WO2021203702A1 - Use of pyroglutamic acid in preparation of drugs for preventing and treating novel coronavirus of novel coronavirus pneumonia - Google Patents

Abstract

Disclosed are the use of pyroglutamic acid in the preparation of drugs for preventing and treating novel coronavirus of novel coronavirus pneumonia and the use of pyroglutamic acid or pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs thereof in the preparation of drugs for preventing and/or treating diseases caused by coronaviruses. The coronaviruses are the novel coronaviruses SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV. The median effective concentration of pyroglutamic acid for the novel coronavirus of new coronavirus pneumonia is 9.34 μM. The median effective concentration is low, and the antiviral effect is good.

Description

[Name of invention developed by ISA according to Rule 37.2] Application of pyroglutamic acid in the preparation of drugs for the prevention and treatment of new coronary pneumonia and new coronavirus Technical field

The invention belongs to the field of antiviral drugs, and specifically relates to the application of pyroglutamic acid in the preparation of drugs for the prevention and treatment of new coronaviruses against new coronary pneumonia.

Background technique

Coronavirus pneumonia (Corona Virus Disease 2019) is an infectious disease caused by a new coronavirus (SARS-Cov-2) infecting the human body. Its symptoms mainly include fever, fatigue, dry cough, difficulty breathing, and kidney failure. [The Lancet ,2020,395(10223):507-513; The Lancet,2020,395(10223):497-506]. New coronary pneumonia cases were reported in December 2019, followed by cases in many countries around the world, including South Korea, Japan, Thailand, Iran, Singapore, Germany, France and the United States. Coronaviruses belong to the genus Coronavirus in the family Coronaviridae (Coronaviridae). The virus of the genus Coronavirus is a positive-stranded single-stranded RNA virus with an envelope, with a diameter of about 80-120nm. Its genetic material is the largest among all RNA viruses, and generally only infects humans, mice, pigs, cats, and dogs. , Poultry vertebrates. The coronavirus was first isolated from chickens in 1937. Coronavirus particles are irregular in shape, about 60-220nm in diameter. The virus has an envelope structure with three proteins: Spike Protein (S, Spike Protein), Small Envelope Protein (E, Envelope Protein) and Membrane Protein (M, Membrane Protein), and a few types also have hemagglutination. Glycoprotein (HE protein, Haemaglutinin-esterase) [Nederlands Tijdschrift Voor Geneeskunde, 2014, 158(158): A8119-A8119].

The diameter of SARS-Cov-2 virus particles is between 60nm and 140nm, and there are 9-12nm spikes outside the envelope, resembling a corolla. Genome sequencing showed that SARS-Cov-2 is a single-stranded RNA coronavirus. By comparison with the gene sequences of other virus samples, it is found that SARS-Cov-2 is similar to SARS-CoV (79.5%) [Nature,2020] and bat coronavirus (96%) [bioRxiv,2020,2020.01.22.914952], and speculates The virus may originate from bats [bioRxiv, 2020, 2020.01.24.915157; Nature, 2020]. The 2019-nCoV virus belongs to βCoV, which is the seventh member of the HCoV family that is different from SARS-CoV and MERS-CoV [New England Journal of Medicine, 2020]. The remaining 6 members include HCoV 229E, NL63, OC43, HKU1, SARS -CoV and MERS-CoV.

The new type of coronavirus pneumonia is caused by a new type of coronavirus. It is the same type of coronavirus as SARS-CoV, which is known to cause atypical pneumonia, but of different types. Although its fatality rate is lower than SARS-CoV, it is far more infectious. In SARS-CoV. Since then, South Korea, Japan, Thailand, Iran, Singapore, Germany, France, and the United States have all seen confirmed cases of the disease, indicating that the new coronavirus pneumonia epidemic may still spread. But so far, there is still no specific medicine that can cure the new type of coronavirus pneumonia. Most of the current treatment methods are symptomatic treatment, especially for some critically ill patients. Therefore, the development of effective drugs for the treatment of coronavirus pneumonia has become an urgent issue that needs to be resolved. In the research and development of new coronavirus (SARS-Cov-2) virus drugs, the present invention studies the anti-SARS-Cov-2 virus effect of L-pyroglutamic acid.

Summary of the invention

The purpose of the present invention is to provide the application of L-pyroglutamic acid in the preparation of drugs for the prevention or treatment of new coronaviruses against new coronary pneumonia.

Specifically, in order to solve the technical problems of the present invention, the following technical solutions are adopted:

The present invention provides pyroglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs in the preparation of prevention and/or treatment of coronary Application in medicines for diseases caused by viruses.

In the technical scheme of the present invention, pyroglutamic acid is L-pyroglutamic acid, D-pyroglutamic acid or a mixture thereof.

In the technical solution of the present invention, the coronaviruses are the new coronaviruses SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV.

In the technical solution of the present invention, the disease caused by the coronavirus is pneumonia or its complications caused by any virus of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV.

In the technical scheme of the present invention, L-pyroglutamic acid is represented by structural formula (1)

In the technical scheme of the present invention, pyroglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs are used as the sole active ingredient Application in the preparation of medicines for preventing and/or treating diseases caused by coronaviruses.

In the technical scheme of the present invention, pyroglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs and other antiviral The application of the composition prepared by the medicine as an active ingredient in the preparation of a medicine for preventing and/or treating diseases caused by coronavirus.

In the technical scheme of the present invention, other antiviral drugs are selected from the group consisting of ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemontan, and acyclovir Sodium, adefovir, alovudine, avirsuto, tricyclodecylamine hydrochloride, alanordine, aridone, ateviridine mesylate, afridine, cidofovir, western Panphylline, Emtricitabine, Cytarabine Hydrochloride, Delavirdine Mesylate, Deciclovir, Didanosine, Dioxali, Edouridine, Emivirin, Etrados Citapine, Enviraden, Enviroxime, Hepatin, Famciclovir, Chlorophenhydroisoquine Hydrochloride, Fecitabine, Feauridine, Phosphatide, Sodium Foscarnet, Sodium Phosphoacetate, Ganciclovir Sodium, iodoside, indinavir, ethoxybutanone, lamivudine, lobukavir, lordadenosine, lopinavir, memotine hydrochloride, methyl red thiourea, narfinavir , Nevirapine, Penciclovir, Pirotavir, Ribavirin, Saquinavir Mesylate, Ritonavir, Somantine Hydrochloride, Sorivudine, Penicillin, Stavudine , Tenofovir, Tirolone Hydrochloride, Trifluridine, Valaciclovir Hydrochloride, Vidarabine, Vidarabine Phosphate, Vidarabine Sodium Phosphate, Tiranavir, Verooxime, Zha Citabine, zidovudine, virgin oxime.

Another aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of diseases caused by coronaviruses, which comprises pyroglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, and stereoisomers. Mixtures, tautomers, esters, amides or prodrugs.

In the technical scheme of the present invention, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

In the technical scheme of the present invention, the dosage form of the pharmaceutical composition is oral preparation, pulmonary inhalation preparation, mucosal administration preparation, ophthalmic preparation or injection.

In the technical scheme of the present invention, the oral preparation is selected from granules, powders, pills, tablets, capsules or oral liquids.

Another aspect of the present invention provides the use of pyroglutamic acid as a disinfectant against coronaviruses of the coronavirus family.

Another aspect of the present invention provides a method for treating diseases caused by viruses of the coronavirus family, comprising adding a therapeutically effective amount of pyroglutamic acid or a pharmaceutically acceptable salt, isotope, stereoisomer, stereo A mixture of isomers, tautomers, esters or prodrugs is administered to the subject.

Another aspect of the present invention provides a method for preventing a subject from being infected with coronaviruses of the coronavirus family, which comprises adding a therapeutically effective amount of pyroglutamic acid or a pharmaceutically acceptable salt, isotope, stereoisomer, Administration of mixtures of stereoisomers, tautomers, esters or prodrugs is administered to the subject prior to infection.

Beneficial effect

The present invention proves for the first time that pyroglutamic acid has a high therapeutic index and a low half effective concentration; the pyroglutamic acid can effectively increase the antivirus effect before being infected. Pyroglutamate is used as an effective drug in the treatment of new coronavirus infections caused by new coronary pneumonia.

Detailed ways

In order to make the above objectives, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, but they should not be understood as limiting the scope of implementation of the present invention.

Example 1. Virus amplification

The African green monkey kidney cells (VeroE6) were inoculated into 96-well plates at ^{3×10 5 cells/well.} ; GibcoInvitrogen), incubate at 37°C, 5% CO ₂ and wait until the monolayer grows up. A 100-fold dilution of the clinical isolates of the new coronavirus pneumonia new type coronavirus was inoculated into a 96-well plate full of monolayer cells, and incubated at 37°C and 5% CO _{2 for} two days (including the normal control group).

Two days later, the degree of disease was as high as 75%, placed in a -80℃ ultra-low temperature refrigerator, repeated freezing and thawing once, collecting the amplified virus solution, centrifuged at 3000r/min for 30 minutes, removing the sediment, and placing the small tubes in the -80℃ ultra-low temperature refrigerator Long-term preservation.

Example 2 Evaluation of the toxicity of L-pyroglutamic acid

After dissolving the L-pyroglutamic acid powder in DMSO, add culture broth to dilute it to 20mg/mL, the final concentration of DMSO is 1%, filter through 0.22μm membrane and store at 4℃; after filtration, store at 4℃. Inoculate about 2.5×10 ⁴ cells per well into a 96-well plate. After 24 to 48 hours, when the cells grow into a monolayer, discard the culture medium, add 100μL/well of drugs of different dilutions, and 100μL/well for normal cell control wells. MEM, 37°C and 5% CO ₂ continue to incubate for 2 to 5 days, add 20 μL of CCK8 solution (5 mg/mL) to each well, and place in a 37°C 5% CO ₂ incubator for further incubation for 4 hours. Aspirate and discard the culture supernatant, add 100 μL dimethyl sulfoxide (DMSO) to each well, and shake at low speed for 10 minutes to fully melt the crystals. Choose the wavelength of 490nm, and measure the light absorption value of each hole on the enzyme-linked immunosorbent monitor. Through the toxicity evaluation, it can be seen that when the concentration of the drug added reaches 400 μM, there is still no obvious cytotoxicity, indicating that the cytotoxicity of the drug is low and it has a good therapeutic window.

Example 3 Evaluation of the efficacy of L-pyroglutamic acid against new coronavirus pneumonia and new coronavirus

In order to evaluate the antiviral efficacy of the drug, VeroE6 cells were cultured overnight in a 48-well cell culture dish with a ^{density of 5×10 4 cells/well.} Virus (MOI of 0.05) was added to make it infected for 2 hours. Then add 2 times the gradient dilution of the drug, each concentration is set to 4 replicate wells, with the maximum non-toxic concentration as the initial concentration of the drug, incubate for 2 days _{in a 34°C, 5% CO 2 incubator.} Record Cytopathogenic Effect (CPE). The occurrence of CPE in the cells was recorded according to the level 6 standard. After the CPE was recorded, it was stained with CCK8, and the OD value was measured. L- pyroglutamic new crown anti-pneumonia novel coronavirus efficacy evaluation efficacy evaluation of L- pyroglutamic CCK8 by law, the median effective concentration for the new crown pneumonia novel coronavirus (SARS-Cov-2) of (EC ₅₀ ) Is 9.34μM, the half effective concentration is low, indicating that the antiviral effect is good.

Example 4 Immunofluorescence

Immunofluorescence microscope: In order to detect the expression of viral proteins in VeroE6 cells, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% TritonX-100. The cells were then blocked with 5% bovine serum albumin (BSA) for 2 hours at room temperature. Incubate the cells with the primary antibody (polyclonal antibody against the nucleocapsid protein of the bat SARS-related coronavirus, anti-NP, at a dilution of 1:1000) for 2 hours, and then incubate the antibody with the secondary antibody (488 AffiniPure Donkey Anti-Rabbit) IgG (H+L). The nucleus is stained with Hoechst33258 dye (Beyotime, China). Observation by fluorescence microscope shows that pyroglutamate can effectively kill the virus in the cell, and has a small effect on the cell, which has a good therapeutic window.

Claims (10)

Pyroglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs are used in the preparation of prevention and/or treatment of diseases caused by coronavirus The application of drugs. The use according to claim 1, wherein the coronaviruses are new coronaviruses SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV. The use according to claim 1, the disease caused by the coronavirus is an infectious disease caused by any virus of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, HCoV-OC43, HKU1, or MERS-CoV or its complications The infectious disease is preferably a respiratory tract infection disease. The use according to claim 1, wherein pyroglutamic acid or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomers, esters, amides or prodrugs thereof are used as the sole active The application of the ingredients in the preparation of drugs for the prevention and/or treatment of diseases caused by coronavirus; or Pyroglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs, in combination with other antiviral drugs as active ingredients Application in the preparation of medicines for the prevention and/or treatment of diseases caused by coronavirus; Preferably, the other antiviral drugs are selected from ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemonam, acyclovir sodium, adefo Vir, alovudine, avirsuto, tricyclodecylamine hydrochloride, alanordine, aridone, ateviridine mesylate, afridine, cidofovir, sipantheophylline, en Tritabine, Cytarabine Hydrochloride, Delavirdine Mesylate, Deciclovir, Didanosine, Dioxali, Edouridine, Emivirin, Etrexitapine, Enemy Veraden, Enviroxime, Hepudin, Famciclovir, Chlorophenhydroisoquine Hydrochloride, Fecitabine, Feuridine, Phosphatide, Pyroglutamic Acid, Sodium Phosphonoacetate, Ganciclovir Sodium, Iodine Glycoside, indinavir, ethoxybutanone, lamivudine, lobukavir, lordadenosine, lopinavir, memotine hydrochloride, methyl red thiourea, narfinavir, nevirapine, Penciclovir, Pirotavir, Ribavirin, Saquinavir Mesylate, Ritonavir, Somantine Hydrochloride, Solivudine, Penicillin, Stavudine, Tenol Fovir, Tirolone Hydrochloride, Trifluridine, Valaciclovir Hydrochloride, Vidarabine, Vidarabine Phosphate, Vidarabine Sodium Phosphate, Tiranavir, Verooxime, Zalcitabine , Zidovudine, Jingwei oxime. A pharmaceutical composition for treating or preventing diseases caused by coronavirus, the pharmaceutical composition comprising pyroglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, and interconversion Isomer, ester, amide or prodrug. The pharmaceutical composition according to claim 5, further comprising pharmaceutically acceptable excipients. The pharmaceutical composition according to claim 5, wherein the dosage form of the pharmaceutical composition is an oral preparation, a pulmonary inhalation preparation, a mucosal administration preparation, an ophthalmic preparation or an injection; preferably, the oral preparation is selected from granules, powders, and pills , Tablets, capsules or oral liquid. A disinfectant for eliminating the pollution of the coronavirus family virus, the disinfectant includes pyroglutamic acid. A method for the treatment or prevention of diseases caused by coronaviruses in the coronavirus family, which comprises adding a therapeutically effective amount of pyroglutamic acid or a pharmaceutically acceptable salt, isotope, stereoisomer, or mixture of stereoisomers , Tautomers, esters, amides or prodrugs are administered to the subject. The pharmaceutical composition according to any one of claims 5-7 or the disinfectant according to claim 8 or the method according to claim 9, wherein the coronavirus is a new coronavirus SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV.