WO2021203703A1 - Use of carglumic acid in preparation of drugs for preventing and treating coronaviruses - Google Patents

Abstract

Disclosed are the use of carglumic acid in the preparation of drugs for preventing and treating coronaviruses and the use of carglumic 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 for the novel coronavirus of novel coronavirus pneumonia is 13.92 μM, the toxicity is low and a good therapeutic window is achieved.

Description

Application of kalglutamic acid in preparation of drugs for prevention and treatment of coronavirus Technical field

The present invention belongs to the field of antiviral drugs, and specifically relates to the application of carboglutamate in the preparation of drugs for the prevention and treatment of anti-coronaviruses.

Background technique

New coronary pneumonia (COVID-19) is an infectious disease caused by a new type of coronavirus (SARS-Cov-2) infecting the human body. Its symptoms mainly include fever, fatigue, dry cough, dyspnea, and kidney failure. [The Lancet, 2020, 395(10223):507-513; The Lancet, 2020,395(10223):497-506]. New crown pneumonia began to report cases in December 2019, and then many countries around the world, including South Korea, Japan, Thailand, Iran, Singapore, Germany, France, and the United States, have also seen cases one after another. 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. Prime 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]. SARS-Cov-2 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, and 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 is of a different type. Although its fatality rate is lower than SARS-CoV, it is far more infectious. In SARS-CoV. The global spread shows that the new coronavirus pneumonia epidemic still has the possibility of spreading. The development of effective antiviral drugs and vaccines has become the most urgent task at the moment. At present, the treatment of COVID-19 mainly relies on symptomatic adjuvant treatment, and there is still a lack of effective specific drugs and vaccines. However, the development of new drugs and the preparation of vaccines is a time-consuming process. Not only is the research and development cycle long, mass production after market approval is also a time-consuming project. Searching for "old drugs" with anti-SARS-Cov-2 virus infection effects from marketed drugs is an effective strategy for the treatment and prevention of COVID-19, an explosive infectious disease. The anti-SARS-Cov-2 virus drugs currently under study mainly include RNA polymerase inhibitors, beta interferon, monoclonal antibodies, and vaccines. However, so far, there are no definite and effective antiviral drugs and vaccines. Data from the analysis of antiviral activity of these drugs in vitro, the majority of IC ₅₀ antiviral drugs is in between the low micromolar to micromolar, still observed in vivo efficacy.

卡谷氨酸是一种甲酰磷酸合成酶1(CPS1)激活剂，治疗由N-乙酰酶合成障碍，异戊酸血症和甲基丙二酸血症以及丙酸血症引起的高血氨症(又称尿素循环代谢病)。鉴于药 物虚拟筛选的结果我们开展了卡谷氨酸在新冠肺炎新型冠状病毒抗病毒药效的应用性研究。 Caglutamic acid was developed by Orphan Europe. It was approved by the European EMA on January 24, 2003, and was approved by the US FDA on March 18, 2010. It was approved by Japan PMDA on September 28, 2016. It was approved by Orphan Europe and Pola Pharma is responsible for its marketing and sales under the trade name

Calglutamate is an activator of formyl phosphate synthase 1 (CPS1), which treats hyperemia caused by N-acetylase synthesis disorder, isovaleric acidemia, methylmalonic acidemia, and propionic acidemia Ammonia (also known as urea cycle metabolic disease). In view of the results of the virtual screening of drugs, we have carried out an applied research on the antiviral efficacy of carglutamic acid in the new coronavirus of new coronary pneumonia.

The development of effective drugs for the treatment of coronavirus pneumonia has become an urgent issue that needs to be resolved. In the aspect of research and development of new coronavirus (SARS-Cov-2) virus drugs, the present invention studies the anti-SARS-Cov-2 virus effect of carglutamic acid.

Summary of the invention

The purpose of the present invention is to provide the application of carglutamic acid in the preparation of drugs for preventing or treating diseases caused by coronavirus.

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

The present invention provides carglutamic 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 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 an infectious disease or its complications caused by any virus of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, HCoV-OC43, HKU1 or MERS-CoV ; Preferably it is a respiratory infection disease, for example, severe acute respiratory syndrome, severe acute respiratory syndrome coronavirus type 2, and Middle East respiratory syndrome.

In the technical scheme of the present invention, calglutamic acid is as shown in structural formula (1)

In the technical solution of the present invention, calglutamic 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 solution of the present invention, carglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs, and other anti- The application of the composition prepared by the virus 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 aldehyde, lamivudine, lobukavir, lordadenosine, lopinavir, memotine hydrochloride, methyl red thiourea, narfinavir , Nevirapine, Penciclovir, Pirodavir, 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 of the coronavirus family. Mixtures of stereoisomers, 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 calglutamic acid as a disinfectant against coronaviruses of the coronavirus family.

Another aspect of the present invention provides a disinfectant for eliminating contamination by coronaviruses of the coronavirus family. The disinfectant includes cardglutamic acid.

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 carglutamic acid or a pharmaceutically acceptable salt, isotope, stereoisomer, stereo A mixture of isomers, tautomers, esters, amides 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 applying a therapeutically effective amount of carglutamic 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 discloses the application of a medicine with carboglutamic acid as the main component and its pharmaceutically acceptable salt in anti-new coronary pneumonia (COVID-19) new type coronavirus (SARS-Cov-2) infection. The present invention finds for the first time that carboglutamic acid has an antiviral effect on the new coronavirus, can block the infection of host cells by the new coronavirus, and can be used as a disease treatment for the treatment of the new coronavirus infection of the new coronary pneumonia.

_{The half effective concentration (EC 50} ) of kallu-glutamic acid in African green monkey kidney cells (VeroE6) against the new type of coronavirus (SARS-Cov-2) is 13.92μM, with low toxicity and a good therapeutic window.

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, 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, and placed in the minimum Eagle's medium (MEM; GibcoInvitrogen) containing 10% fetal bovine serum (FBS; GibcoInvitrogen). Incubate at 37°C with 5% CO ₂ 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 kaglutamic acid

Calglutamic acid powder was dissolved in DMSO, and then diluted to 20mg/mL by adding culture solution. The final concentration of DMSO was 1%. After filtering through a 0.22μm membrane, it was stored at 4°C; after filtration, it was stored at 4°C. Inoculate about 2.5×10 ⁴ cells per well into a 96-well plate. After 24 to 48 hours, after the cells have grown 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 Efficacy evaluation of kaglutamic acid against new coronary 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 with 4 replicate wells, with 200 μM 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 cells is recorded according to the 6-level standard. After recording CPE, stain with CCK8 method to determine OD value. Use Reed-Muench method to calculate the half effective concentration (EC ₅₀ ) of the drug. Cardglutamine _{The half effective concentration (EC 50} ) of acid for the new coronavirus (SARS-Cov-2) of new coronary pneumonia (SARS-Cov-2) is 13.92μM. The experimental results show that the half effective concentration of foscarnet sodium is low in antiviral and has good antiviral effect.

Example 4 Immunofluorescence detection

Immunofluorescence microscope: In order to detect the expression of viral proteins in VeroE6 cells, the cells of Example 3 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 kaglutamic acid can effectively kill the virus in the cell, and has a small effect on the cell, which has a good therapeutic window.

Claims (10)

Calglutamic 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 Application of the drug. 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 calglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs 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 Calglutamic 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, sipanphylline, en Tritabine, Cytarabine Hydrochloride, Delaviridine Mesylate, Deciclovir, Didanosine, Dioxali, Edouridine, Emivirin, Etracitapine, Enemy Veraden, Enviroxime, Hepudin, Famciclovir, Chlorophenhydroisoquine Hydrochloride, Fecitabine, Feiridine, Phosphatide, Sodium Foscarnet, Phosphonoacetate Sodium, Ganciclovir Sodium, Iodoside, Indinavir, ethoxybutanone, lamivudine, lobukavir, lordadenosine, lopinavir, memotine hydrochloride, methyl red thiourea, narfinavir, nevirapine, penxix Lovir, Pirotavir, Ribavirin, Saquinavir Mesylate, Ritonavir, Somantine Hydrochloride, Sorivudine, Penicillin, Stavudine, Tenofovir , Tirolone Hydrochloride, Trifluridine, Valacyclovir Hydrochloride, Vidarabine, Vidarabine Phosphate, Vidarabine Sodium Phosphate, Tiranavir, Verooxime, Zalcitabine, Qi Dovudine, Jingwei oxime. A pharmaceutical composition for the treatment or prevention of diseases caused by coronavirus, said pharmaceutical composition comprising carboglutamic acid or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, and interconversions 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 coronaviruses of the coronavirus family, the disinfectant includes cardglutamic acid. A method for the treatment or prevention of diseases caused by coronaviruses in the coronavirus family, comprising adding a therapeutically effective amount of carglutamic 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.