Use of dibenzoylmethane for inhibiting coronavirus and treating related diseases caused thereby

Abstract

The present application relates to the use of dibenzoylmethane for inhibiting coronavirus and treating related diseases caused by the same. In the present application, in vitro cell experiments show that dibenzoylmethane can inhibit the proliferation of SARS-CoV-2, HCoV-229E and HCoV-OC43 in cells, and good inhibition effect can be achieved at a low dose; in vivo mouse experiments show that dibenzoylmethane can effectively inhibit the load of HCoV-OC43 in brain, spine, lung and kidney and other tissues and organs, and has good therapeutic effect on the nervous and respiratory system diseases caused by HCoV-OC43 infection. Dibenzoylmethane has the advantages of good curative effect, high safety and the like, and therefore can be developed into a new coronavirus inhibitor for treating diseases caused by coronavirus infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and specifically relates to the use of dibenzoylmethane for inhibiting coronavirus or in the preparation of a drug for treating or preventing a disease caused by coronavirus. BACKGROUND

[0002] Coronaviruses are a class of single-stranded positive-sense RNA viruses with envelopes, belonging to the Coronaviridae family. The Coronaviridae family is divided into four genera, including alpha coronaviruses, beta coronaviruses, gamma coronaviruses, and delta coronaviruses. Among them, alpha coronaviruses and beta coronaviruses mainly infect mammals, while gamma coronaviruses and delta coronaviruses mainly infect birds. Coronaviruses have high genetic diversity and variability, which makes them have strong ability to spread across species and adapt to new hosts.

[0003] Currently, effective treatment methods for coronavirus infection are still limited. Especially in the face of the virus's variability, the effectiveness of existing antiviral drugs and vaccines may be weakened or even ineffective as the virus mutates. For example, multiple mutations of SARS-CoV-2 have produced a variety of variants, which have shown significant differences in transmission, pathogenicity, and resistance to vaccines. Although the current vaccines and treatment programs are effective to some extent, coping with future possible new coronavirus variants remains a great challenge.

[0004] In this context, the development of broad-spectrum antiviral drugs is particularly important. Broad-spectrum antiviral drugs not only can inhibit a variety of known coronaviruses, but also have the potential to combat future possible coronavirus variants. The main advantage of broad-spectrum antiviral drugs is that they do not depend on specific genetic sequences or protein structures of the virus, but rather interfere with the virus's key biological processes or target related mechanisms of host cells, thereby achieving inhibition of a variety of coronaviruses. This broad-spectrum characteristic makes this class of drugs more adaptable and effective in dealing with coronavirus mutations and new outbreaks.

[0005] Developing broad-spectrum anti-coronavirus drugs not only has important significance for current and future coronavirus prevention and control, but also provides scientific basis and technical reserves for coping with other potential viral pandemics. In the context of globalization, cross-border virus transmission has become the norm, and the development and stockpiling 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, providing more possibilities for human resistance to various new and re-emerging viral diseases.

[0006] In summary, as the threat of coronavirus to human health is becoming increasingly serious, developing new antiviral compounds with broad inhibitory effects to cope with the challenge of multiple coronaviruses and their variants has become a key direction of current drug research and development. SUMMARY

[0007] The inventors of the present application unexpectedly found that dibenzoylmethane has antiviral activity against multiple coronaviruses and can be used for the treatment or prevention of diseases related to infection with multiple coronaviruses, thereby completing the present application.

[0008] The present application provides the use of dibenzoylmethane, a solvate or hydrate thereof, or a pharmaceutical composition comprising any of the foregoing in the preparation of a medicament selected from:

[0009] i. a medicament for preventing or treating diseases related to coronavirus infection;

[0010] ii. a medicament for inhibiting the replication or proliferation of coronavirus in mammalian cells, tissues or organs.

[0011] In a specific embodiment, the coronavirus is selected from:

[0012] (1) human coronaviruses: 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);

[0013] (2) animal coronaviruses: porcine epidemic diarrhea virus (PEDV), feline infectious peritonitis virus (FIFV).

[0014] In a specific embodiment, the coronavirus is HCoV-OC43, SARS-CoV-2 Omicron variant BA.5, or HCoV-229E.

[0015] In a specific embodiment, the coronavirus is HCoV-OC43.

[0016] In a specific embodiment, the disease related to coronavirus infection is selected from respiratory diseases caused by coronavirus, encephalitis, encephalomyelitis, sepsis, septic shock, arrhythmia, myocarditis, liver function impairment, and kidney function impairment; porcine epidemic diarrhea caused by porcine epidemic diarrhea virus; and feline infectious peritonitis caused by feline infectious peritonitis virus.

[0017] In specific embodiments, the respiratory disease caused by the coronavirus is selected from the group consisting of simple infection caused by the coronavirus, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure, and acute respiratory distress syndrome.

[0018] In specific embodiments, the simple infection caused by the coronavirus includes fever, cough, and sore throat.

[0019] In specific embodiments, in ii, the mammal includes bovine, equine, ovine, porcine, canine, feline, rodent, primate, wherein the preferred mammal is human, cat, dog, or pig.

[0020] In specific embodiments, in ii, inhibiting the coronavirus includes reducing the nucleic acid load of the coronavirus in the cell culture, tissue, or organ of the mammal cell.

[0021] In specific embodiments, the pharmaceutical composition comprises a therapeutically effective amount of dibenzoylmethane, a solvate or hydrate thereof, and a pharmaceutically acceptable excipient.

[0022] In specific embodiments, the dosage form of the pharmaceutical composition includes, but is not limited to, tablets, capsules, aqueous solutions or aqueous suspensions or micronized suspensions or solutions, suppositories, ointments, sprays, lotions or creams, rectal suppository formulations or enemas, topical transdermal patches, sterile injection solutions or oil suspensions.

[0023] In the present application, the pharmaceutical composition can be prepared into a desired dosage form according to the suitable administration mode of the drug by means known in the art.

[0024] Advantages

[0025] In vitro cell experiments of the present application show that dibenzoylmethane can inhibit the proliferation of SARS-CoV-2, HCoV-229E, and HCoV-OC43 in cells, and good inhibition effect can be achieved at low doses.

[0026] In vivo mouse experiments of the present application show that dibenzoylmethane can effectively inhibit the load of HCoV-OC43 in tissues and organs such as brain, spine, lung, and kidney, and has good therapeutic effect on neurological and respiratory diseases caused by HCoV-OC43 infection.

[0027] Therefore, dibenzoylmethane can be used as a coronavirus inhibitor for treating diseases caused by coronavirus infection, which has the advantages of good curative effect, high safety, etc. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1Results of the in vitro inhibition activity test of different concentrations of dibenzoylmethane on HCoV-OC43 in Example 1 are shown.

[0029] Figure 2 Results of the in vitro inhibition activity test of different concentrations of dibenzoylmethane on HCoV-229E in Example 2 are shown.

[0030] Figure 3 Results of the in vitro inhibition activity test of different concentrations of dibenzoylmethane on SARS-CoV-2 Omicron variant (BA.5) in Example 3 are shown.

[0031] Figure 4 Results of the in vivo inhibition activity test of different concentrations of dibenzoylmethane on HCoV-OC43 on a mouse model infected with HCoV-OC43 in Example 4 are shown.

[0032] Figure 5 Results of the efficacy of different concentrations of dibenzoylmethane on organ damage caused by HCoV-OC43 on a mouse model infected with HCoV-OC43 in Example 4 are shown.

[0033] Figure 6 Results of the cytotoxicity test of different concentrations of dibenzoylmethane on five different cell lines including Huh-7 in Example 5 are shown. DETAILED DESCRIPTION

[0034] Terms:

[0035] In the present application, dibenzoylmethane (DBM for short) has the structure shown below:

[0036]

[0037] In the present application, unless otherwise explicitly stated, the terms used in the present application have the meanings defined below. The terms not explicitly defined in the present application have the general meanings generally understood by those skilled in the art.

[0038] As used herein, "pharmaceutically acceptable excipient" includes 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, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0039] As used herein, "inhibit" means to reduce or suppress a particular condition, symptom, or disorder or disease, or to significantly decrease the baseline activity of a biological activity or process.

[0040] As used herein, the term "treatment" of any disease or disorder, in one embodiment, means ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, "treatment" means ameliorating at least one physical parameter including, e.g., not perceptible to the patient. In another embodiment, "treatment" means modulating the disease or disorder, either physically, (e.g., stabilization of a perceptible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

[0041] As used herein, "prevention" means administering to an individual having a predisposition for the disease one or more pharmaceutical substances, in particular a compound according to the application and / or a pharmaceutically acceptable salt thereof, in order to prevent the individual from contracting the disease.

[0042] As used herein, "individual" means a mammal, for example, a primate (e.g., human), a cow, a sheep, a goat, a horse, a dog, a cat, a rabbit, a rat, a mouse, and the like. In a preferred embodiment, the individual is a human, a pig, and a cat. In a preferred embodiment, the individual is a human.

[0043] Pharmaceutical compositions of the application

[0044] According to the application, the pharmaceutical compositions can be administered in any way possible, orally, by spray inhalation, rectally, nasally, buccally, vaginally, topically, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, intraventricularly, intrasternally and intracranially, by injection or infusion, or by means of an external reservoir. Among these, oral, intraperitoneal or intravenous administration is preferred.

[0045] When administered orally, the dibenzoylmethanes, solvates and / or hydrates thereof, can be formulated in any orally acceptable dosage form including, but not limited to, tablets, capsules, aqueous or non-aqueous solutions or suspensions. In such embodiments, tablets are typically formulated using carriers including, but not limited to, lactose and corn starch. Lubricating agents, such as magnesium stearate, can also be added. Capsules are typically formulated using diluents including, but not limited to, lactose and dried corn starch. Aqueous or non-aqueous suspensions are typically formulated using a mixture of an active ingredient with a pharmaceutically acceptable diluent or carrier, such as water or a suitable liquid excipient. If desired, sweetening, flavoring or coloring agents can be added.

[0046] When administered rectally, the dibenzoylmethanes, solvates and / or hydrates thereof, are typically formulated in the form of suppositories, which can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature but liquid at body temperature and therefore melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.

[0047] When administered topically, particularly for the treatment of local external application to easily accessible affected areas or organs, such as the eye, skin or lower intestinal nervous disorders, the dibenzoylmethanes, solvates and / or hydrates thereof, can be formulated into a variety of topically administrable dosage forms depending on the affected area or organ, as described below:

[0048] When administered topically to the eye, the dibenzoylmethanes, solvates and / or hydrates thereof, can be formulated in the form of a micronized suspension or solution using a carrier which is isotonic, pH adjusted, sterile saline, where a preservative can or can not be added. In addition, for ophthalmic use, the compounds can be made into a gel, such as a petrolatum gel.

[0049] When administered topically to the skin, the dibenzoylmethanes, solvates and / or hydrates thereof, can be formulated into a suitable ointment, lotion or cream containing suitable emulsifiers or suspending agents. Such carriers include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax and water. Alternatively, the compounds can be formulated into a suitable lotion or cream containing suitable emulsifiers or suspending agents. Such carriers include, but are not limited to, mineral oil, sorbitan monostearate, isopropyl alcohol, lanolin, lanolin alcohols, cetyl esters wax, cetearyl, 2-octyldodecanol, benzyl alcohol and water.

[0050] When administered topically to the lower intestinal tract, the dibenzoylmethanes, solvates and / or hydrates thereof, can be formulated in a rectal suppository or suitable enema formulation as described above, or alternatively, as a transdermal patch.

[0051] The dibenzoylmethane, solvate thereof and / or hydrate thereof can also be administered in the form of a sterile injectable preparation, including sterile injectable water or oil suspensions or sterile injectable solutions. Among the carriers and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils can be used as a solvent or suspending medium, such as for example, glycerin, propylene glycol or glyceryl triester.

[0052] The pharmaceuticals of the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

[0053] In the present application, the term "therapeutically effective amount" or "prophylactically effective amount" means an amount sufficient to treat or prevent a disease in a patient, but low enough to avoid serious side effects (at a reasonable benefit / risk ratio) within the scope of sound medical judgment. The therapeutically effective amount of the compound will vary depending on the specific compound chosen (for example, taking into account the potency, efficacy and half-life of the compound), the chosen route of administration, the disease being treated, the severity of the disease being treated, the age, size, body weight, and physical health of the patient being treated, the medical history of the patient being treated, the duration of treatment, the nature of concurrent therapy, the desired therapeutic effect, and the like, but can be routinely determined by one of skill in the art.

[0054] It should be further noted that the specific dosage and method of use of the dibenzoylmethane, solvate thereof and / or hydrate thereof for different patients are determined by many factors, including the age, body weight, sex, natural health status, nutritional status, activity strength of the drug, taking time, metabolic rate, severity of the disease and subjective judgment of the treating physician. Here, the preferred dosage is 0.001-1000 mg / kg body weight / day.

[0055] The present inventors have made extensive and in-depth research and, through experiments, unexpectedly found that the dibenzoylmethane has antiviral activity against a variety of coronaviruses and can be used for the treatment of diseases caused by infection with the same.

[0056] Experiments have shown that the dibenzoylmethane has significant anti-coronavirus activity and is expected to have obvious advantages in the treatment of a variety of coronavirus infections such as SARS-CoV-2 and Human coronavirus OC43, and on this basis, the present application has been completed.

[0057] In particular, the present application discloses the use of dibenzoylmethane in anti-coronavirus, which can be used for preparing a medicine 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 2 (SARS-CoV-2), Middle east respiratory syndrome coronavirus (MERS-CoV), Human coronavirus OC43, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1, Porcine epidemic diarrhea virus (PEDV) or Feline infectious peritonitis virus (FIFV).

[0058] In particular, the present application provides the use of dibenzoylmethane in preparing a coronavirus inhibitor, such as in preparing a medicine for treating and / or preventing, alleviating a disease caused by infection of Severe acute respiratory syndrome coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle east respiratory syndrome coronavirus (MERS-CoV), Human coronavirus OC43, Human coronavirus 229E, Human coronavirus NL63, 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 coronavirus such as SARS-CoV-2 and HCoV-OC43 in cells in vitro and in vivo, and a low dose can achieve good inhibition effect, and has good clinical application prospect.

[0059] The application will be described in detail below with reference to examples. It should be noted that the examples of the application are only for illustration and have no limiting effect. The test methods and other various experimental operations involved in the examples are conventional techniques in the art, and the parts not specifically described herein can be implemented by referring to various commonly used tool books, scientific and technical literature or related instructions, manuals, etc. before the application date of the application.

[0060] Materials and reagents

[0061] Dibenzoylmethane was purchased from MedChemExpress with a purity of more than 98%; WVI 16 was purchased from MedChemExpress with a purity of more 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; Cell Counting Kit-8 (CCK-8) reagent was purchased from Glpbio.

[0062] Human hepatocellular carcinoma cell line (Huh-7), human rhabdomyosarcoma cell (RD), human diploid fibroblast cell (MRC-5) and African monkey kidney cell (Vero E6) were purchased from American Type Culture Collection (ATCC); ACE2 and TMPRSS2 stable transgenic strain of human embryonic kidney cell 293 expressing SV40 large T antigen (HEK293T-ACE2-TMPRSS2, HEK293T-AT) was a gift from Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0063] In vitro and in vivo activity of anti-SARS-CoV-2 (Omicron variant BA.5) was tested in a biosafety level 3 laboratory. SARS-CoV-2 (Omicron variant BA.5) was obtained from the National Virus Resource Bank.

[0064] In vitro and in vivo activity of anti-HCoV-OC43 and HCoV-229E was tested in a biosafety level 2 laboratory. HCoV-OC43 and HCoV-229E viruses were obtained from the National Virus Resource Bank.

[0065] Example 1

[0066] Test method

[0067] 1) Human rhabdomyosarcoma cells (RD) were evenly spread in a 48-well plate, 50-60 thousand cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a 37°C cell incubator for 12 hours;

[0068] 2) Discard the cell supernatant, replace the culture medium with DMEM containing 2% (V / V) FBS containing dibenzoylmethane, and incubate in a 37°C cell incubator for 1 hour. Drug concentration design scheme: 7 dibenzoylmethane concentrations are designed, and the concentrations are 5.00 μM, 1.67 μM, 0.56 μM, 0.19 μM, 0.06 μM, 0.02 μM, 0.00 μM in turn;

[0069] 3) Add coronavirus HCoV-OC43, MOI = 0.1 per well, gently mix, and incubate in a 37°C cell incubator for 24 hours;

[0070] 4) Collect the cell supernatant, extract the viral RNA in the supernatant, and detect the viral copy number using qRT-PCR.

[0071] Test results:

[0072] The results are shown in Figure 1 . The inhibition rate of virus was quantitatively evaluated by determining the viral copy number by real-time quantitative polymerase chain reaction (qRT-PCR), which can reflect the inhibition effect of dibenzoylmethane on HCoV-OC43. It can be seen from Figure 1 that dibenzoylmethane has obvious inhibition effect on HCoV-OC43 and is dose-dependent, and can achieve significant therapeutic effect as low as 0.56 μM.

[0073] Example 2:

[0074] Test method:

[0075] 1) Human diploid fibroblasts (MRC-5) were uniformly plated in a 48-well plate at 5-6 million cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a 37°C cell incubator for 12 hours;

[0076] 2) Discard the cell supernatant, replace the culture medium with DMEM containing 2% (V / V) FBS containing dibenzoylmethane, and incubate in a 37°C cell incubator for 1 hour. Drug concentration design scheme: 7 dibenzoylmethane concentrations are designed, and the concentrations are 10.00 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.04 μM, 0.00 μM in turn;

[0077] 3) Add coronavirus HCoV-229E, MOI = 0.1 per well, gently mix, and incubate in a 37°C cell incubator for 48 hours;

[0078] 4) Collect the cell supernatant, extract the viral RNA in the supernatant, and detect the viral copy number using qRT-PCR.

[0079] Test results:

[0080] Results are shown in Figure 2 . The inhibitory rate of virus was quantitatively evaluated by determining the viral copy number by real-time quantitative polymerase chain reaction (qRT-PCR), which can reflect the inhibitory effect of dibenzoylmethane on HCoV-229E. From Figure 2 it can be seen that dibenzoylmethane has a significant inhibitory effect on HCoV-229E and is dose-dependent, and a significant therapeutic effect can be achieved at as low as 1.11 μM.

[0081] Example 3:

[0082] Test method:

[0083] 1) African monkey kidney cells (Vero E6) were uniformly plated in a 48-well plate at 50-60 thousand cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a 37°C cell incubator for 12 hours;

[0084] 2) Discard the cell supernatant, replace it with 2% (V / V) FBS-containing DMEM containing dibenzoylmethane, and incubate in a 37°C cell incubator for 1 hour. Drug concentration design: 7 dibenzoylmethane concentrations are designed, with concentrations of 20.00 μM, 6.67 μM, 2.22 μM, 0.74 μM, 0.24 μM, 0.08 μM, and 0.00 μM, respectively;

[0085] 3) Add the new coronavirus SARS-CoV-2 Omicron variant (BA.5) at an MOI of 0.01 per well, gently mix, and incubate in a 37°C cell incubator for 24 hours;

[0086] 4) Collect the cell supernatant, extract the viral RNA in the supernatant, and detect the viral copy number using qRT-PCR.

[0087] Test results:

[0088] Results are shown in Figure 3 . The inhibitory rate of virus was quantitatively evaluated by determining the viral copy number by real-time quantitative polymerase chain reaction (qRT-PCR), which can reflect the inhibitory effect of dibenzoylmethane on SARS-CoV-2 Omicron variant (BA.5). From Figure 3 it can be seen that dibenzoylmethane has a significant inhibitory effect on SARS-CoV-2 Omicron variant (BA.5) and is dose-dependent, and a significant therapeutic effect can be achieved at as low as 6.67 μM.

[0089] Example 4:

[0090] Test method:

[0091] Five groups of 5-6 day old Balb / c mice, 4-6 in each group, were infected with HCoV-OC43 virus (10000 TCID 50 / each) by intranasal infection, and 1 hour later, the mice were administered by gavage (on day 0) with dibenzoylmethane 500 mpk (milligrams per kilogram), dibenzoylmethane 200 mpk, dibenzoylmethane 100 mpk, respectively, for each group of Balb / c mice, while a vehicle control group (Vehicle group, the vehicle was 40% (V / V) PEG400 + 10% (V / V) Solutol HS-15 + 50% (V / V) ultrapure water) and a positive control group (VV116 50 mpk) were set up, and then the animals in each group were administered by gavage once a day, the behavior and weight changes of the mice were observed, and the mice were dissected on the 5th day, the brain, spinal cord, lung and kidney of the mice were taken for qPCR to detect the viral copy number (viral load) in the tissues, and hematoxylin-eosin (HE) staining was performed on the brain and lung tissue sections of the mice.

[0092] Test results:

[0093] The results of qPCR to detect the viral copy number in the tissues are shown in Figure 4 In the HCoV-OC43 infected mouse model, the viral copy number in the brain, spinal cord, lung and kidney of the mice was determined by real-time quantitative polymerase chain reaction (qRT-PCR) to quantitatively evaluate the inhibition rate of the virus, which could reflect the inhibitory effect of dibenzoylmethane on HCoV-OC43. As can be seen from Figure 4 compared with the vehicle control group, the administration of 500 mpk dibenzoylmethane could reduce the viral load in the brain, spinal cord, lung and kidney of the mice to about 1 / 1400, 1 / 40, 1 / 20 and 1 / 60, respectively, of the vehicle control group; the above results showed that dibenzoylmethane had a significant inhibitory effect on HCoV-OC43 in mice.

[0094] The results of hematoxylin-eosin (HE) staining are shown in Figure 5 In the HCoV-OC43 infected mouse model, oral administration of DBM can significantly reduce the damage caused by viral infection in organs such as brain and lung. Compared with the control group, DBM treatment reduces the accumulation of immune effector cells in the brain and the death of nerve cells in the brain, and reduces pulmonary fibrosis, pulmonary edema and the formation of sputum in the lungs. Therefore, it can be considered that DBM has a good therapeutic effect on the neurological and respiratory diseases caused by HCoV-OC43 infection.

[0095] Example 5:

[0096] Test method:

[0097] 1) Human hepatocellular carcinoma cell line (Huh-7) was evenly spread in 96-well plates, 2-3 million cells per well, cultured in DMEM medium containing 10% (V / V) FBS, and placed in a 37°C cell incubator for 12 hours;

[0098] 2) Discard the cell supernatant, replace the culture medium containing 2% (V / V) FBS with DBM, and incubate in a 37°C cell incubator for 24 hours. Drug concentration design: 7 DBM concentrations were designed, with concentrations of 500.00 μM, 166.67 μM, 55.56 μM, 18.52 μM, 6.17 μM, 2.06 μM, and 0.00 μM, respectively;

[0099] 3) Discard the cell supernatant, add 100 microliters of fresh DMEM medium containing 2% (V / V) FBS to each well, add 10 microliters of Cell Counting Kit-8 (CCK-8) reagent to each well, mix gently, and incubate in a 37°C cell incubator for 1 hour;

[0100] 4) Gently shake the 96-well plate, then use a microplate reader to read the absorbance value of each well sample at 450 nm.

[0101] The Huh-7 cell line was replaced with human rhabdomyosarcoma cells (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 green monkey kidney cells (Vero E6), and the experiment was performed under the same conditions, and the drug toxicity detection experiment data of the corresponding cell lines were obtained.

[0102] Experimental results:

[0103] The results are shown in Figure 6 The absorbance value of the cell culture sample after drug treatment at 450 nm can quantitatively evaluate the effect of DBM on the viability of different cell lines, thus reflecting the toxicity of DBM to different cell lines and the safety of DBM. From Figure 6 it can be seen that the cell toxicity index CC 50 of DBM on Huh-7, RD, MRC-5, 293T-AT, and Vero E6 cell lines is greater than 500.00 μM, indicating that DBM still has low toxicity and good safety at a working concentration of up to 500.00 μM.

Claims

1. Use of dibenzoylmethane, or a pharmaceutical composition comprising therein, in the preparation of a medicament for inhibiting the replication or multiplication of a coronavirus in mammalian cells, tissues, or organs, wherein the coronavirus is HCoV-OC43, SARS-CoV-2 Omeprón variant BA.5, or HCoV-229E.

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

3. The use according to claim 1, wherein, The mammals mentioned include bovines, equines, sheep, pigs, canines, felines, rodents, and primates.

4. The use according to claim 3, wherein, The mammals referred to are humans, cats, dogs, or pigs.

5. The use according to claim 1, wherein, The pharmaceutical composition comprises a therapeutically effective amount of dibenzoylmethane, and pharmaceutically acceptable excipients.

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

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