culture incubator for 24 hours. Drug concentration design plan: Design 7 dibenzoylmethane concentrations, which are 500.00μM, 166.67μM, 55.56μM, 18.52μM, 6.17μM, 2.06μM, and 0.00μM respectively;

[0100] 3) Discard the cell supernatant, add 100 μl of fresh DMEM medium containing 2% (V / V) FBS to each well, add 10 μl of Cell Counting Kit-8 (CCK-8) reagent to each well, tap to mix, and incubate in a 37°C cell culture incubator for 1 hour;

[0101] 4) Gently shake the 96-well plate, and then read the absorbance of each well sample at 450 nm using a microplate reader.

[0102] The Huh-7 cell line was replaced with human rhabdomyosarcoma cells (Rhabdomyosarcoma, RD), human diploid fibroblasts (MRC-5), ACE2 and TMPRSS2 stable strains of human embryonic kidney cells 293 expressing SV40 large T antigen (HEK293T-ACE2-TMPRSS2, HEK293T-AT) or African monkey kidney cells (Vero E6), and experiments were carried out while keeping other conditions unchanged, and drug toxicity test experimental data of the corresponding cell lines were obtained.

[0103] Experimental results:

[0104] Results Figure 6 The absorbance value of the cell culture samples after drug treatment at 450nm can quantitatively evaluate the effect of dibenzoylmethane on the vitality of different cell lines, thereby reflecting the toxicity of dibenzoylmethane to different cell lines and the safety of dibenzoylmethane. Figure 6 As can be seen in the figure, the cytotoxicity index CC of dibenzoylmethane on Huh-7, RD, MRC-5, 293T-AT and Vero E6 cell lines is 50 All of them were greater than 500.00 μM, indicating that dibenzoylmethane still had low toxicity and good safety at a working concentration of up to 500.00 μM.

Claims

1. Use of dibenzoylmethane, its solvate or hydrate, or a pharmaceutical composition comprising any of the foregoing in the preparation of a drug, wherein the drug is selected from: i. Drugs used to prevent or treat diseases caused by coronavirus; ii. Drugs used to inhibit the replication or reproduction of coronaviruses in mammalian cells, tissues or organs.

2. The use according to claim 1, wherein The coronavirus is selected from: (1) Coronaviruses that infect humans: severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus MERS-CoV (MERS-CoV), human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1); (2) Coronaviruses that infect animals: porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIFV).

3. The use according to claim 1, wherein The coronavirus is HCoV-OC43, SARS-CoV-2 Omicron variant BA.5 or HCoV-229E.

4. The use according to claim 1, wherein The coronavirus is HCoV-OC43.

5. The use according to claim 1, wherein The related diseases caused by the coronavirus are selected from: respiratory diseases, encephalitis, encephalomyelitis, sepsis, septic shock, arrhythmia, myocarditis, liver function damage and renal function damage caused by coronavirus; porcine epidemic diarrhea caused by porcine epidemic diarrhea virus; and feline infectious peritonitis caused by feline infectious peritonitis virus.

6. The use according to claim 5, wherein The respiratory disease caused by the coronavirus is selected from simple infection, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome caused by coronavirus.

7. The use according to claim 6, wherein Simple infections caused by the coronavirus include fever, cough and sore throat.

8. The use according to claim 1, wherein In ii, the mammals include bovines, equines, ovines, porcines, canines, felines, rodents, and primates, wherein the preferred mammals are humans, cats, dogs, or pigs.

9. The use according to claim 1, wherein The pharmaceutical composition comprises a therapeutically effective amount of dibenzoylmethane, a solvate or a hydrate thereof, and a pharmaceutically acceptable auxiliary material.

10. The use according to claim 1, wherein The dosage forms of the pharmaceutical composition include tablets, capsules, aqueous solutions or suspensions or micronized suspensions or solutions, suppositories, ointments, sprays, lotions or creams, rectal suppository preparations or enemas, local transdermal patches, sterile injections or oil suspensions.

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