WO2024088403A1 - 异喹啉类生物碱在预防和治疗冠状病毒感染所致疾病中的应用 - Google Patents

异喹啉类生物碱在预防和治疗冠状病毒感染所致疾病中的应用 Download PDF

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WO2024088403A1
WO2024088403A1 PCT/CN2023/127237 CN2023127237W WO2024088403A1 WO 2024088403 A1 WO2024088403 A1 WO 2024088403A1 CN 2023127237 W CN2023127237 W CN 2023127237W WO 2024088403 A1 WO2024088403 A1 WO 2024088403A1
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acid
virus
cov
sars
isoquinoline
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French (fr)
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童贻刚
陈士林
范华昊
冷梁
徐志超
宋驰
邹忠梅
洪碧霞
刘珂
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Beijing University of Chemical Technology
Northeast Forestry University
Institute of Medicinal Plant Development of CAMS and PUMC
Chengdu University of Traditional Chinese Medicine
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Beijing University of Chemical Technology
Northeast Forestry University
Institute of Medicinal Plant Development of CAMS and PUMC
Chengdu University of Traditional Chinese Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4748—Quinolines; Isoquinolines forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
    • A61K8/4926—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having six membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts

Definitions

  • the present invention belongs to the field of medical technology and relates to a new use of isoquinoline alkaloids in medicine, specifically to the use of isoquinoline alkaloids in preventing or treating SARS-CoV-2 viral infectious diseases.
  • the oral anti-new crown drug Molnupiravir jointly developed by Merck and Ridgeback Biotherapeutics and Paxlovid tablets/ritonavir tablets developed by Pfizer were conditionally approved for marketing.
  • These two drugs target the virus's RdRp (RNA-dependent RNA polymerase) and 3CLpro, respectively, but there is drug resistance.
  • RdRp RNA-dependent RNA polymerase
  • 3CLpro 3CLpro
  • the threat of infection, and the possibility of monopivir causing DNA mutations requires further study for its safety.
  • the combination of namatevir tablets/ritonavir tablets and drug interactions require further study, and the clinical effects of these two drugs in the vaccinated population are still unknown.
  • azithromycin was conditionally approved as my country's first independently developed oral small molecule COVID-19 treatment drug with completely independent intellectual property rights. Its target is similar to that of monopivir, targeting the RdRp of the virus. Similarly, its safety as a nucleoside analog still needs further study.
  • Vaccination is the most effective method in the long-term strategy of preventing and controlling COVID-19.
  • Different SARS-CoV-2 vaccines have been developed. As of March 28, 2022, 153 vaccines have been approved for clinical trials, 196 vaccines are in the preclinical trial stage, 10 vaccines (including 3 Indian vaccines) have been approved for emergency use by the World Health Organization, and 4 domestically developed new crown vaccines have been approved for marketing in China (https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape/).
  • vaccines mainly include inactivated vaccines (14% of the total), live attenuated vaccines (1%), viral vector vaccines (replicating and non-replicating; 17% of the total), RNA vaccines (18%), DNA vaccines (11%), protein subunit vaccines (34%), and VLP vaccines (4%).
  • inactivated vaccines (14% of the total
  • live attenuated vaccines 1%
  • viral vector vaccines replicating and non-replicating; 17% of the total
  • RNA vaccines (18%)
  • DNA vaccines (11%
  • protein subunit vaccines 34%
  • VLP vaccines 4%
  • novel coronavirus vaccine and "special medicines” continue to "fail", so there is an urgent need to develop new antiviral drugs targeting different targets, or use combination therapy to inhibit viral infection from multiple aspects.
  • drugs that block viral infection or inhibit viral replication for the novel coronavirus itself, and consider combined medication on the other hand, target the interaction between the novel coronavirus and the host, block a series of clinical symptoms and complications caused by the novel coronavirus, and reduce the mortality rate. Therefore, it is of great significance to develop safe, effective and broad-spectrum antiviral drugs for the novel coronavirus epidemic and possible coronavirus diseases in the future.
  • Alkaloids are a class of nitrogen-containing alkaline organic compounds that exist in nature. Most of them have a complex ring structure with nitrogen atoms bound inside the ring. Among them, isoquinoline alkaloids are the most numerous. Isoquinoline alkaloids are derived from phenylalanine and tyrosine, are widely distributed in plants, are numerous, and have a variety of biological activities. Isoquinoline alkaloids can be divided into simple isoquinolines, benzyl isoquinolines, and phenylisoquinolines according to the different connecting groups.
  • the benzophenanthridines are benzophenanthridines, aporphenanthridines, bisbenzylisoquinolines, berberines, protopines, morphines and benzophenanthridines.
  • the phenyl group is attached to the skeleton of isoquinoline, the derivative is called a benzylisoquinoline alkaloid.
  • Monobenzylisoquinolines are alkaloids with a benzyl group attached to the 1st position of the isoquinoline nucleus.
  • Papaverine which has an antispasmodic effect in the genus Papaver, belongs to this category.
  • Bisbenzylisoquinolines are a class of alkaloids in which two benzylisoquinolines are connected by 1 to 3 ether bonds, such as daurine, the main phenolic alkaloid found in the root of the Menispermaceae family, and tetrandrine A and tetrandrine B in Stephania tetrandria.
  • Isoquinoline alkaloids are widely distributed in the Papaveraceae, Babbittaceae, Ranunculaceae and Menispermaceae.
  • Isoquinoline and isoquinoline derivatives are an important class of pharmaceutical and chemical intermediates, widely used in the synthesis of a series of important drugs.
  • the isoquinoline skeleton is often used to design active structural groups in drug molecules, such as atracurium, nomifensine, moxaverine, demoxicillin (methyl papaverine), isoquincaine, etc.
  • isoquinoline alkaloid drugs have many aspects of biological activity, including anti-tumor, antibacterial, analgesic, immune function regulation, anti-platelet aggregation, anti-arrhythmia, and hypotension.
  • Most of the isoquinoline alkaloid drugs have basic structures such as isoquinoline or isoquinoline derivatives. Such drugs can basically be derived from simple isoquinoline derivatives by further synthesis.
  • the object of the present invention is to provide an effective inhibitor of SARS-CoV-2, which can prevent, alleviate and/or treat infection caused by the new coronavirus.
  • the present invention first provides an isoquinoline alkaloid and a pharmaceutically acceptable salt thereof as a novel coronavirus inhibitor, and uses the same in the preparation of a product for preventing, alleviating and/or treating an infection caused by a coronavirus;
  • the isoquinoline alkaloids are selected from at least one of cyclopentane, dauricine, dauricine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine (22864-92-2).
  • the pharmaceutically acceptable salt is selected from the acid addition salt of the nitrogen atom with sufficient basicity in the structure of the isoquinoline alkaloid, and the acid addition salt includes the isoquinoline alkaloid and an inorganic acid such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; an organic acid Such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicot
  • an inorganic acid such
  • the product is medicine, food, skin care product, cosmetics, daily necessities and health care products, preferably medicine.
  • the coronavirus is the 2019 novel coronavirus (SARS-CoV-2).
  • the coronavirus infection is novel coronavirus pneumonia.
  • the isoquinoline alkaloids and pharmaceutically acceptable salts thereof as described above can be configured into various suitable pharmaceutical preparation forms. They can be used alone, or mixed with pharmaceutical excipients (such as excipients, diluents, etc.) to be configured into tablets, capsules, granules, syrups, etc. for oral administration or powder injections, solutions for injection, and developed into functional foods, functional drinks, medicinal wines, nasal drops and mouthwashes, etc.
  • pharmaceutical excipients such as excipients, diluents, etc.
  • the present invention also provides a pharmaceutical composition for preventing, alleviating and/or treating infection caused by coronavirus, which comprises at least one of cyclopentane, dauricine, dauricine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine (22864-92-2).
  • the present invention also provides a method for preventing, alleviating and/or treating infection caused by coronavirus, comprising administering at least one of isoquinoline alkaloids and pharmaceutically acceptable salts thereof to an individual in need thereof.
  • the isoquinoline alkaloids are selected from at least one of cyclotine, dauricine, dauricine, thalidomide, thalidomide or 6-acetonyldihydrochelerythrine (22864-92-2).
  • the isoquinoline alkaloids of the present invention can effectively inhibit the infection and replication of pangolin coronavirus strain GX_P2V, SARS-CoV-2 virus-like particles (trVLP) and VSV pseudovirus, and can exert inhibitory effects before and after GX_P2V and SARS-CoV-2 virus-like particles (trVLP) enter the cell and at all times.
  • the EC 50 of cyclotine at the cellular level is 1.64 ⁇ M, CC 50 >50 ⁇ M, SI>30.49; the EC 50 of dauricine at the cellular level is 2.84 ⁇ M, CC 50 >50 ⁇ M, SI>17.61; the EC 50 of dauricine at the cellular level is ⁇ 1.52 ⁇ M, CC 50 >50 ⁇ M, SI> ⁇ 32.89; the EC 50 of thalidomide at the cellular level is ⁇ 25.29 ⁇ M, CC 50 >50 ⁇ M, SI> ⁇ 1.98; the EC 50 of thalidomide at the cellular level is 6.20 ⁇ M, CC 50 >50 ⁇ M, SI>8.06; the EC 50 of 6-acetonyldihydrochelerythrine at the cellular level is 2.25 ⁇ M, CC 50 >50 ⁇ M, SI>22.22.
  • the EC 50 of cyclinine at the cellular level is 6.54 ⁇ M; the EC 50 of batryticatine at the cellular level is 5.91 ⁇ M; the EC 50 of batryticatine at the cellular level is 8.48 ⁇ M; the EC 50 of thalidomide at the cellular level is 2.83 ⁇ M; the EC 50 of thalidomide at the cellular level is 2.73 ⁇ M; the EC 50 of 6-acetonyldihydrochelerythrine at the cellular level is 11.76 ⁇ M.
  • cyclinine, batryticatine, batryticatine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine can be used for the prevention, relief and/or treatment of COVID-19 infection.
  • isoquinoline alkaloids such as cyclopentane, daurine, daurine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine can block the entry of coronavirus into cells at the cellular level, and also have the effect of effectively inhibiting the replication of coronavirus, thereby achieving prevention, relief and/or treatment.
  • isoquinoline alkaloids such as cyclopentane, daurine, daurine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine
  • Figure 1 shows the test results of isoquinoline alkaloids such as cyclopentane, daurine, daurine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine inhibiting GX_P2V virus infection in Vero E6 cells;
  • Figure 2 shows the test results of different action stages of isoquinoline alkaloids such as cyclopentane, daurine, daurine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine in inhibiting GX_P2V virus infection in Vero E6 cells;
  • Figure 3 shows the test results of isoquinoline alkaloids cyclopentane, daurine, daurine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine inhibiting SARS-CoV-2 virus-like particles (trVLP) from infecting Caco-2-N cells;
  • Figure 4 shows the test results of different stages (viral RNA levels in cells) of the isoquinoline alkaloids cyclopentane, dauricine, dauricine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine in inhibiting the infection of SARS-CoV-2 virus-like particles (trVLP) in Caco-2-N cells;
  • Figure 5 shows the test results of isoquinoline alkaloids cyclopentane, daurine, daurine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine inhibiting the infection of SARS-CoV-2 wild-type pseudovirus in BHK21-ACE2 cells.
  • Isoquinoline alkaloids inhibit pangolin coronavirus xCoV (GX_P2V)
  • the African green monkey kidney cell line Vero E6 was obtained from the American Type Culture Collection (ATCC, No. 1586) and cultured in a 37°C, 5% CO 2 incubator using DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen).
  • Pangolin coronavirus xCoV (GX_P2V) (GISAID: EPI_ISL_410539) is a novel coronavirus xCoV isolated and cultured by the inventor from a dead pangolin seized by the customs. GX_P2V was propagated in Vero E6 cells, and the virus titer was determined by plaque assay using Vero E6 cells (ATCC, No. 1586). All infection experiments were performed in a biosafety level 2 (BLS-2) laboratory.
  • EC 50 assay 2.5 ⁇ 10 4 Vero E6 cells were inoculated into 96-well plates and cultured in an incubator at 37°C and 5% CO 2 for 24 h. Then, isoquinoline alkaloids such as cyclopentane, dauricine, dauricine, thalicyline, thalicyline, and 6-acetonyldihydrochelerythrine were added to the cell culture wells at final concentrations of 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, 3.125 ⁇ M, 1.56 ⁇ M, 0.78 ⁇ M, 0.39 ⁇ M, and 0 ⁇ M, respectively. The cells were pre-incubated at 37°C for 1 h.
  • isoquinoline alkaloids such as cyclopentane, dauricine, dauricine, thalicyline, thalicyline, and 6-acetonyldihydrochelerythrine
  • CC 50 assay CellTiter-Blue assay was used to assay CC 50. Vero E6 cells were seeded at 96 The test was performed when the cell density reached 60%-80%. After the Vero E6 cells were replaced with diluted drugs, the final concentrations were 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, 3.125 ⁇ M, 1.56 ⁇ M, 0.78 ⁇ M, 0.39 ⁇ M, and 0 ⁇ M. The cells were cultured at 37°C and 5% CO2 for 48h, and the fluorescence intensity was detected by CellTiter-Blue reagent, with an excitation wavelength of 554nm and an emission wavelength of 593nm. GraphPad-Prism 8 software was used for data analysis and calculation of CC50 .
  • Vero E6 cells were inoculated into a 24-well plate and cultured in an incubator at 37°C and 5% CO 2 for 24 hours.
  • the final concentration of isoquinoline alkaloids determined according to the EC 50 of the drug was added to the cell culture wells of the full-time administration group and the pre-intracellular administration group, respectively, and pre-incubated at 37°C for 1 hour.
  • the cells were infected with the above virus-drug mixture virus. After 2 hours of infection, PBS was washed 3 times and the medium was replaced.
  • the culture medium containing the same concentration of drugs was added to the cell culture wells of the full-time administration group and the post-intracellular administration group, and the culture medium without drugs was added to the cell culture wells of the pre-intracellular administration group and the control group, and cultured in an incubator at 37°C and 5% CO 2. 12 hours after virus infection, the expression of intracellular viral RNA and the intracellular reference gene GAPDH was quantitatively detected by qRT-PCR. At the same time, intracellular proteins were collected, and Western Blot was used to quantitatively analyze the SARS-CoV-2 N protein and GAPDH protein.
  • Isoquinoline alkaloids inhibit SARS-CoV-2 virus-like particles (trVLP)
  • Caco-2-N cells were cultured in high-glucose DMEM + 10% fetal bovine serum in a 37°C 5% CO 2 incubator. keep.
  • SARS-CoV-2 virus-like particles are a system with transcription and replication capabilities developed by Professor Qiang Ding's team at Tsinghua University.
  • the reporter gene (GFP) expressed by SARS-CoV-2trVLP replaces the nucleoprotein gene (N) required for viral genome packaging and viral particle assembly (SARS-CoV-2-GFP/ ⁇ NtrVLP).
  • the virus can complete the entire life cycle in Caco-2-N cells that ectopically express the SARS-CoV-2N protein. Since there is no N gene in the nucleic acid of the produced viral particles, the virus-like particles can only infect cells that overexpress the N gene, so they are not infectious to humans and animals.
  • EC 50 assay 2.5 ⁇ 10 4 Caco-2-N cells were seeded into 96-well plates and incubated at 37°C and 5% CO After culturing in an incubator at 2 for 24 h, isoquinoline alkaloids such as cyclotine, dauricine, dauricine, thalidomide, thalidomide and 6-acetonyldihydrochelerythrine were added to the cell culture wells at final concentrations of 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, 3.125 ⁇ M, 1.56 ⁇ M, 0.78 ⁇ M, 0.39 ⁇ M and 0 ⁇ M, respectively, and pre-incubated at 37 ° C for 1 h.
  • isoquinoline alkaloids such as cyclotine, dauricine, dauricine, thalidomide, thalidomide and 6-acetonyldihydrochelerythrine
  • CC 50 detection was detected using the CellTiter-Blue method. Caco-2-N cells were inoculated into 96-well cell culture plates, and the test was performed when the cell density reached 60%-80%. After the Caco-2-N cells were replaced with diluted drugs, the final concentrations were 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 6.25 ⁇ M, 3.125 ⁇ M, 1.56 ⁇ M, 0.78 ⁇ M, 0.39 ⁇ M, 0 ⁇ M. The cells were cultured at 37°C and 5% CO 2 for 48h, and the fluorescence intensity was detected using CellTiter-Blue reagent, with an excitation wavelength of 554nm and an emission wavelength of 593nm. GraphPad-Prism 8 software was used for data analysis and calculation of CC 50 .
  • Caco-2-N cells were inoculated into 24-well plates and cultured in an incubator at 37°C and 5% CO 2 for 24 hours.
  • the final concentration of isoquinoline alkaloids determined according to the EC 50 of the drug was added to the cell culture wells of the full-time administration group and the pre-intracellular administration group, respectively, and pre-incubated at 37°C for 1 hour.
  • the cells were infected with the above virus-drug mixture. After 2 hours of infection, the PBS was washed three times and the medium was replaced.
  • the culture medium containing the same concentration of drugs was added to the cell culture wells of the full-time administration group and the post-intracellular administration group, and the culture medium without drugs was added to the cell culture wells of the pre-intracellular administration group and the control group, and cultured in an incubator at 37°C and 5% CO 2. 12 hours after virus infection, the expression of intracellular viral RNA and the intracellular reference gene GAPDH was quantitatively detected by qRT-PCR. Data analysis was performed using GraphPad-Prism 8 software.
  • EC50 refers to the drug concentration that can effectively inhibit 50% of cells from being infected with the virus. The smaller the value, the better the drug's inhibitory effect on the virus.
  • CC50 is the drug concentration that causes cytotoxicity in 50% of cells. The higher the value, the lower the toxicity of the drug to cells.
  • SI Selectivity index, which is the ratio of CC 50 to EC 50. The larger the value, the higher the possibility of drug development.
  • BHK21-ACE2 and HEK-293T cells were cultured in high-glucose DMEM + 10% fetal bovine serum and incubated in a 37°C 5% CO2 incubator.
  • SARS-CoV-2 wild-type S protein pseudovirus adopts the VSV pseudovirus packaging system.
  • the assembly and determination process of the pseudovirus is based on the paper published by Nie et al. (Nie et al., Nat Protoc. 2020; 15(11): 3699-3715.).
  • the plasmid of SARS-CoV-2 wild-type S protein was transfected into HEK-293T cells to provide membrane protein on the cell surface, and G* ⁇ G-VSV was used to provide VSV genome.
  • the cell supernatant was collected 24h and 48h after infection and transfection to obtain SARS-CoV-2 wild-type S protein pseudovirus.
  • the corresponding final concentration of isoquinoline alkaloids was incubated with the cells at 37°C for 60-90 minutes. After the incubation, the pseudovirus-isoquinoline alkaloid mixture was added to the BHK21-ACE2 cells with the corresponding drug concentration. After culturing at 37°C for 24 hours, the expression of firefly luciferase in the cell lysate was detected using the Luciferase Assay System.
  • isoquinoline alkaloids cyclopentane, dauricine, dauricine, Thalictrine, thalictrine and 6-acetonyldihydrochelerythrine have good inhibitory effects on GX_P2V virus in a dose-dependent manner.
  • the above-mentioned isoquinoline alkaloids (cyclotine, daurine, daurine, thalictine, thalictine, 6-acetonyldihydrochelerythrine) have inhibitory effects on GX_P2V infection of Vero E6 cells before, after and during the whole period at the selected concentrations.
  • the above-mentioned isoquinoline alkaloids have inhibitory effects on GX_P2V infection of Vero E6 cells before, after and during the whole period at the nucleic acid and protein levels.
  • isoquinoline alkaloids (cyclotine, daurine, daurine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine) have good inhibitory effects on SARS-CoV-2 virus-like particles (trVLPs) on Caco-2-N cells, and have a dose-dependent effect.
  • the EC 50 of isoquinoline alkaloid cyclotine for SARS-CoV-2 virus-like particles (trVLP) at the cellular level is 1.64 ⁇ M, CC 50 >50 ⁇ M, and SI>30.49;
  • the EC 50 of dauricine for SARS-CoV-2 virus-like particles (trVLP) at the cellular level is 2.84 ⁇ M, CC 50 >50 ⁇ M, and SI>17.61;
  • the EC 50 of dauricine for SARS-CoV-2 virus-like particles (trVLP) at the cellular level is ⁇ 1.52 ⁇ M, CC 50 >50 ⁇ M, and SI> ⁇ 32.89;
  • the EC 50 of ophiopogonine for SARS-CoV-2 virus-like particles (trVLP) at the cellular level is ⁇ 25.29 ⁇ M, CC 50 >50 ⁇ M, SI> ⁇ 1.98;
  • the above-mentioned isoquinoline alkaloids (cyclamenine, dauricine, dauricine, thalidomide, thalidomide, and 6-acetonyldihydrochelerythrine) at the selected concentrations showed inhibitory effects on the nucleic acid level before, after and all time periods of SARS-CoV-2 virus-like particles (trVLP) infection of Caco-2-N cells.
  • trVLP SARS-CoV-2 virus-like particles
  • the EC 50 of isoquinoline alkaloids cyclamenine for SARS-CoV-2 wild-type pseudovirus at the cell level on BHK21-ACE2 cells is 6.54 ⁇ M; the EC 50 of batryticatine for SARS-CoV-2 wild-type pseudovirus at the cell level is 5.91 ⁇ M; the EC 50 of batryticatine for SARS-CoV-2 wild-type pseudovirus at the cell level is 8.48 ⁇ M; the EC 50 of thalmineline for SARS-CoV-2 wild-type pseudovirus at the cell level is 2.83 ⁇ M; the EC 50 of thalmineline for SARS-CoV-2 wild-type pseudovirus at the cell level is 2.73 ⁇ M; the EC 50 of 6-acetonyldihydrochelerythrine for SARS-CoV-2 wild-type pseudovirus at the cell level is 11.76 ⁇ M.
  • the above-mentioned isoquinoline alkaloids all have good inhibitory effects on SARS-CoV-2 wild-type pseudovirus
  • isoquinoline alkaloids are very likely to become specific drugs for novel coronavirus pneumonia. Therefore, the inventors believe that as potential drugs for the treatment of SARS-CoV-2 and other coronavirus infections, isoquinoline alkaloids have significant medicinal value and are a promising candidate drug for the treatment of SARS-CoV-2 virus and other coronavirus infectious diseases.

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Abstract

本发明涉及异喹啉生物碱及其药学上可接受的盐在医药方面的新用途，具体为异喹啉生物碱类化合物及其药学上可接受的盐在预防，缓解和/或治疗SARS-CoV-2病毒感染性疾病中的应用。实验结果表明异喹啉类生物碱在抗SARS-CoV-2病毒药物筛选模型穿山甲冠状病毒毒株xCoV、SARS-CoV-2病毒样颗粒(trVLP)系统和VSV假病毒系统上，都明显抑制了病毒的感染和复制。异喹啉类生物碱通过抑制病毒的整个生命周期来抑制病毒复制，这些结果强烈提示异喹啉类生物碱对于目前流行的新冠病毒以及其他冠状病毒感染引起的病症的潜在临床应用价值。

Description

异喹啉类生物碱在预防和治疗冠状病毒感染所致疾病中的应用
本申请要求2022年10月27日向中国国家知识产权局提交的，专利申请号为2022113286285，发明名称为“异喹啉类生物碱在预防和治疗冠状病毒中的应用”在先申请的优先权。该申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于医药技术领域，涉及异喹啉类生物碱在医药方面的新用途，具体为异喹啉类生物碱在预防或治疗SARS-CoV-2病毒感染性疾病中的应用。
背景技术
随着各种突变株大浪潮的出现，尤其是omicron各亚型突变株(包括BA.4、BA.5、BF.7、XBB等)具有更快的传播速度以及更强的免疫逃逸能力，疫苗接种所诱导的中和抗体的保护活性似乎也在随着时间降低，新的有效药物仍然是治疗COVID-19的重点。
从COVID-19以来，包括病毒S蛋白中和抗体、瑞德西韦、洛匹那韦/利托那韦(克力芝)、I型干扰素等多种药物在体外或体内实验中表现出了抗病毒活性，但是药物的临床疗效和安全性仍存在争议。目前美国FDA还批准了瑞德西韦和几款中和抗体如Bamlanivimab、Etesevimab、Casirivimab、Imdevimab用于COVID-19住院患者的治疗，但是这些药物需要静脉滴注且价格昂贵，有效性和安全性也尚需进一步临床验证，限制了其进一步应用。2021年底，Merck和Ridgeback Biotherapeutics联合开发的口服抗新冠药物莫努匹韦(Molnupiravir)以及辉瑞公司研发的奈玛特韦片/利托那韦片(Paxlovid)有条件获批上市，这两种药物分别针对病毒的RdRp(RNA依赖RNA聚合酶)和3CLpro，但存在耐药 性的威胁，且莫努匹韦可能引起DNA突变，安全性尚待进一步研究。奈玛特韦片/利托那韦片的联合用药和药物相互作用尚需进一步研究，而且这两种药物在疫苗接种人群中的临床效果还未可知。近期，阿兹夫定作为我国首款自主研发、具有完全自主知识产权的口服小分子新冠治疗药物附条件获批，其作用靶点与莫努匹韦相似，针对病毒的RdRp，同样的，其作为核苷类似物的安全性仍然需要进一步研究。
疫苗接种是预防和控制COVID-19的长期策略中最有效的方法。不同的SARS-CoV-2疫苗已被开发，截至2022年3月28日，已有153种疫苗获批进行临床试验，196种疫苗处于临床前试验阶段，10种疫苗(含3种印度疫苗)获世卫组织批准紧急使用，目前国内已有4款自主研发的新冠疫苗获批上市(https：//vac-lshtm.shinyapps.io/ncov_vaccine_landscape/)。这些疫苗主要包括灭活疫苗(占总数的14％)、减毒活疫苗(1％)、病毒载体疫苗(复制和非复制；占总数的17％)、RNA疫苗(18％)、DNA疫苗(11％)、蛋白质亚单位疫苗(34％)和VLP疫苗(4％)。然而随着世界多地报道SARS-CoV-2的突变体，目前研究显示疫苗所产生的抗体对SARS-CoV-2突变株的中和能力表现出不同程度的下降甚至完全丧失。由于新冠病毒不断升级，新冠疫苗和“特效药”不断“失效”，因此迫切需要开发针对不同靶点的抗病毒新药物，或者使用联合疗法从多方面抑制病毒感染，一方面针对新冠病毒本身开发阻断病毒感染或抑制病毒复制的药物，同时考虑联合用药；另一方面针对新冠病毒与宿主的相互作用，阻断新冠引起的一系列临床症状和并发症，降低病死率。因此，针对新冠肺炎疫情和未来可能出现的冠状病毒病，研发安全有效且具有广谱性的抗病毒药物意义重大。
生物碱是存在于自然界的一类含氮的碱性有机化合物，大多有较复杂的环状结构，氮原子结合在环内。其中异喹啉生物碱的种类最多，异喹啉生物碱来源于苯丙氨酸和酪氨酸系，在植物中分布广泛，数目较多，具有多方面的生物活性。异喹啉生物碱根据连接基团的不同可分为：简单异喹啉类、苄基异喹啉 类、阿朴菲类、双苄基异喹啉类、小檗碱类、普罗托品类、吗啡类和苯菲啶类。当苯基连接在异喹啉的骨架上时该衍生物便称为苄基异喹啉生物碱，单苄基异喹啉类为异喹啉母核1位连有苄基，罂粟属中具解痉作用的罂粟碱即属此类。双苄基异喹啉则为两个苄基异喹啉通过1～3个醚键相连接的一类生物碱，如存在于防己科北豆根中的主要酚性生物碱蝙蝠葛碱，汉防己中的汉防己甲素和汉防己乙素。
异喹啉生物碱广泛分布于罂粟科、巴比特科、毛茛科和防己科中。异喹啉及异喹啉衍生物是一类重要的医药化工中间体，广泛应用于一系列重要药物的合成过程，异喹啉骨架常用于设计药物分子中的活性结构基团，如阿曲库铵、诺米芬辛、莫沙维林、地莫昔林(甲基罂粟碱)、异喹卡因等。同时，异喹啉生物碱类药物具有多方面的生物活性，包括抗肿瘤、抗菌、镇痛、调节免疫功能、抗血小板凝聚、抗心律失常、降压等。大部分的异喹啉类生物碱药物都存在着异喹啉或异喹啉衍生物等基本结构，此类药物基本可以从简单的异喹啉衍生物进一步合成而衍生出来。
但异喹啉类生物碱是否可用于治疗新冠病毒肺炎，还有待进一步研究。
发明内容
本发明的目的在于提供SARS-CoV-2的有效抑制剂，其可以预防，缓解和/或治疗新型冠状病毒所致感染。
本发明首先提供了异喹啉类生物碱及其药学上可接受的盐作为新型冠状病毒抑制剂，在制备预防、缓解和/或治疗冠状病毒所致感染的产品中的应用；
所述异喹啉类生物碱选自轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱(22864-92-2)中的至少一种。
根据本发明的实施方案，所述药学上可接受的盐选自异喹啉类生物碱结构中具有足够碱性的氮原子的酸加成盐，所述酸加成盐包括异喹啉类生物碱与无机酸如盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸；有机酸 如甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸所形成的盐。
根据本发明的实施方案，所述产品为药品、食品、护肤品、化妆品、日用品和保健品，优选为药品。
根据本发明的实施方案，其中冠状病毒为2019新型冠状病毒(SARS-CoV-2)。
根据本发明的实施方案，冠状病毒感染为新型冠状病毒肺炎。
根据本发明的实施方案，如上所述异喹啉类生物碱及其药学上可接受的盐可以配置成各种适合的药物制剂形式。可以单独使用，或者将其与药物辅料(例如赋形剂、稀释剂等)混合，配置成口服给药的片剂、胶囊剂、颗粒剂、糖浆剂等或注射给药的粉针剂、溶液剂，以及研制为功能食品，功能饮品、药酒，滴鼻液及漱口水等。
本发明还提供一种可预防，缓解和/或治疗冠状病毒所致感染的药物组合物，其包括轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱(22864-92-2)中的至少一种。
本发明还提供一种预防，缓解和/或治疗冠状病毒所致感染的方法，包括将异喹啉类生物碱及其药学上可接受的盐中的至少一种施用于有此需要的个体。
所述异喹啉类生物碱选自轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱或6-丙酮基二氢白屈菜红碱(22864-92-2)中的至少一种。
本发明人经过大量研究发现异喹啉类生物碱的新用途，通过利用新冠病毒药物筛选替代模型GX_P2V、SARS-CoV-2病毒样颗粒(trVLP)模型及VSV假 病毒模型对异喹啉类生物碱进行抗SARS-CoV-2病毒活性评价。结果表明本发明的异喹啉类生物碱能有效抑制穿山甲冠状病毒毒株GX_P2V、SARS-CoV-2病毒样颗粒(trVLP)及VSV假病毒的感染和复制，并且在GX_P2V、SARS-CoV-2病毒样颗粒(trVLP)入胞前和入胞后、全时段均能发挥抑制作用。
其中，在新冠病毒药物筛选替代模型GX_P2V上，轮环藤宁在细胞水平的EC50＝1.37μM，CC50＞50μM，SI＞36.50；蝙蝠葛碱在细胞水平的EC50＝17.04μM，CC50＞50μM，SI＞2.93；蝙蝠葛苏林碱在细胞水平的EC50＝14.08μM，CC50＞50μM，SI＞3.55；峨眉唐松草碱在细胞水平的EC50＝3.31μM，CC50＝49.85μM，SI＝15.06；唐松明灵碱在细胞水平的EC50＝1.29μM，CC50＞50μM，SI＞38.76；6-丙酮基二氢白屈菜红碱在细胞水平的EC50＝2.47μM，CC50＞50μM，SI＞24.24。在SARS-CoV-2病毒样颗粒(trVLP)模型上，轮环藤宁在细胞水平的EC50＝1.64μM，CC50＞50μM，SI＞30.49；蝙蝠葛碱在细胞水平的EC50＝2.84μM，CC50＞50μM，SI＞17.61；蝙蝠葛苏林碱在细胞水平的EC50＝～1.52μM，CC50＞50μM，SI＞～32.89；峨眉唐松草碱在细胞水平的EC50＝～25.29μM，CC50＞50μM，SI＞～1.98；唐松明灵碱在细胞水平的EC50＝6.20μM，CC50＞50μM，SI＞8.06；6-丙酮基二氢白屈菜红碱在细胞水平的EC50＝2.25μM，CC50＞50μM，SI＞22.22。在VSV假病毒模型上，轮环藤宁在细胞水平的EC50＝6.54μM；蝙蝠葛碱在细胞水平的EC50＝5.91μM；蝙蝠葛苏林碱在细胞水平的EC50＝8.48μM；峨眉唐松草碱在细胞水平的EC50＝2.83μM；唐松明灵碱在细胞水平的EC50＝2.73μM；6-丙酮基二氢白屈菜红碱在细胞水平的EC50＝11.76μM。因此，发明人认为轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱可应用于针对COVID-19感染的预防，缓解和/或治疗。
与现有技术相比，本发明的技术效果如下：
本申请发明人发现异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱在细胞水平能够阻断冠状病毒进入细胞，也具有有效抑制冠状病毒的复制从而达到预防，缓解和/或治疗 COVID-19的效果。
附图说明
图1为异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱抑制GX_P2V病毒感染Vero E6细胞的测试结果；
图2为异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱抑制GX_P2V病毒感染Vero E6细胞的不同作用阶段测试结果；
A.异喹啉类生物碱显著降低了Vero E6细胞中病毒RNA水平；
B.异喹啉类生物碱显著抑制了Vero E6细胞中病毒蛋白的表达。
图3为异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱抑制SARS-CoV-2病毒样颗粒(trVLP)感染Caco-2-N细胞的测试结果；
图4为异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱抑制SARS-CoV-2病毒样颗粒(trVLP)感染Caco-2-N细胞的不同作用阶段(细胞中病毒RNA水平)测试结果；
图5为异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱抑制SARS-CoV-2野生型假病毒的感染BHK21-ACE2细胞的测试结果。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
一、实验方法
1.异喹啉类生物碱抑制穿山甲冠状病毒xCoV(GX_P2V)
1)细胞病毒培养
非洲绿猴肾细胞系Vero E6是从美国模式培养物集存库(ATCC，1586号)中获得的，用含有10％胎牛血清(FBS；Gibco Invitrogen)的DMEM培养基(Gibco)，在37℃、5％CO2的培养箱中培养。
穿山甲冠状病毒xCoV(GX_P2V)(GISAID：EPI_ISL_410539)为本发明人从海关查获的死亡的穿山甲中分离并培养的一株新型冠状病毒xCoV。将GX_P2V在Vero E6细胞中繁殖，用Vero E6细胞(ATCC，1586号)做噬斑实验测定病毒滴度。所有感染实验均在生物安全2级(BLS-2)实验室进行。
2)异喹啉类生物碱抑制穿山甲冠状病毒xCoV(GX_P2V)的EC50和CC50测定
EC50检测：将2.5×104个Vero E6细胞接种到96孔板中，在37℃、5％CO2的培养箱中培养24h后，在细胞培养孔中分别加入终浓度为50μM、25μM、12.5μM、6.25μM、3.125μM、1.56μM、0.78μM、0.39μM、0μM的异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱，37℃预孵育1h，同时将病毒(MOI＝0.01)与终浓度为50μM、25μM、12.5μM、6.25μM、3.125μM、1.56μM、0.78μM、0.39μM、0μM的异喹啉类生物碱4℃预孵育1h，孵育结束后将上述病毒-药物混合物加入含对应浓度药物的细胞中，感染2h后PBS清洗3遍换液，加入含相同浓度药物的培养基，37℃、5％CO2的培养箱中进行培养。病毒感染后的48h，通过qRT-PCR的方法对细胞内病毒RNA以及细胞内参基因GAPDH的表达情况进行定量检测。用GraphPad-Prism 8软件进行数据分析计算EC50。
CC50检测：使用CellTiter-Blue法进行CC50的检测。Vero E6细胞接种至96 孔细胞培养板，细胞密度达到60％-80％时进行试验。Vero E6细胞换液后加入稀释后的药物，终浓度分别为50μM、25μM、12.5μM、6.25μM、3.125μM、1.56μM、0.78μM、0.39μM、0μM。37℃ 5％CO2培养48h，利用CellTiter-Blue试剂检测荧光强度，激发波长554nm，发射波长593nm，GraphPad-Prism 8软件进行数据分析计算CC50。
3)加药时间实验
将1×105个Vero E6细胞接种到24孔板中，在37℃、5％CO2的培养箱中培养24h后，在全时段给药组和入胞前给药组细胞培养孔中分别加入根据药物EC50确定的终浓度的异喹啉类生物碱，37℃预孵育1h，同时将病毒(MOI＝0.01)与相同浓度的异喹啉类生物碱4℃预孵育1h，孵育结束后用上述病毒-药物混合物病毒感染细胞，感染2h后PBS清洗3遍换液，在全时段给药组和入胞后给药组细胞培养孔中加入含相同浓度药物的培养基，入胞前给药组和对照组细胞培养孔加入不含药物培养基，37℃、5％CO2的培养箱中进行培养。病毒感染后的12h，通过qRT-PCR的方法对细胞内病毒RNA以及细胞内参基因GAPDH的表达情况进行定量检测。同时收集细胞内蛋白，利用Western Blot对新冠病毒N蛋白和GAPDH蛋白进行定量分析。
4)病毒样本处理和检测
按照制造商的说明，使用细胞/组织RNA提取试剂盒(北京诺贝莱生物科技有限公司，R11202109JN)提取总RNA。反转录使用可消化gDNA的Hifair II 1st strand cDNA合成试剂盒(上海翊圣生物科技，货号：11121ES60)，qRT-PCR使用Quantstudio实时PCR检测试剂(Applied biosystems，Foster City，CA，USA)，SYBR-Green法的qPCR扩增：95℃ 5min，40个循环，95℃ 10s，55℃ 20s，72℃ 31s。通过对GAPDH基因的检测实现均一化。
2.异喹啉类生物碱抑制SARS-CoV-2病毒样颗粒(trVLP)
1)细胞培养
Caco-2-N细胞用高糖DMEM+10％胎牛血清，在37℃ 5％CO2培养箱中培 养。
SARS-CoV-2病毒样颗粒(trVLP)为清华大学丁强教授团队开发的具有转录和复制能力的系统，将SARS-CoV-2trVLP表达的报告基因(GFP)取代病毒基因组包装和病毒粒子组装所必需的核蛋白基因(N)(SARS-CoV-2-GFP/ΔNtrVLP)，该病毒可以在异位表达SARS-CoV-2N蛋白的Caco-2-N细胞中完成完整的生命周期。由于产生的病毒颗粒核酸中没有N基因，该病毒样颗粒只能感染过表达N基因的细胞，所以对人和动物不具有感染性。该系统在保留了SARS-CoV-2生物学特性的同时保证了安全性，这一成果加快了研究人员在BSL-2实验室对新冠病毒的研究。我们在本申请中使用了丁强教授馈赠的SARS-CoV-2trVLP系统，用于异喹啉类生物碱对SARS-CoV-2的抑制作用研究。
2)异喹啉类生物碱抑制SARS-CoV-2病毒样颗粒(trVLP)的EC50和CC50测定
EC50检测：将2.5×104个Caco-2-N细胞接种到96孔板中，在37℃、5％CO2的培养箱中培养24h后，在细胞培养孔中分别加入终浓度为50μM、25μM、12.5μM、6.25μM、3.125μM、1.56μM、0.78μM、0.39μM、0μM的异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱，37℃预孵育1h，同时将SARS-CoV-2trVLP病毒(MOI＝0.001)与终浓度为50μM、25μM、12.5μM、6.25μM、3.125μM、1.56μM、0.78μM、0.39μM、0μM的异喹啉类生物碱4℃预孵育1h，孵育结束后用上述病毒-药物混合物感染细胞。感染2h后PBS清洗3遍换液，加入含相同浓度药物的培养基，37℃、5％CO2的培养箱中进行培养。病毒感染后的48h通过qRT-PCR的方法对细胞内病毒RNA以及细胞内参基因GAPDH的表达情况进行定量检测。用GraphPad-Prism 8软件进行数据分析计算EC50。
CC50检测：使用CellTiter-Blue法进行CC50的检测。Caco-2-N细胞接种至96孔细胞培养板，细胞密度达到60％-80％时进行试验。Caco-2-N细胞换液后加入稀释后的药物，终浓度分别为50μM、25μM、12.5μM、6.25μM、3.125μM、 1.56μM、0.78μM、0.39μM、0μM。37℃ 5％CO2培养48h，利用CellTiter-Blue试剂检测荧光强度，激发波长554nm，发射波长593nm，GraphPad-Prism 8软件进行数据分析计算CC50。
3)加药时间实验
将1×105个Caco-2-N细胞接种到24孔板中，在37℃、5％CO2的培养箱中培养24h后，在全时段给药组和入胞前给药组细胞培养孔中分别加入根据药物EC50确定的终浓度的异喹啉类生物碱，37℃预孵育1h，同时将SARS-CoV-2trVLP病毒(MOI＝0.001)与相同浓度的异喹啉类生物碱4℃预孵育1h，孵育结束后用上述病毒-药物混合物感染细胞，感染2h后PBS清洗3遍换液，在全时段给药组和入胞后给药组细胞培养孔中加入含相同浓度药物的培养基，入胞前给药组和对照组细胞培养孔加入不含药物培养基，在37℃、5％CO2的培养箱中进行培养。病毒感染后12h，通过qRT-PCR的方法对细胞内病毒RNA以及细胞内参基因GAPDH的表达情况进行定量检测。用GraphPad-Prism 8软件进行数据分析。
4)病毒样本处理和检测
按照制造商的说明，使用Axygen TM多用途总RNA小提试剂盒(Axygene，货号AP-MN-MS-RNA-250G)进行RNA提取。反转录使用可消化gDNA的Hifair II 1st strand cDNA合成试剂盒(上海翊圣生物科技，货号：11121ES60)，qRT-PCR使用Quantstudio实时PCR检测试剂(Applied biosystems，Foster City，CA，USA)，SYBR-Green法的qPCR扩增：95℃ 5min，40个循环，95℃ 10s，55℃ 20s，72℃ 31s。通过对GAPDH基因的检测实现均一化。
EC50是指能够有效抑制50％细胞感染病毒的药物浓度，数值越小说明药物对病毒的抑制效果越好。
CC50是使得50％的细胞发生细胞毒性的药物浓度，数值越高说明药物对细胞的毒性越低。
SI：选择性指数，为CC50与EC50的比值，数值越大说明成药的可能性越高。
3.异喹啉类生物碱抑制SARS-CoV-2野生型假病毒
1)细胞培养
BHK21-ACE2、HEK-293T细胞使用高糖DMEM+10％胎牛血清培养，在37℃5％CO2温箱中孵育。
2)假病毒包装
SARS-CoV-2野生型S蛋白假病毒的制备采用VSV假病毒包装系统，假病毒的组装和测定过程基于Nie等人发表的论文中(Nie et al.，Nat Protoc.2020；15(11)：3699-3715.)。将SARS-CoV-2野生型S蛋白的质粒转染到HEK-293T细胞中在细胞表面提供膜蛋白，而利用G*ΔG-VSV感染提供VSV的基因组，感染和转染后24h及48h分别收集细胞上清液以获得SARS-CoV-2野生型的S蛋白假病毒。
3)异喹啉类生物碱抑制SARS-CoV-2野生型假病毒的感染复制
将5×104个重悬的BHK21-ACE2细胞接种到96孔板中，在37℃、5％CO2的培养箱中培养24h后，使用添加10％胎牛血清和1％青霉素-链霉素的DMEM分别稀释异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱，然后分别加入野生型S蛋白假病毒，4℃下孵育60-90min，使轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱的终浓度为25μM、8.33μM、2.78μM、0.93μM、0.31μM、0.1μM。同时对应终浓度的异喹啉类生物碱分别与细胞在37℃下孵育60-90min，孵育结束后，假病毒-异喹啉类生物碱混合物加入对应药物浓度的BHK21-ACE2细胞中。37℃培养24h后利用萤光素酶检测系统(Luciferase Assay System)检测细胞裂解液中的萤火虫荧光素酶的表达量。
实验结果
结果见图1-5。
由图1、图2可知，异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、 峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱对GX_P2V病毒具有良好的抑制作用，且具有剂量依赖效应。由图1可知，在Vero E6细胞上异喹啉类生物碱轮环藤宁对于GX_P2V在细胞水平的EC50＝1.37μM，CC50＞50μM，SI＞36.50；蝙蝠葛碱对于GX_P2V在细胞水平的EC50＝17.04μM，CC50＞50μM，SI＞2.93；蝙蝠葛苏林碱对于GX_P2V在细胞水平的EC50＝14.08μM，CC50＞50μM，SI＞3.55；峨眉唐松草碱对于GX_P2V在细胞水平的EC50＝3.31μM，CC50＝49.85μM，SI＝15.06；唐松明灵碱(Thalmineline)对于GX_P2V在细胞水平的EC50＝1.29μM，CC50＞50μM，SI＞38.76；6-丙酮基二氢白屈菜红碱对于GX_P2V在细胞水平的EC50＝2.47μM，CC50＞50μM，SI＞24.24。可见，上述异喹啉类生物碱均对GX_P2V具有较好的抑制效果，且存在剂量依赖，毒副作用较小。
由图2可知，上述异喹啉类生物碱(轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱)在选定浓度下对GX_P2V感染Vero E6细胞入胞前、入胞后及全时段均有抑制作用。且上述异喹啉生物碱均在核酸和蛋白水平上对GX_P2V感染Vero E6细胞入胞前、入胞后及全时段均有抑制作用。
由图3、图4可知，在Caco-2-N细胞上异喹啉类生物碱(轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱)对SARS-CoV-2病毒样颗粒(trVLP)均具有良好的抑制作用，且具有剂量依赖效应。
由图3可知，在Caco-2-N细胞上异喹啉类生物碱轮环藤宁对于SARS-CoV-2病毒样颗粒(trVLP)在细胞水平的EC50＝1.64μM，CC50＞50μM，SI＞30.49；蝙蝠葛碱对于SARS-CoV-2病毒样颗粒(trVLP)在细胞水平的EC50＝2.84μM，CC50＞50μM，SI＞17.61；蝙蝠葛苏林碱对于SARS-CoV-2病毒样颗粒(trVLP)在细胞水平的EC50＝～1.52μM，CC50＞50μM，SI＞～32.89；峨眉唐松草碱对于SARS-CoV-2病毒样颗粒(trVLP)在细胞水平的EC50＝～25.29μM，CC50＞50μM， SI＞～1.98；唐松明灵碱对于SARS-CoV-2病毒样颗粒(trVLP)在细胞水平的EC50＝6.20μM，CC50＞50μM，SI＞8.06；6-丙酮基二氢白屈菜红碱对于SARS-CoV-2病毒样颗粒(trVLP)在细胞水平的EC50＝2.25μM，CC50＞50μM，SI＞22.22。可见，上述异喹啉类生物碱均对SARS-CoV-2病毒样颗粒(trVLP)具有较好的抑制效果，且存在剂量依赖，毒副作用较小。
由图4可知，上述异喹啉类生物碱(轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱)在选定浓度下，对SARS-CoV-2病毒样颗粒(trVLP)感染Caco-2-N细胞入胞前、入胞后及全时段在核酸水平均显示出抑制作用。
由图5可知，在BHK21-ACE2细胞上异喹啉类生物碱轮环藤宁对于SARS-CoV-2野生型假病毒在细胞水平的EC50＝6.54μM；蝙蝠葛碱对于SARS-CoV-2野生型假病毒在细胞水平的EC50＝5.91μM；蝙蝠葛苏林碱对于SARS-CoV-2野生型假病毒在细胞水平的EC50＝8.48μM；峨眉唐松草碱对于SARS-CoV-2野生型假病毒在细胞水平的2.83μM；唐松明灵碱(Thalmineline)对于SARS-CoV-2野生型假病毒在细胞水平的EC50＝2.73μM；6-丙酮基二氢白屈菜红碱对于SARS-CoV-2野生型假病毒在细胞水平的EC50＝11.76μM。上述异喹啉类生物碱均对SARS-CoV-2野生型假病毒具有较好的抑制效果，存在剂量依赖。
利用SARS-CoV-2病毒进行研究需要高等级的生物防护设施，这与目前研究的迫切需要相冲突。发明人在本申请中采用了穿山甲冠状病毒毒株xCoV、SARS-CoV-2病毒样颗粒(trVLP)及基于VSV骨架的假病毒系统对几种异喹啉类生物碱进行了活性评价，在保证了安全性的前提下也实现了对SARS-CoV-2研究的有效性和可靠性，并且通过多种验证手段保证了本发明内容的真实性。
在本申请中，实验数据显示异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱具有显著地抑制冠状病毒的活性。发明人认为异喹啉类生物碱轮环藤宁、蝙蝠葛碱、蝙蝠葛苏 林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱是潜在的治疗SARS-CoV-2感染性疾病的药物。异喹啉类生物碱在抗SARS-CoV-2病毒药物筛选模型穿山甲冠状病毒毒株xCoV、SARS-CoV-2病毒样颗粒(trVLP)系统、VSV假病毒系统上，都明显抑制了病毒的感染和复制。异喹啉类生物碱在病毒的整个生命周期均能抑制病毒复制，这些结果强烈提示异喹啉类生物碱对于目前流行的新冠病毒以及其他冠状病毒感染引起的病症的潜在临床应用价值。
鉴于所观察到的异喹啉类生物碱对病毒的强烈抑制作用，异喹啉类生物碱极有可能成为新型冠状病毒肺炎的特效药。因此，发明人认为，作为潜在的治疗SARS-CoV-2及其他冠状病毒感染的药物，异喹啉类生物碱具有重大的药用价值，是治疗SARS-CoV-2病毒及其他冠状病毒感染性疾病的一类有前途的候选药物。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 异喹啉类生物碱及其药学上可接受的盐在制备预防、缓解和/或治疗冠状病毒感染所致疾病的产品中的应用；
    所述异喹啉类生物碱选自轮环藤宁、蝙蝠葛碱、蝙蝠葛苏林碱、峨眉唐松草碱、唐松明灵碱、6-丙酮基二氢白屈菜红碱中的至少一种。
  2. 根据权利要求1所述的应用，其特征在于，所述药学上可接受的盐选自异喹啉类生物碱结构中具有足够碱性的氮原子的酸加成盐，所述酸加成盐包括异喹啉类生物碱与无机酸或有机酸所形成的盐。
  3. 根据权利要求2所述的应用，其特征在于，所述无机酸选自盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸。
  4. 根据权利要求2所述的应用，其特征在于，所述有机酸选自甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
  5. 根据权利要求1-4任一项所述的应用，其特征在于，所述产品为药品、食品、化妆品、护肤品、日用品和保健品。
  6. 根据权利要求1-5任一项所述的应用，其特征在于，所述冠状病毒为2019新型冠状病毒。
  7. 根据权利要求1-6任一项所述的应用，其特征在于，异喹啉类生物碱及其药学上可接受的盐可以配置成各种适合的药物制剂形式；可以单独使用，或 者将其与药物辅料混合，配置成口服给药的片剂、胶囊剂、颗粒剂、糖浆剂；或注射给药的粉针剂、溶液剂，以及研制为功能食品，功能饮品、药酒，滴鼻液及漱口水。
  8. 根据权利要求1-5任一项所述的应用，其特征在于，所述药物辅料为赋形剂或稀释剂。
PCT/CN2023/127237 2022-10-27 2023-10-27 异喹啉类生物碱在预防和治疗冠状病毒感染所致疾病中的应用 Ceased WO2024088403A1 (zh)

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