Application of ethyl parahydroxybenzoate in preventing coronavirus infection

Abstract

The present invention belongs to the field of drug therapy, and specifically relates to the use of ethyl parahydroxybenzoate in the preparation of drugs for preventing or treating viral infectious diseases. The present invention discloses the use of ethyl parahydroxybenzoate and its inhalation preparation in the treatment of coronavirus infectious diseases. By verifying the new use of ethyl parahydroxybenzoate inhalation solution in a mouse test infected with human coronavirus HCoV-229E, the technical solution has novelty, and an efficacy test of meta-cresol inhalation solution in the treatment of pneumonia caused by HCoV-229E virus infection was conducted. The results showed that ethyl parahydroxybenzoate inhalation solution showed good efficacy in the lung index and inhibition rate of pneumonia mice infected with HCoV-229E virus, and could significantly reduce the lung index of mice.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of ethyl p-hydroxybenzoate in preparing medicines for preventing or treating coronavirus infectious diseases. Background Art

[0002]

[0003] Ethyl p-hydroxybenzoate (C9H 10 O3) is also known as ethyl paraben, with a molecular weight of 166.18.

[0004] Ethylparaben, as a preservative and antibacterial agent, is widely used in cosmetics, foods, oral preparations, and topical formulations. It can be used alone or in combination with other parabens and other antimicrobial preservatives, making it one of the most commonly used antimicrobial preservatives in cosmetics. Ethylparaben exhibits antimicrobial activity within the pH range of 4 to 8. Due to the formation of phenolate anions, its preservative efficacy decreases with increasing pH. It possesses broad-spectrum antimicrobial activity, being more potent against yeasts and molds than bacteria, and more potent against Gram-positive bacteria than Gram-negative bacteria. The stability of aqueous solutions of ethylparaben is pH-dependent. Aqueous solutions of ethylparaben can be autoclaved without degradation within the pH range of 3 to 6 and remain stable at room temperature for up to four years (with degradation less than 10%). However, at pH ≥ 8, aqueous solutions rapidly hydrolyze, with hydrolysis reaching 10% or more within 60 days at room temperature. Currently, there are no reports of ethylparaben treating coronaviruses.

[0005] Coronaviruses are widely present in nature and are divided into four genera (α, β, γ, and δ). They were first isolated from poultry in 1937 and first discovered in humans in 1965. Currently, seven coronaviruses have been identified that can infect humans. Four of these coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1) are relatively common in the human population and have low pathogenicity, generally causing mild respiratory illnesses similar to the common cold. The other three coronaviruses (SARS-CoV, SARS-CoV-2, and MERS-CoV) are highly pathogenic and contagious, posing a significant threat to human health.

[0006] Human coronaviruses cause 10% to 30% of upper respiratory tract infections worldwide. Over the past two decades, new coronavirus variants have emerged, increasing in pathogenicity and spreading globally from localized outbreaks, posing a significant threat to global health. In the absence of a vaccine, antiviral drug research is crucial for viral prevention and control. However, there are currently no approved, effective drugs for treating coronavirus infections, necessitating the urgent need for a drug to prevent and treat human coronaviruses.

[0007] To address these clinical needs, we screened a large number of compounds for efficacy. The present invention provides the use of ethyl parahydroxybenzoate in the preparation of a medicament for preventing or treating coronavirus infections. Currently, there are no reports on the aforementioned applications, particularly the use of ethyl parahydroxybenzoate in the treatment of human coronavirus HCoV-229E infections. Summary of the Invention

[0008] The present invention aims to provide an application of ethyl p-hydroxybenzoate in preparing a medicament for preventing or treating coronavirus infectious diseases.

[0009] Wherein, the molecular formula of ethyl p-hydroxybenzoate is C9H 10 O3, molecular weight is 166.18.

[0010] Preferably, the coronavirus infectious disease is a disease caused by infection with human coronavirus HCoV-229E.

[0011] Preferably, the diseases caused by human coronavirus HCoV-229E infection include upper respiratory tract infections, lower respiratory tract infections, gastrointestinal diseases, nervous system diseases, nephritis, and hepatitis, wherein upper respiratory tract infections include the common cold, viral pharyngitis and laryngitis, herpetic pharyngitis, pharyngoconjunctivitis, and tonsillitis, and lower respiratory tract infections include bronchitis and pneumonia. More preferably, they are pneumonia, nephritis, and hepatitis.

[0012] Preferably, the drug for preventing or treating coronavirus infectious diseases is a pharmaceutical preparation containing ethyl parahydroxybenzoate.

[0013] Wherein, the pharmaceutical preparation is a liquid preparation or a solid preparation prepared from ethyl p-hydroxybenzoate and appropriate pharmaceutical excipients.

[0014] The preparation may be an inhalation preparation, an injection, an oral preparation or a nasal spray.

[0015] More preferably, the mass percentage of ethyl p-hydroxybenzoate in the inhalation preparation is 0.01%-1%, and further preferably 0.01%-0.5%.

[0016] Among them, the inhalation preparation refers to an atomized inhalation solution, and the prescription composition is ethyl parahydroxybenzoate, sodium chloride, propylene glycol and water for injection.

[0017] Preferably, the amount of ethyl p-hydroxybenzoate is 0.1g-1g, the amount of sodium chloride is 8.0g-10.0g, the amount of propylene glycol is 1.0g-20.0g, and the amount of water for injection is added to a solution volume of 1000ml.

[0018] Its specific preparation method is:

[0019] Weigh the prescribed amount of ethyl p-hydroxybenzoate, propylene glycol and sodium chloride, add 1000 ml of water for injection at 20°C ~ 35°C, dilute and stir evenly, filter through a microporous filter membrane, and prepare according to the aseptic production process to obtain the product.

[0020] Beneficial Effects of the Invention: The technical solution of the present invention demonstrates novelty by verifying the novel use of ethyl parahydroxybenzoate in a mouse experiment infected with the human coronavirus HCoV-229E. Ethyl parahydroxybenzoate was also tested for its efficacy in treating pneumonia caused by HCoV-229E infection. The test results showed that ethyl parahydroxybenzoate exhibited good efficacy in terms of lung index and inhibition rate in pneumonia-infected mice. The present solution also demonstrates the use of ethyl parahydroxybenzoate as an antiviral drug through examples. DETAILED DESCRIPTION

[0021] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below.

[0022] Test Example 1

[0023] The pneumonia model of mice infected with human coronavirus HCoV-229E was used to evaluate the therapeutic effects of different compounds on the pneumonia model of mice infected with human coronavirus HCoV-229E from two aspects: lung index and inhibition rate.

[0024] 1. Experimental Materials

[0025] 1.1 The cell line was mouse mononuclear macrophage RAW264.7, purchased from Beijing Beina Chuanglian Biotechnology Research Institute, passaged in our laboratory, and stored in liquid nitrogen for future use.

[0026] 1.2 The virus strain is human coronavirus HCoV-229E, provided by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, passaged in our laboratory, and stored in a -80°C refrigerator for future use.

[0027] 1.3 The concentration of the test drugs was 0.02% for butyl parahydroxybenzoate, 0.02% for propyl parahydroxybenzoate, and 0.02% for ethyl parahydroxybenzoate.

[0028] 1.4 Experimental reagents are shown in Table 1.

[0029] Table 1:

[0030] Reagent name batch number Manufacturer Reagent use Ether 20150417 Sinopharm Chemical Reagent Co., Ltd. Mouse anesthesia

[0031] 1.5 Experimental instruments are shown in Table 2.

[0032] Table 2:

[0033]

[0034]

[0035] 2. Experimental Methods

[0036] 2.1 Drug preparation

[0037] Preparation of the test drug: refer to Examples 1-3, and administer by aerosol inhalation according to the above concentrations, 10 minutes each time, once a day, for 4 consecutive days.

[0038] 2.2 Virus passage

[0039] Take a 25cm2 monolayer of RAW264.7 cells 2 The culture flask was cultured, the culture medium was discarded, the cell surface was rinsed three times with cell maintenance medium, 5 ml of cell maintenance medium was added, and then 200 μl of HCoV-229E virus solution was added. The cells were cultured in a 37°C 5% CO2 incubator for 72 to 96 hours. The cell pathological changes were observed under an inverted microscope every day until 80% of the cells showed obvious pathological changes (CPE). The cell culture flask was placed in a -80°C low-temperature refrigerator for freezing. The virus solution was repeatedly frozen and thawed three times and then used for virus titer determination.

[0040] 2.3 Virus titer determination

[0041] A 96-well plate containing RAW264.7 cells grown as a monolayer was prepared. The culture medium was discarded and the cells were rinsed three times with cell maintenance medium. Eight dilutions (10-1 to 10-8) of HCoV-229E virus were inoculated at different titers, 100 μl per well. Four replicates of each dilution were prepared. A normal cell control was also included. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 72 to 96 hours. Cytopathic effects were observed daily under an inverted microscope and the cytopathic effect was recorded for each well. The 50% cytopathic concentration (TCID50) was calculated using the Reed-Muench method.

[0042] 2.4 Construction and drug administration of a mouse model of human coronavirus pneumonia

[0043] Forty ICR mice were randomly divided according to body weight into the following groups: a normal control group, a model control group, a butylparaben group, a propylparaben group, and an ethylparaben group. Each group consisted of eight mice, half male and half female. Except for the normal control group, mice in all other groups were lightly anesthetized with ether and then intranasally infected with 100 TCID50 HCoV-229E (50 μl / mouse). On the day of infection, each group received nebulized inhalation for 10 minutes daily for four consecutive days. Body weight was measured one day after the last dose, and lungs were dissected and weighed. Lung index and lung index inhibition rate were calculated.

[0044] 2.5 Calculation of mouse lung index and inhibition rate

[0045] After weighing the mice, the lung tissue was dissected and weighed, and the lung index and inhibition rate of the mice were calculated. The specific calculation formula is as follows:

[0046] Lung index = lung wet weight (g) × 100 / body weight (g)

[0047]

[0048] 2.6 Statistical analysis

[0049] The data were expressed as (MEAN±SD), and the differences between the groups were tested using T test. P<0.05 was considered statistically significant.

[0050] 3. Experimental Results

[0051] Table 3 Effects of the test drugs on the mouse model of human coronavirus pneumonia (lung index and inhibition rate)

[0052]

[0053] The results in Table 3 show that compared with the normal control group, the lung index of the mice in the model control group was significantly increased (P<0.01); compared with the model control group, the lung index of the mice in the ethylparaben group was significantly decreased (P<0.01). Compared with the butylparaben group and the propylparaben group, the ethylparaben group had a better therapeutic effect.

[0054] Test Example 2

[0055] A pneumonia model of mice infected with human coronavirus HCoV-229E was used to evaluate the therapeutic effect of ethyl paraben on the pneumonia model of mice infected with human coronavirus HCoV-229E from the aspects of lung index and inhibition rate.

[0056] 1. Experimental Materials

[0057] 1.1 The cell line was mouse mononuclear macrophage RAW264.7, purchased from Beijing Beina Chuanglian Biotechnology Research Institute, passaged in our laboratory, and stored in liquid nitrogen for future use.

[0058] 1.2 The virus strain is human coronavirus HCoV-229E, provided by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, passaged in our laboratory, and stored in a -80°C refrigerator for future use.

[0059] 1.3 The test drug was ethyl p-hydroxybenzoate (C9H 10 O3), also known as ethyl paraben, molecular weight 166.18.

[0060] 1.4 Experimental reagents are shown in Table 4.

[0061] Table 4 Experimental reagents

[0062] Reagent name batch number Manufacturer Reagent use Ether 20150417 Sinopharm Chemical Reagent Co., Ltd. Mouse anesthesia

[0063] 1.5 Experimental instruments are shown in Table 5.

[0064] Table 5 Experimental instruments

[0065]

[0066] 2. Experimental Methods

[0067] 2.1 Drug preparation

[0068] Preparation of the test drugs: According to Examples 4-7, ethyl p-hydroxybenzoate group 1 (0.005%), ethyl p-hydroxybenzoate group 2 (0.01%), ethyl p-hydroxybenzoate group 3 (0.025%), and ethyl p-hydroxybenzoate group 4 (0.05%) were prepared and administered directly by aerosol inhalation for 10 minutes each time, once a day, for 4 consecutive days.

[0069] 2.2 Virus passage

[0070] Take a 25cm2 monolayer of RAW264.7 cells 2 The culture flask was cultured, the culture medium was discarded, the cell surface was rinsed three times with cell maintenance medium, 5 ml of cell maintenance medium was added, and then 200 μl of HCoV-229E virus solution was added. The cells were cultured in a 37°C 5% CO2 incubator for 72 to 96 hours. The cell pathological changes were observed under an inverted microscope every day until 80% of the cells showed obvious pathological changes (CPE). The cell culture flask was placed in a -80°C low-temperature refrigerator for freezing. The virus solution was repeatedly frozen and thawed three times and then used for virus titer determination.

[0071] 2.3 Virus titer determination

[0072] A 96-well plate containing RAW264.7 cells grown as a monolayer was prepared. The culture medium was discarded and the cells were rinsed three times with cell maintenance medium. Eight dilutions (10-1 to 10-8) of HCoV-229E virus were inoculated at different titers, 100 μl per well. Four replicates of each dilution were prepared. A normal cell control was also included. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 72-96 hours. Cytopathic effects were observed daily under an inverted microscope and the cytopathic effect was recorded for each well. The 50% cytopathic concentration (TCID50) was calculated using the Reed-Muench method.

[0073] 2.4 Construction and drug administration of a mouse model of human coronavirus pneumonia

[0074] Seventy-two ICR mice were randomly divided according to body weight into the following groups: a normal control group, a model control group, and seven ethyl parahydroxybenzoate-administered groups. Each group consisted of eight mice, half male and half female. Except for the normal control group, mice in all other groups were lightly anesthetized with ether and then intranasally infected with 100 TCID50 HCoV-229E (50 μl / mouse). On the day of infection, each group received nebulized inhalation for 10 minutes daily for four consecutive days. Body weight was measured one day after the last dose, and lungs were dissected and weighed. Lung index and lung index inhibition rate were calculated.

[0075] 2.5 Calculation of mouse lung index and inhibition rate

[0076] After weighing the mice, the lung tissue was dissected and weighed, and the lung index and inhibition rate of the mice were calculated. The specific calculation formula is as follows:

[0077] Lung index = lung wet weight (g) × 100 / body weight (g)

[0078]

[0079] 2.6 Statistical analysis

[0080] The data were expressed as (MEAN±SD), and the differences between the groups were tested using T test. P<0.05 was considered statistically significant.

[0081] 3. Experimental Results

[0082] Table 6 Effects of the test drugs on the mouse model of human coronavirus pneumonia (lung index and inhibition rate)

[0083]

[0084] Note: Compared with the normal control group ## P<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0085] As shown in Table 6, compared with the normal control group, the lung index of mice in the model control group was significantly increased (P<0.01); compared with the model control group, the lung index of mice in groups 2, 3, and 4 of ethyl paraben was significantly reduced (P<0.05, P<0.01), with the best being group 4. Group 1 (0.005%) had no significant effect in inhibiting pneumonia in mice caused by human coronavirus 229E.

[0086] Example 1 Butyl p-hydroxybenzoate aerosol inhalation preparation

[0087] Butyl paraben 0.2g

[0088] Sodium chloride 9.0g

[0089] Add water for injection to 1000ml

[0090] Preparation process:

[0091] Step 1: Weigh 0.2 g of butyl parahydroxybenzoate and 9.0 g of sodium chloride, add 1000 ml of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane;

[0092] Step 2: Fill the ampoule and sterilize it with high-pressure steam at 115°C for 30 minutes.

[0093] Example 2 Propyl p-hydroxybenzoate aerosol inhalation preparation

[0094] Propyl paraben 0.2g

[0095] Sodium chloride 9.0g

[0096] Add water for injection to 1000ml

[0097] Preparation process:

[0098] Step 1: Weigh 0.2 g of propyl paraben and 9.0 g of sodium chloride, add 1000 ml of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane;

[0099] Step 2: Fill the ampoule and sterilize it with high-pressure steam at 115°C for 30 minutes.

[0100] Example 3 Ethyl p-hydroxybenzoate aerosol inhalation preparation

[0101] Ethyl parahydroxybenzoate 0.2g

[0102] Sodium chloride 9.0g

[0103] Add water for injection to 1000ml

[0104] Preparation process:

[0105] Step 1: Weigh 0.2 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 ml of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane;

[0106] Step 2: Fill the mixture into a glass ampoule and sterilize it by high-pressure steam at 115°C for 30 minutes.

[0107] Example 4 Ethyl p-hydroxybenzoate aerosol inhalation preparation

[0108] Ethyl parahydroxybenzoate 0.05g

[0109] Sodium chloride 9.0g

[0110] Add water for injection to 1000ml

[0111] Preparation process:

[0112] Step 1: Weigh 0.05 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 ml of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane;

[0113] Step 2: Fill and seal the mixture into a glass ampoule under a sterile environment.

[0114] Example 5 Ethyl p-hydroxybenzoate aerosol inhalation preparation

[0115] Ethyl parahydroxybenzoate 0.1g

[0116] Sodium chloride 9.0g

[0117] Add water for injection to 1000ml

[0118] Preparation process:

[0119] Step 1: Weigh 0.1 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 ml of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane;

[0120] Step 2: Fill the mixture into a brown ampoule under a sterile environment and sterilize it with high-pressure steam at 115°C for 30 minutes.

[0121] Example 6 Ethyl p-hydroxybenzoate aerosol inhalation preparation

[0122] Ethyl parahydroxybenzoate 0.25g

[0123] Sodium chloride 9.0g

[0124] Add water for injection to 1000ml

[0125] Preparation process:

[0126] Step 1: Weigh 0.25 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 ml of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane;

[0127] Step 2: Fill the mixture into a brown ampoule under a sterile environment and sterilize it with high-pressure steam at 115°C for 30 minutes.

[0128] Example 7 Ethyl p-hydroxybenzoate aerosol inhalation preparation

[0129] Ethyl parahydroxybenzoate 0.5g

[0130] Sodium chloride 9.0g

[0131] Add water for injection to 1000ml

[0132] Preparation process:

[0133] Step 1: Weigh 0.5 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 ml of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane;

[0134] Step 2: Fill the mixture into a brown ampoule under a sterile environment and sterilize it with high-pressure steam at 115°C for 30 minutes.

Claims

1. The use of ethyl p-hydroxybenzoate for the preparation of a drug for preventing or treating coronavirus infectious diseases, wherein the molecular formula of the ethyl p-hydroxybenzoate is C9H 10 O3, with a molecular weight of 166.18, the coronavirus infectious disease is pneumonia caused by infection with human coronavirus HCoV-229E, and the drug for preventing or treating coronavirus infectious diseases refers to an inhalation preparation containing ethyl parahydroxybenzoate, wherein the mass percentage of ethyl parahydroxybenzoate in the inhalation preparation is 0.01%-0.05%.

2. The use according to claim 1, characterized in that The inhalation preparation refers to an atomized inhalation solution, the prescription composition of which is 0.1g of ethyl parahydroxybenzoate, 9.0g of sodium chloride, and water for injection added to 1000mL; The preparation process of the atomized inhalation solution is as follows: Step 1: Weigh 0.1 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 mL of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane; Step 2: Fill the mixture into a brown ampoule under a sterile environment and sterilize it with high-pressure steam at 115°C for 30 minutes.

3. The use according to claim 1, characterized in that The inhalation preparation refers to an atomized inhalation solution, the prescription composition of which is 0.25g of ethyl parahydroxybenzoate, 9.0g of sodium chloride, and water for injection added to 1000mL; The preparation process of the atomized inhalation solution is as follows: Step 1: Weigh 0.25 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 mL of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane; Step 2: Fill the mixture into a brown ampoule under a sterile environment and sterilize it with high-pressure steam at 115°C for 30 minutes.

4. The use according to claim 1, characterized in that The inhalation preparation refers to an atomized inhalation solution, the prescription composition of which is 0.5g of ethyl parahydroxybenzoate, 9.0g of sodium chloride, and water for injection added to 1000mL; The preparation process of the atomized inhalation solution is as follows: Step 1: Weigh 0.5 g of ethyl p-hydroxybenzoate and 9.0 g of sodium chloride, add 1000 mL of water for injection at 20°C to 35°C, dilute, stir evenly, and filter through a 0.22 μm microporous membrane; Step 2: Fill the mixture into a brown ampoule under a sterile environment and sterilize it with high-pressure steam at 115°C for 30 minutes.

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