WO2023149204A1 - Covid-19 therapeutic, oral composition for treating covid-19, and use of compounds for manufacturing covid-19 therapeutic - Google Patents

Covid-19 therapeutic, oral composition for treating covid-19, and use of compounds for manufacturing covid-19 therapeutic Download PDF

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Publication number
WO2023149204A1
WO2023149204A1 PCT/JP2023/001263 JP2023001263W WO2023149204A1 WO 2023149204 A1 WO2023149204 A1 WO 2023149204A1 JP 2023001263 W JP2023001263 W JP 2023001263W WO 2023149204 A1 WO2023149204 A1 WO 2023149204A1
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covid
protopanaxadiol
therapeutic
sc2r
sars
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PCT/JP2023/001263
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French (fr)
Japanese (ja)
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正徳 池田
緑 武田
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国立大学法人 鹿児島大学
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Publication of WO2023149204A1 publication Critical patent/WO2023149204A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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

Definitions

  • the present invention relates to COVID-19 therapeutic agents, oral compositions for COVID-19 therapeutic agents, and uses of compounds for the manufacture of COVID-19 therapeutic agents.
  • Non-Patent Document 1 quantitative evaluation of the amount of phosphorylation and protein due to SARS-CoV-2 infection in order to elucidate the mechanism of viral pathogenicity, and profiles such as changes in phosphorylation that disrupt kinases and pathways Based on this, promising compounds for the treatment of COVID-19 are being investigated.
  • the present invention has been made in view of the above circumstances, and has a high degree of safety.
  • the object is to provide the use of the compounds for the manufacture of
  • the COVID-19 therapeutic agent according to the first aspect of the present invention is Protopanaxadiol or a pharmacologically acceptable salt thereof is included.
  • the protopanaxadiol is is (20R)-protopanaxadiol; You can do it.
  • the oral composition for treating COVID-19 comprises Protopanaxadiol or a pharmacologically acceptable salt thereof is included.
  • the use according to the third aspect of the invention is Use of protopanaxadiol or a pharmacologically acceptable salt thereof for the manufacture of a COVID-19 therapeutic agent.
  • COVID-19 therapeutic drug and COVID-19 therapeutic oral composition according to the present invention are highly safe and can be applied clinically at an early stage.
  • FIG. 1 shows the organization of the genome of SARS-CoV-2.
  • B is a diagram showing the structure of the replicon DNA according to the example.
  • C shows fragments F1 to F10 obtained by dividing the replicon DNA shown in (B) into a plurality of fragments.
  • FIG. 4 is a diagram showing the effect of each compound contained in a compound library on the replication level of replicon RNA and cell viability.
  • FIG. 10 shows the response of the replication level of replicon RNA to the concentration of (20R)-protopanaxadiol. (20R)-Protopanaxadiol cell viability.
  • Figure 2 shows the antiviral activity of (20R)-protopanaxadiol and (20S)-protopanaxadiol.
  • COVID-19 therapeutic agents include protopanaxadiol and pharmacologically acceptable salts thereof.
  • Protopanaxadiol is abundantly contained in the roots of ginseng, a perennial herbaceous plant (Araliaceae) native to Northeast China and the Korean Peninsula, more specifically Panax ginseng CA Meyer.
  • Panax ginseng is also known as Panax ginseng (Korean ginseng) or Asian ginseng (Korean ginseng).
  • the root of Panax ginseng has long been used for medicinal or food purposes, particularly as a crude drug.
  • the protopanaxadiol is (20R)-protopanaxadiol, but it may be its optical isomer (20S)-protopanaxadiol.
  • COVID-19 therapeutics may include (20R)-protopanaxadiol and (20S)-protopanaxadiol.
  • the structures of (20R)-protopanaxadiol and (20S)-protopanaxadiol are shown in Formulas 1 and 2, respectively.
  • Protopanaxadiol may be synthesized by a known method, or may be obtained from Araliaceae ginseng such as Panax ginseng.
  • ginseng of the family Araliaceae When protopanaxadiol is obtained from ginseng of the family Araliaceae, protopanaxadiol may be obtained, for example, by extracting from ginseng of the family Araliaceae, may be obtained by enzymatic fermentation of ginseng of the family Araliaceae, or may be obtained by It may be obtained by hydrolyzing carrots.
  • Araliaceae ginseng When protopanaxadiol is obtained from Araliaceae ginseng, Araliaceae ginseng may be used as it is as it is collected from nature. and rhizome parts, or powder obtained by pulverizing roots and rhizome parts.
  • a method of extraction with a water-ethanol solution there are no particular restrictions on the method of obtaining by extraction, and a method of extraction with a water-ethanol solution, a supercritical extraction method, an HPLC extraction method, etc. may be selected as appropriate.
  • the mixing ratio of the water-ethanol solution is not particularly limited.
  • water: ethanol (V/V) is preferably 9:1 to 2:1, 3:1. more preferred.
  • the COVID-19 therapeutic drug according to the present embodiment may contain protopanaxadiol as an extract of Araliaceae ginseng, particularly Panax ginseng.
  • the COVID-19 therapeutic drug according to this embodiment may contain a salt of protopanaxadiol as an active ingredient as long as it exhibits antiviral activity against SARS-CoV-2.
  • Salts are not particularly limited, and examples include alkali metal salts such as lithium salts, sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, hydrochlorides, hydrobromides, sulfates, and nitrates.
  • inorganic acid salts such as oxalates and phosphates, as well as acetates, propionates, hexanoates, cyclopentanepropionates, glycolates, pyruvates, lactates, malonates, succinates , malate, fumarate, tartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, p-toluenesulfonate, camphorsulfonate, 4- Methylbicyclo[2.2.2]oct-2-ene-1-carboxylate, glucoheptanoate, 3-
  • the COVID-19 therapeutic drug according to the present embodiment is manufactured by a known method, and contains % by mass, 0.001 to 99.6% by mass, 0.01 to 99.5% by mass, 0.1 to 99% by mass, 0.5 to 60% by mass, 1 to 50% by mass, or 1 to 20% by mass of protopanaxadiol or a salt thereof.
  • a COVID-19 therapeutic agent may be a solid or liquid formulation.
  • the COVID-19 therapeutic agent may contain any pharmacologically acceptable ingredient in addition to protopanaxadiol or its salt.
  • Optional ingredients include, for example, carriers, excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, tonicity agents, buffers and soothing agents. Additives such as preservatives, antioxidants, coloring agents and sweetening agents may also be incorporated into the COVID-19 therapeutic if desired.
  • the dosage of the COVID-19 therapeutic drug according to this embodiment is appropriately determined according to the sex, age, weight, symptoms, etc. of the subject.
  • the COVID-19 therapeutic agent is administered in an effective amount of protopanaxadiol or a salt thereof.
  • the effective amount is the amount of protopanaxadiol or a salt thereof necessary to obtain the desired result, suppressing the growth of SARS-CoV-2, or delaying the progression of symptoms due to SARS-CoV-2 infection. , the amount necessary to effect inhibition, prevention, reversal or cure.
  • the dosage of the COVID-19 therapeutic agent is, for example, 0.01 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, more preferably 0.2 mg/kg to 20 mg/kg; It can be administered in one or more divided doses per day.
  • the COVID-19 therapeutic agent is administered 1 to 4 times per day.
  • the COVID-19 therapeutic may be administered at different dosing frequencies, such as daily, every other day, once a week, every other week and once a month. Amounts outside the above ranges can also be used, if desired.
  • COVID-19 therapeutics may be administered, for example, parenterally or orally.
  • parenteral administration intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, pulmonary administration, enteral administration, transmucosal administration, and the like may be used.
  • COVID-19 therapeutics may be administered via an infusion.
  • COVID-19 therapeutic agent can be formulated in any form.
  • COVID-19 therapeutic agents include tablets such as dragees, buccal tablets, coated tablets and chewable tablets, lozenges, pills, powders and capsules including soft capsules, granules, suspensions, emulsions and dry syrups. It may be a syrup containing and a liquid such as an elixir.
  • COVID-19 therapeutic agents may be injections, inhalants, transdermal absorption tapes, aerosols, suppositories, and the like.
  • the COVID-19 therapeutic drug will be administered to any subject as long as the subject is infected with SARS-CoV-2.
  • the COVID-19 therapeutic agent is preferably administered to vertebrates, more preferably mammals. Mammals include, for example, humans, chimpanzees and other primates, pigs and horses, as well as birds such as ducks and chickens.
  • a particularly preferred administration subject is a human.
  • the COVID-19 therapeutic agent according to the present embodiment is administered to subjects in whom SARS-CoV-2 is detected for the purpose of preventing the onset of symptoms during the period without clear symptoms or the incubation period after infection.
  • Protopanaxadiol contained in the COVID-19 therapeutic drug according to this embodiment has been confirmed to be safe in humans. Indeed, as shown in the examples below, the CC50 (50% cytotoxic concentration) of protopanaxadiol was well above the EC50 (50% effective concentration). Therefore, the COVID-19 therapeutic agent according to this embodiment has high antiviral activity and high safety.
  • Also provided in another embodiment is the use of protopanaxadiol or a salt thereof for the manufacture of a COVID-19 therapeutic agent.
  • methods of treating, ameliorating or preventing COVID-19 are provided. The method includes administering protopanaxadiol or a salt thereof to the subject. Also provided in another embodiment is protopanaxadiol or a salt thereof for use in treating COVID-19.
  • an anti-SARS-CoV-2 agent according to another embodiment comprises protopanaxadiol or a salt thereof.
  • the treatment of COVID-19 in this embodiment also includes improvement and prevention of COVID-19.
  • the COVID-19 therapeutic agent in this embodiment may be used as a COVID-19 prophylactic agent.
  • an oral composition for treating COVID-19 comprising protopanaxadiol or a pharmacologically acceptable salt thereof is provided.
  • Specific examples of the oral composition include supplements, food compositions, food and drink, functional foods, and food additives.
  • the form of the supplement is not particularly limited, and may be in any form such as tablets, powders, granules, capsules, sugar-coated tablets, films, troches, chewables, solutions, emulsions and suspensions.
  • a supplement may contain any ingredient normally used as a supplement.
  • “Functional food” means food or beverage that is ingested for the purpose of maintaining health.
  • the functional food food with specified health uses or food with nutrient function claims, which are foods with health claims, are preferable.
  • various additives used in food specifically, coloring agents, preservatives, thickening stabilizers, antioxidants, bleaching agents, antibacterial antifungal agents , acidulants, sweeteners, flavor enhancers, emulsifiers, enhancers, manufacturing agents, flavoring agents and the like may be added to the oral compositions.
  • Functional foods may be foods or beverages, and are not particularly limited as long as they can be taken orally.
  • functional foods include beverages, confectionery, processed grain products, paste products, dairy products, and seasonings.
  • beverages include nutritional drinks, soft drinks, black tea, green tea, and the like.
  • confectionery include candy, cookies, tablet confectionery, chewing gum and jelly.
  • Bread, rice, biscuits and the like are exemplified as processed grain noodles. Sausages, hams, fish cakes, and the like are examples of paste products.
  • Dairy products include butter and yogurt.
  • the oral composition may be added to food as a food additive.
  • the food additives may be pastes, gels, powders, liquids, suspensions, emulsions, granules, etc. so as to be easily added to foods.
  • the oral composition may contain water, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, functional ingredients, food additives, etc., to the extent that the anti-SARS-CoV-2 action is maintained. good.
  • the oral composition can be produced by a known method, optionally adding other ingredients than protopanaxadiol or a salt thereof.
  • the oral composition may be divided and stored in one or more containers so that the daily intake is the above-mentioned intake, in which case it is preferable that one container contains a daily dose of The oral composition is contained.
  • the oral composition contains protopanaxadiol or a salt thereof, and is provided in a manner distinguishable from other products as a product in that it is used for COVID-19.
  • at least one of the product packaging, instructions, and promotional material for the oral composition is labeled as having anti-SARS-CoV-2 activity.
  • BsaI (-) vector The restriction enzyme BsaI site (ggtctc) in the ampicillin resistant gene of pUC19 was transfected into "gAtctc" using QuickChange mutagenesis (manufactured by Stratagene) to construct a BsaI-deficient plasmid pUC19b.
  • FIG. 1(A) shows the organization of the genome of SARS-CoV-2.
  • the genome of SARS-CoV-2 consists of nonstructural regions including nonstructural protein gene 1a (ORF1a) and nonstructural protein gene 1b (ORF1b), S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF7b, and a structural region containing ORF8, the N gene and ORF10.
  • ORF1a nonstructural protein gene 1a
  • ORF1b nonstructural protein gene 1b
  • the replicon DNA (hereinafter referred to as "SC2R") according to this example is a DNA construct constructed based on the nucleotide sequence of the genome of SARS-CoV-2.
  • the nucleotide sequence of SC2R is shown in SEQ ID NO:2.
  • a viral replicon maintains a nonstructural region and lacks a structural region, so that it does not have infectivity but propagates autonomously.
  • the structure of SC2R is shown in FIG. 1(B).
  • SC2R has the non-structural region of SARS-CoV-2 and has no genes contained in the structural region except the N gene.
  • SC2R consists of cytomegalovirus (CMV) promoter, ORF1a, ORF1b, secretory luciferase gene (sNLuc), N gene, poly A region (pA), hepatitis delta virus ribozyme gene (Rz ) and bovine growth hormone poly A signal (BGH).
  • CMV cytomegalovirus
  • ORF1a ORF1a
  • ORF1b secretory luciferase gene
  • N gene N gene
  • pA poly A region
  • Rz hepatitis delta virus ribozyme gene
  • BGH bovine growth hormone poly A signal
  • the N gene has the function of improving the replication level of genomic RNA.
  • SC2R is transcribed into RNA (replicon RNA) in cells, translated, and replicated. Duplicated SC2R can be quantified through the activity of sNLuc.
  • Neo is a drug resistance gene as a marker gene.
  • pA contributes to transcript stability.
  • Rz is required for excision by self-cleavage after replication. BGH terminates transcription.
  • SC2R was synthesized by artificial synthesis without using a natural virus as a template, by the golden gate method using type IIS restriction enzyme, as follows.
  • Ggacc was used to synonymously replace the base sequence corresponding to the BsaI site (gagacc) from 17972nd to 17977th from the 5' end of the base sequence shown in SEQ ID NO: 1.
  • a BsaI site was introduced at the 5' and 3' ends of each of the 10 fragments.
  • F1-4 and F6-10 were introduced into pUC19b (pUC19b/SC2R-F1, F2, F3, F4, F6, F7, F8, F9, F10).
  • F5 was introduced into the pCC1-4k vector (pCC1-4k/SC2R-F5).
  • PCR fragments A and B containing BsaI inside EcoRI and BamHI were prepared.
  • PCR fragment A was performed using the HRP gene shown in SEQ ID NO: 4 as a template and a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6).
  • the nucleotide sequence of PCR fragment A is shown in SEQ ID NO:7.
  • PCR was performed using the HRP gene shown in SEQ ID NO: 4 as a template and a forward primer (SEQ ID NO: 8) and a reverse primer (SEQ ID NO: 9).
  • the nucleotide sequence of PCR fragment B is shown in SEQ ID NO:10.
  • PCR fragment C containing BsaI inside HindIII and BamHI was prepared.
  • PCR was performed using the HRP gene shown in SEQ ID NO: 4 as a template and a forward primer (SEQ ID NO: 11) and a reverse primer (SEQ ID NO: 12).
  • the nucleotide sequence of PCR fragment C is shown in SEQ ID NO:13.
  • pHSG298 was cleaved with EcoRI and BamHI, and PCR fragment A was ligated to construct pHSG298c vector A. Further, pHSG298 was cleaved with EcoRI and BamHI, and PCR fragment B was ligated to construct pHSG298c vector B. pCC1-4k was cleaved with HindIII and BamHI and PCR fragment C was ligated to construct pCC1-4kc vector.
  • PCR was performed using pSMART BACbc/SC2R as a template to amplify the N gene of SARS-CoV-2.
  • the nucleotide sequences of the forward primer and reverse primer used for the PCR are shown in SEQ ID NOs: 14 and 15, respectively.
  • a PCR fragment was prepared by introducing MluI and NotI sites at the 5' end and 3' end, and the PCR fragment was ligated to the pCX4bsr vector cleaved with MluI and NotI to construct the pCX4bsr/SARS-CoV-2-N vector. .
  • the cured cell line has a higher level of HCV RNA replication than the parent HuH-7 cell line (Masanori Ikeda, et al., ⁇ Efficient replication of a full-length hepatitis C virus genome, strain O,''). in cell culture, and development of a luciferase reporter system,” Biochem Biophys Res Commun., 2005, 329(4):1350-9).
  • HuH-7.6c cells were seeded in a 6-well plate at 1 ⁇ 10 5 cells/well, and the next day, 2 ⁇ g of pSMART BACbc/SC2R was transfected using Fugene HD reagent and introduced into the cells. After 24 hours, cells were detached using trypsin and seeded at 3 x 104 cells/well in 24-well plates. After an additional 24 hours, compounds were added to the indicated concentrations. After culturing for 48 hours, the culture supernatant was collected and luciferase activity was measured using the Nano-Glo Luciferase Assay System.
  • HuH-7.6c cells were seeded in 96-well plates at 5 ⁇ 10 3 cells. Twenty-four hours after the initiation of culture, compounds were added to the cells at the given concentrations. After culturing for 48 hours, 10 ⁇ l of Premix WST-1 Cell Proliferation Assay System (manufactured by Takara Bio Inc.) was added to the medium, cultured at 37° C. for 2 hours, and absorbance at 450 nm was measured using a microplate reader.
  • Premix WST-1 Cell Proliferation Assay System manufactured by Takara Bio Inc.
  • FIG. 3 shows the SC2R assay results for (20R)-protopanaxadiol and (20S)-protopanaxadiol.
  • (20R)-protopanaxadiol inhibited luciferase activity in a concentration-dependent manner with an EC50 of 5.46 ⁇ M.
  • (20S)-protopanaxadiol was also found to inhibit luciferase activity.
  • FIG. 4 shows cell viability at each concentration of (20R)-protopanaxadiol.
  • the CC 50 of (20R)-protopanaxadiol was 26.7 ⁇ M.
  • Fig. 5 shows the results of Western blot analysis and the relative values of luciferase activity. It was confirmed that (20R)-protopanaxadiol and (20S)-protopanaxadiol suppress the expression of N protein.
  • the present invention is useful for treating COVID-19.

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Abstract

This COVID-19 therapeutic contains protopanaxadiol or a pharmacologically acceptable salt thereof.

Description

ＣＯＶＩＤ－１９治療薬、ＣＯＶＩＤ－１９治療用経口組成物及びＣＯＶＩＤ－１９治療薬の製造のための化合物の使用Use of compounds for the manufacture of COVID-19 therapeutics, oral compositions for COVID-19 therapeutics and COVID-19 therapeutics
　本発明は、ＣＯＶＩＤ－１９治療薬、ＣＯＶＩＤ－１９治療用経口組成物及びＣＯＶＩＤ－１９治療薬の製造のための化合物の使用に関する。 The present invention relates to COVID-19 therapeutic agents, oral compositions for COVID-19 therapeutic agents, and uses of compounds for the manufacture of COVID-19 therapeutic agents.
　重症急性呼吸器症候群（ＳＡＲＳ）を引き起こす新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）感染による新型コロナウイルス感染症（ＣＯＶＩＤ－１９）の流行は医療、経済、文化及び生活様式等、広範囲に影響を及ぼし世界が総力をあげて取り組むべき課題となっている。ワクチンの臨床応用が進んでいるが、ワクチンの有効性を低減する変異株が出現しており、今後も新たな変異株が出現するおそれがある。このような現状を打破するためには、ワクチンに加えて安全な治療薬の開発が重要である。 The novel coronavirus infectious disease (COVID-19) epidemic caused by the novel coronavirus (SARS-CoV-2) that causes severe acute respiratory syndrome (SARS) has a wide-ranging impact on medical care, the economy, culture and lifestyle. This is an issue that the world must make a concerted effort to address. Clinical application of vaccines is progressing, but mutant strains that reduce the effectiveness of vaccines have emerged, and there is a risk that new mutant strains will emerge in the future. In order to overcome such a situation, it is important to develop safe therapeutic agents in addition to vaccines.
　非特許文献１では、ウイルス病原性の機序を解明するためにＳＡＲＳ－ＣｏＶ－２感染によるリン酸化及びタンパク質の量の定量的な評価、並びにキナーゼ及びパスウェイを乱すリン酸化の変化等のプロファイルに基づいて、ＣＯＶＩＤ－１９の治療に有望な化合物が検討されている。 In Non-Patent Document 1, quantitative evaluation of the amount of phosphorylation and protein due to SARS-CoV-2 infection in order to elucidate the mechanism of viral pathogenicity, and profiles such as changes in phosphorylation that disrupt kinases and pathways Based on this, promising compounds for the treatment of COVID-19 are being investigated.
　免疫による排除が困難である慢性感染症の場合、ウイルスを完全に排除する強力な抗ウイルス剤が求められている。これに対し、ＣＯＶＩＤ－１９を含む急性感染症の原因となるウイルスは、自然免疫により最終的に排除されるため、ウイルス量を抑制することが重要となる。急性感染症では、ウイルス量をある程度減少させることで、ウイルスの排出量を減らし周囲への感染を抑制でき、さらには重症化も抑制できる。 In the case of chronic infections that are difficult to eliminate by immunity, there is a demand for powerful antiviral agents that completely eliminate the virus. In contrast, viruses that cause acute infections, including COVID-19, are ultimately eliminated by innate immunity, so it is important to suppress viral load. In acute infectious diseases, by reducing the amount of virus to some extent, it is possible to reduce the amount of virus excretion, suppress the infection to the surroundings, and further suppress the severity of the infection.
　一刻も早いＣＯＶＩＤ－１９治療薬の開発が求められている。新規の化合物をＣＯＶＩＤ－１９治療薬として開発する場合、安全性試験等を要し、開発に時間がかかってしまう。これに対し、既にヒトに投与又は摂取されて安全性が担保されている化合物であれば、開発の時間を短縮することができる。 There is a demand for the development of a COVID-19 therapeutic drug as soon as possible. When developing a new compound as a COVID-19 treatment drug, safety tests and the like are required, and development takes time. On the other hand, if the compound has already been administered or ingested by humans and is guaranteed to be safe, the development time can be shortened.
　本発明は上述の事情に鑑みてなされたものであり、高い安全性を有し、早期に臨床応用することができるＣＯＶＩＤ－１９治療薬、ＣＯＶＩＤ－１９治療用経口組成物及びＣＯＶＩＤ－１９治療薬の製造のための化合物の使用を提供することを目的とする。 The present invention has been made in view of the above circumstances, and has a high degree of safety. A therapeutic agent for COVID-19, an oral composition for treating COVID-19, and a therapeutic agent for COVID-19 that can be clinically applied at an early stage. The object is to provide the use of the compounds for the manufacture of
　本発明の第１の観点に係るＣＯＶＩＤ－１９治療薬は、
　プロトパナキサジオール又は薬理学的に許容されるその塩を含む。 The COVID-19 therapeutic agent according to the first aspect of the present invention is
Protopanaxadiol or a pharmacologically acceptable salt thereof is included.
　前記プロトパナキサジオールは、
　（２０Ｒ）－プロトパナキサジオールである、
　こととしてもよい。 The protopanaxadiol is
is (20R)-protopanaxadiol;
You can do it.
　本発明の第２の観点に係るＣＯＶＩＤ－１９治療用経口組成物は、
　プロトパナキサジオール又は薬理学的に許容されるその塩を含む。 The oral composition for treating COVID-19 according to the second aspect of the present invention comprises
Protopanaxadiol or a pharmacologically acceptable salt thereof is included.
　本発明の第３の観点に係る使用は、
　ＣＯＶＩＤ－１９治療薬の製造のためのプロトパナキサジオール又は薬理学的に許容されるその塩の使用である。 The use according to the third aspect of the invention is
Use of protopanaxadiol or a pharmacologically acceptable salt thereof for the manufacture of a COVID-19 therapeutic agent.
　本発明に係るＣＯＶＩＤ－１９治療薬及びＣＯＶＩＤ－１９治療用経口組成物は、高い安全性を有し、早期に臨床応用することができる。 The COVID-19 therapeutic drug and COVID-19 therapeutic oral composition according to the present invention are highly safe and can be applied clinically at an early stage.
（Ａ）はＳＡＲＳ－ＣｏＶ－２のゲノムの構成を示す図である。（Ｂ）は実施例に係るレプリコンＤＮＡの構成を示す図である。（Ｃ）は（Ｂ）に示すレプリコンＤＮＡを複数に分割したフラグメントＦ１～Ｆ１０を示す図である。(A) shows the organization of the genome of SARS-CoV-2. (B) is a diagram showing the structure of the replicon DNA according to the example. (C) shows fragments F1 to F10 obtained by dividing the replicon DNA shown in (B) into a plurality of fragments. 化合物ライブラリに含まれる各化合物のレプリコンＲＮＡの複製レベルへの影響及び細胞生存率を示す図である。FIG. 4 is a diagram showing the effect of each compound contained in a compound library on the replication level of replicon RNA and cell viability. レプリコンＲＮＡの複製レベルの（２０Ｒ）－プロトパナキサジオールの濃度に対する応答性を示す図である。FIG. 10 shows the response of the replication level of replicon RNA to the concentration of (20R)-protopanaxadiol. （２０Ｒ）－プロトパナキサジオールの細胞生存率を示す図である。(20R)-Protopanaxadiol cell viability. （２０Ｒ）－プロトパナキサジオール及び（２０Ｓ）－プロトパナキサジオールの抗ウイルス活性を示す図である。Figure 2 shows the antiviral activity of (20R)-protopanaxadiol and (20S)-protopanaxadiol.
　本発明に係る実施の形態について図面を参照して説明する。なお、本発明は下記の実施の形態及び図面によって限定されるものではない。なお、下記の実施の形態において、“有する”、“含む”又は“含有する”といった表現は、“からなる”又は“から構成される”という意味も包含する。 An embodiment according to the present invention will be described with reference to the drawings. The present invention is not limited by the following embodiments and drawings. In the following embodiments, expressions such as "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".
　（実施の形態）
　本実施の形態に係るＣＯＶＩＤ－１９治療薬は、プロトパナキサジオール及び薬理学的に許容されるその塩を含む。プロトパナキサジオールは、中国東北部から朝鮮半島原産の多年生草本植物（ウコギ科）の人参、より詳細にはオタネニンジン（Ｐａｎａｘ　ｇｉｎｓｅｎｇ　Ｃ．Ａ．Ｍｅｙｅｒ）の根に多く含まれる。オタネニンジンは、チョウセンニンジン（朝鮮人参）又はコウライニンジン（高麗人参）としても知られている。オタネニンジンの根は、古くから薬用又は食用に用いられ、特に、生薬として使用されている。 (Embodiment)
COVID-19 therapeutic agents according to this embodiment include protopanaxadiol and pharmacologically acceptable salts thereof. Protopanaxadiol is abundantly contained in the roots of ginseng, a perennial herbaceous plant (Araliaceae) native to Northeast China and the Korean Peninsula, more specifically Panax ginseng CA Meyer. Panax ginseng is also known as Panax ginseng (Korean ginseng) or Asian ginseng (Korean ginseng). The root of Panax ginseng has long been used for medicinal or food purposes, particularly as a crude drug.
　好ましくは、プロトパナキサジオールは、（２０Ｒ）－プロトパナキサジオールであるが、その光学異性体の（２０Ｓ）－プロトパナキサジオールであってもよい。ＣＯＶＩＤ－１９治療薬は、（２０Ｒ）－プロトパナキサジオール及び（２０Ｓ）－プロトパナキサジオールを含んでもよい。（２０Ｒ）－プロトパナキサジオール及び（２０Ｓ）－プロトパナキサジオールの構造をそれぞれ式１及び式２に示す。 Preferably, the protopanaxadiol is (20R)-protopanaxadiol, but it may be its optical isomer (20S)-protopanaxadiol. COVID-19 therapeutics may include (20R)-protopanaxadiol and (20S)-protopanaxadiol. The structures of (20R)-protopanaxadiol and (20S)-protopanaxadiol are shown in Formulas 1 and 2, respectively.
　プロトパナキサジオールは、公知の方法で合成してもよいし、オタネニンジン等のウコギ科人参から取得してもよい。プロトパナキサジオールをウコギ科人参から取得する場合、プロトパナキサジオールは、例えば、ウコギ科人参から抽出して得てもよいし、ウコギ科人参を酵素発酵して得てもよいし、ウコギ科人参を加水分解して得てもよい。 Protopanaxadiol may be synthesized by a known method, or may be obtained from Araliaceae ginseng such as Panax ginseng. When protopanaxadiol is obtained from ginseng of the family Araliaceae, protopanaxadiol may be obtained, for example, by extracting from ginseng of the family Araliaceae, may be obtained by enzymatic fermentation of ginseng of the family Araliaceae, or may be obtained by It may be obtained by hydrolyzing carrots.
　プロトパナキサジオールをウコギ科人参から取得する場合、ウコギ科人参は、天然から採取されたそのままの状態で用いてもよいが、その根や根茎に特に有効成分を多く含んでいるので、その根及び根茎部分の部分、あるいは根及び根茎部分を粉砕した粉末を用いることが好ましい。 When protopanaxadiol is obtained from Araliaceae ginseng, Araliaceae ginseng may be used as it is as it is collected from nature. and rhizome parts, or powder obtained by pulverizing roots and rhizome parts.
　抽出により得る方法としては、特に制限はなく、水－エタノール溶液で抽出する方法、超臨界抽出法及びＨＰＬＣ抽出法等を適宜選択すればよい。水－エタノール溶液で抽出する場合、水－エタノール溶液の混合比としては、特に制限はなく、例えば、水：エタノール（Ｖ／Ｖ）が、９：１～２：１が好ましく、３：１がより好ましい。 There are no particular restrictions on the method of obtaining by extraction, and a method of extraction with a water-ethanol solution, a supercritical extraction method, an HPLC extraction method, etc. may be selected as appropriate. When extracting with a water-ethanol solution, the mixing ratio of the water-ethanol solution is not particularly limited. For example, water: ethanol (V/V) is preferably 9:1 to 2:1, 3:1. more preferred.
　なお、本実施の形態に係るＣＯＶＩＤ－１９治療薬は、プロトパナキサジオールを、ウコギ科人参、特にはオタネニンジンの抽出物として含有してもよい。 The COVID-19 therapeutic drug according to the present embodiment may contain protopanaxadiol as an extract of Araliaceae ginseng, particularly Panax ginseng.
　本実施の形態に係るＣＯＶＩＤ－１９治療薬は、ＳＡＲＳ－ＣｏＶ－２に対して抗ウイルス活性を示す限り、プロトパナキサジオールの塩を有効成分として含んでもよい。塩は特に限定されず、例えば、リチウム塩、ナトリウム塩及びカリウム塩等のアルカリ金属塩、マグネシウム塩及びカルシウム塩等のアルカリ土類金属塩等、塩酸塩、臭化水素酸塩、硫酸塩、硝酸塩、シュウ酸塩及びリン酸塩等の無機酸塩、並びに酢酸塩、プロピオン酸塩、ヘキサン酸塩、シクロペンタンプロピオン酸塩、グリコール酸塩、ピルビン酸塩、乳酸塩、マロン酸塩、コハク酸塩、リンゴ酸塩、フマル酸塩、酒石酸塩、クエン酸塩、安息香酸塩、ｏ－（４－ヒドロキシベンゾイル）安息香酸塩、桂皮酸塩、マンデル酸塩、メタンスルホン酸塩、エタンスルホン酸塩、１，２－エタンジスルホン酸塩、２－ヒドロキシエタンスルホン酸塩、ベンゼンスルホン酸塩、ｐ－クロロベンゼンスルホン酸塩、２－ナフタレンスルホン酸塩、ｐ－トルエンスルホン酸塩、カンファースルホン酸塩、４－メチルビシクロ［２．２．２］オクト－２－エン－１－カルボン酸塩、グルコヘプタン酸塩、３－フェニルプロピオン酸塩、トリメチル酢酸塩、第三級ブチル酢酸塩、ラウリル硫酸塩、グルコン酸塩、グルタミン酸塩、ヒドロキシナフトエ酸塩、サリチル酸塩、ステアリン酸塩、及びムコン酸塩等の有機酸塩等である。なお、抗ウイルス活性とは、ＳＡＲＳ－ＣｏＶ－２の感染性、増殖能又は免疫回避能を低下させる活性を意味する。 The COVID-19 therapeutic drug according to this embodiment may contain a salt of protopanaxadiol as an active ingredient as long as it exhibits antiviral activity against SARS-CoV-2. Salts are not particularly limited, and examples include alkali metal salts such as lithium salts, sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, hydrochlorides, hydrobromides, sulfates, and nitrates. , inorganic acid salts such as oxalates and phosphates, as well as acetates, propionates, hexanoates, cyclopentanepropionates, glycolates, pyruvates, lactates, malonates, succinates , malate, fumarate, tartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, p-toluenesulfonate, camphorsulfonate, 4- Methylbicyclo[2.2.2]oct-2-ene-1-carboxylate, glucoheptanoate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconic acid salts, organic acid salts such as glutamate, hydroxynaphthoate, salicylate, stearate, and muconate; The antiviral activity means an activity that reduces the infectivity, proliferative ability, or immune evasion ability of SARS-CoV-2.
　本実施の形態に係るＣＯＶＩＤ－１９治療薬は、既知の方法で製造され、有効成分として０．０００００１～９９．９質量％、０．００００１～９９．８質量％、０．０００１～９９．７質量％、０．００１～９９．６質量％、０．０１～９９．５質量％、０．１～９９質量％、０．５～６０質量％、１～５０質量％又は１～２０質量％のプロトパナキサジオール又はその塩を含む。ＣＯＶＩＤ－１９治療薬は、固形製剤であっても、液状製剤であってもよい。 The COVID-19 therapeutic drug according to the present embodiment is manufactured by a known method, and contains % by mass, 0.001 to 99.6% by mass, 0.01 to 99.5% by mass, 0.1 to 99% by mass, 0.5 to 60% by mass, 1 to 50% by mass, or 1 to 20% by mass of protopanaxadiol or a salt thereof. A COVID-19 therapeutic agent may be a solid or liquid formulation.
　ＣＯＶＩＤ－１９治療薬はプロトパナキサジオール又はその塩に加え、薬理学的に許容される任意の成分を含んでもよい。任意の成分は、例えば、担体、賦形剤、滑沢剤、結合剤、崩壊剤、溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤及び無痛化剤等である。また、必要に応じて、防腐剤、抗酸化剤、着色剤及び甘味剤等の添加物がＣＯＶＩＤ－１９治療薬に配合されてもよい。 The COVID-19 therapeutic agent may contain any pharmacologically acceptable ingredient in addition to protopanaxadiol or its salt. Optional ingredients include, for example, carriers, excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, tonicity agents, buffers and soothing agents. Additives such as preservatives, antioxidants, coloring agents and sweetening agents may also be incorporated into the COVID-19 therapeutic if desired.
　本実施の形態に係るＣＯＶＩＤ－１９治療薬の投与量は、投与対象の性別、年齢、体重及び症状等によって適宜決定される。当該ＣＯＶＩＤ－１９治療薬は、プロトパナキサジオール又はその塩が有効量となるように投与される。有効量とは、所望の結果を得るために必要なプロトパナキサジオール又はその塩の量であり、ＳＡＲＳ－ＣｏＶ－２の増殖の抑制、あるいはＳＡＲＳ－ＣｏＶ－２の感染による症状の進行の遅延、阻害、予防、逆転又は治癒をもたらすのに必要な量である。 The dosage of the COVID-19 therapeutic drug according to this embodiment is appropriately determined according to the sex, age, weight, symptoms, etc. of the subject. The COVID-19 therapeutic agent is administered in an effective amount of protopanaxadiol or a salt thereof. The effective amount is the amount of protopanaxadiol or a salt thereof necessary to obtain the desired result, suppressing the growth of SARS-CoV-2, or delaying the progression of symptoms due to SARS-CoV-2 infection. , the amount necessary to effect inhibition, prevention, reversal or cure.
　ＣＯＶＩＤ－１９治療薬の投与量は、例えば、０．０１ｍｇ／ｋｇ～１０００ｍｇ／ｋｇ、好ましくは０．１ｍｇ／ｋｇ～２００ｍｇ／ｋｇ、より好ましくは０．２ｍｇ／ｋｇ～２０ｍｇ／ｋｇであり、１日に１回、又はそれ以上に分割して投与することができる。ＣＯＶＩＤ－１９治療薬を分割して投与する場合、ＣＯＶＩＤ－１９治療薬は、１日に１～４回投与される。また、ＣＯＶＩＤ－１９治療薬は、毎日、隔日、１週間に１回、隔週及び１ヶ月に１回等の様々な投与頻度で投与してもよい。なお、必要に応じて、上記の範囲外の量を用いることもできる。 The dosage of the COVID-19 therapeutic agent is, for example, 0.01 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, more preferably 0.2 mg/kg to 20 mg/kg; It can be administered in one or more divided doses per day. When administering the COVID-19 therapeutic agent in divided doses, the COVID-19 therapeutic agent is administered 1 to 4 times per day. Also, the COVID-19 therapeutic may be administered at different dosing frequencies, such as daily, every other day, once a week, every other week and once a month. Amounts outside the above ranges can also be used, if desired.
　ＣＯＶＩＤ－１９治療薬の投与経路は特に限定されない。ＣＯＶＩＤ－１９治療薬は、例えば非経口で投与されてもよいし、経口で投与されてもよい。非経口投与の場合、静脈注射、皮下注射、腹腔内注射、筋肉内注射、経皮投与、経鼻投与、経肺投与、経腸投与及び経粘膜投与等であってもよい。ＣＯＶＩＤ－１９治療薬は、点滴を介して投与されてもよい。 The route of administration of the COVID-19 therapeutic drug is not particularly limited. COVID-19 therapeutics may be administered, for example, parenterally or orally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, pulmonary administration, enteral administration, transmucosal administration, and the like may be used. COVID-19 therapeutics may be administered via an infusion.
　ＣＯＶＩＤ－１９治療薬は、任意の形態の製剤とすることができる。経口投与の場合、ＣＯＶＩＤ－１９治療薬は、糖衣錠、バッカル錠、コーティング錠及びチュアブル錠等の錠剤、トローチ剤、丸剤、散剤及びソフトカプセルを含むカプセル剤、顆粒剤、懸濁剤、乳剤及びドライシロップを含むシロップ剤、並びにエリキシル剤等の液剤であってもよい。非経口投与の場合、ＣＯＶＩＤ－１９治療薬は、注射剤、吸入剤、経皮吸収テープ、エアゾール剤及び坐剤等であってもよい。 The COVID-19 therapeutic agent can be formulated in any form. When administered orally, COVID-19 therapeutic agents include tablets such as dragees, buccal tablets, coated tablets and chewable tablets, lozenges, pills, powders and capsules including soft capsules, granules, suspensions, emulsions and dry syrups. It may be a syrup containing and a liquid such as an elixir. For parenteral administration, COVID-19 therapeutic agents may be injections, inhalants, transdermal absorption tapes, aerosols, suppositories, and the like.
　ＣＯＶＩＤ－１９治療薬は、ＳＡＲＳ－ＣｏＶ－２が感染する対象であれば、任意の対象に投与される。例えば、ＣＯＶＩＤ－１９治療薬の投与対象は、脊椎動物が好ましく、哺乳類動物がより好ましい。哺乳類動物としては、例えば、ヒト、チンパンジー及びその他の霊長類、ブタ及びウマに加え、カモ及びニワトリ等の鳥類等が挙げられる。特に好ましい投与対象はヒトである。 The COVID-19 therapeutic drug will be administered to any subject as long as the subject is infected with SARS-CoV-2. For example, the COVID-19 therapeutic agent is preferably administered to vertebrates, more preferably mammals. Mammals include, for example, humans, chimpanzees and other primates, pigs and horses, as well as birds such as ducks and chickens. A particularly preferred administration subject is a human.
　本実施の形態に係るＣＯＶＩＤ－１９治療薬は、ＳＡＲＳ－ＣｏＶ－２が検出された対象に対して、明確な症状がない期間、あるいは感染後の潜伏期間に症状の発症を予防する目的で投与されてもよい。 The COVID-19 therapeutic agent according to the present embodiment is administered to subjects in whom SARS-CoV-2 is detected for the purpose of preventing the onset of symptoms during the period without clear symptoms or the incubation period after infection. may be
　本実施の形態に係るＣＯＶＩＤ－１９治療薬に含まれるプロトパナキサジオールは、ヒトでの安全性が確認されている。実際に下記実施例に示されるように、プロトパナキサジオールのＣＣ５０（５０％細胞毒性濃度）はＥＣ５０（５０％有効濃度）よりも十分に高かった。よって、本実施の形態に係るＣＯＶＩＤ－１９治療薬は高い抗ウイルス活性と高い安全性とを有する。 Protopanaxadiol contained in the COVID-19 therapeutic drug according to this embodiment has been confirmed to be safe in humans. Indeed, as shown in the examples below, the CC50 (50% cytotoxic concentration) of protopanaxadiol was well above the EC50 (50% effective concentration). Therefore, the COVID-19 therapeutic agent according to this embodiment has high antiviral activity and high safety.
　また、他の実施の形態では、ＣＯＶＩＤ－１９治療薬の製造のためのプロトパナキサジオール又はその塩の使用が提供される。別の実施の形態では、ＣＯＶＩＤ－１９を治療、改善又は予防する方法が提供される。当該方法は、プロトパナキサジオール又はその塩を対象に投与する工程を含む。また、他の実施の形態では、ＣＯＶＩＤ－１９の治療における使用のためのプロトパナキサジオール又はその塩が提供される。また、別の実施の形態に係る抗ＳＡＲＳ－ＣｏＶ－２剤は、プロトパナキサジオール又はその塩を含む。なお、本実施の形態におけるＣＯＶＩＤ－１９の治療には、ＣＯＶＩＤ－１９の改善及び予防も含まれる。また、本実施の形態におけるＣＯＶＩＤ－１９治療薬はＣＯＶＩＤ－１９予防薬として使用されてもよい。 Also provided in another embodiment is the use of protopanaxadiol or a salt thereof for the manufacture of a COVID-19 therapeutic agent. In another embodiment, methods of treating, ameliorating or preventing COVID-19 are provided. The method includes administering protopanaxadiol or a salt thereof to the subject. Also provided in another embodiment is protopanaxadiol or a salt thereof for use in treating COVID-19. Also, an anti-SARS-CoV-2 agent according to another embodiment comprises protopanaxadiol or a salt thereof. The treatment of COVID-19 in this embodiment also includes improvement and prevention of COVID-19. In addition, the COVID-19 therapeutic agent in this embodiment may be used as a COVID-19 prophylactic agent.
　別の実施の形態では、プロトパナキサジオール又は薬理学的に許容されるその塩を含む、ＣＯＶＩＤ－１９治療用経口組成物が提供される。当該経口組成物としては、具体的には、サプリメント、食品組成物、飲食品、機能性食品及び食品添加剤が挙げられる。 In another embodiment, an oral composition for treating COVID-19 comprising protopanaxadiol or a pharmacologically acceptable salt thereof is provided. Specific examples of the oral composition include supplements, food compositions, food and drink, functional foods, and food additives.
　サプリメントの形態は、特に制限されず、錠剤、散剤、顆粒剤、カプセル剤、糖衣錠、フイルム剤、トローチ剤、チュアブル剤、溶液、乳濁液及び懸濁液等の任意の形態でよい。サプリメントは、サプリメントとして通常使用される任意の成分を含んでもよい。 The form of the supplement is not particularly limited, and may be in any form such as tablets, powders, granules, capsules, sugar-coated tablets, films, troches, chewables, solutions, emulsions and suspensions. A supplement may contain any ingredient normally used as a supplement.
　“機能性食品”とは、健康の維持の目的で摂取する食品又は飲料を意味し、保健機能食品である特定保健用食品、機能性表示食品、栄養機能食品、健康食品及び栄養補助食品等を含む。機能性食品としては、保健機能食品である特定保健用食品又は栄養機能食品が好ましい。なお、機能性食品として製品化する場合には、食品に用いられる様々な添加剤、具体的には、着色料、保存料、増粘安定剤、酸化防止剤、漂白剤、防菌防黴剤、酸味料、甘味料、調味料、乳化剤、強化剤、製造用剤及び香料等を当該経口組成物に添加してもよい。 “Functional food” means food or beverage that is ingested for the purpose of maintaining health. include. As the functional food, food with specified health uses or food with nutrient function claims, which are foods with health claims, are preferable. In addition, when commercializing as a functional food, various additives used in food, specifically, coloring agents, preservatives, thickening stabilizers, antioxidants, bleaching agents, antibacterial antifungal agents , acidulants, sweeteners, flavor enhancers, emulsifiers, enhancers, manufacturing agents, flavoring agents and the like may be added to the oral compositions.
　機能性食品は、食品であっても飲料であってもよく、経口で摂取できれば特に限定されない。機能性食品の態様としては、例えば、飲料、菓子、穀類加工品、練り製品、乳製品及び調味料等が挙げられる。飲料として、栄養ドリンク、清涼飲料水、紅茶及び緑茶等が例示される。菓子として、キャンデー、クッキー、錠菓、チューインガム及びゼリー等が例示される。穀類加工品麺として、パン、米飯及びビスケット等が例示される。練り製品として、ソーセージ、ハム及びかまぼこ等が挙げられる。乳製品として、バター及びヨーグルト等が挙げられる。 Functional foods may be foods or beverages, and are not particularly limited as long as they can be taken orally. Examples of functional foods include beverages, confectionery, processed grain products, paste products, dairy products, and seasonings. Examples of beverages include nutritional drinks, soft drinks, black tea, green tea, and the like. Examples of confectionery include candy, cookies, tablet confectionery, chewing gum and jelly. Bread, rice, biscuits and the like are exemplified as processed grain noodles. Sausages, hams, fish cakes, and the like are examples of paste products. Dairy products include butter and yogurt.
　当該経口組成物は、食品添加剤として食品に添加されてもよい。この場合、当該食品添加剤は、食品に添加しやすいように、ペースト剤、ゲル状剤、散剤、液剤、懸濁剤、乳剤及び顆粒剤等であってもよい。 The oral composition may be added to food as a food additive. In this case, the food additives may be pastes, gels, powders, liquids, suspensions, emulsions, granules, etc. so as to be easily added to foods.
　当該経口組成物は、抗ＳＡＲＳ－ＣｏＶ－２作用を維持する範囲で、水、ビタミン類、ミネラル類、有機酸、有機塩基、果汁、フレーバー、機能性成分及び食品添加物等を含有してもよい。当該経口組成物は、必要に応じてプロトパナキサジオール又はその塩以外の他の成分を添加して、公知の方法によって製造することができる。 The oral composition may contain water, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, functional ingredients, food additives, etc., to the extent that the anti-SARS-CoV-2 action is maintained. good. The oral composition can be produced by a known method, optionally adding other ingredients than protopanaxadiol or a salt thereof.
　当該経口組成物は、１日の摂取量が上述の摂取量となるように１個又は複数個の容器に分けて収容されてもよく、この場合、好ましくは１個の容器に１日分の当該経口組成物が収容される。 The oral composition may be divided and stored in one or more containers so that the daily intake is the above-mentioned intake, in which case it is preferable that one container contains a daily dose of The oral composition is contained.
　当該経口組成物は、プロトパナキサジオール又はその塩を含有し、ＣＯＶＩＤ－１９に用いられる点において、製品として他の製品と区別することができる態様で提供される。例えば、当該経口組成物に係る製品の包装、説明書及び宣伝物の少なくとも１つに、抗ＳＡＲＳ－ＣｏＶ－２作用がある旨が表示される。 The oral composition contains protopanaxadiol or a salt thereof, and is provided in a manner distinguishable from other products as a product in that it is used for COVID-19. For example, at least one of the product packaging, instructions, and promotional material for the oral composition is labeled as having anti-SARS-CoV-2 activity.
　以下の実施例により、本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The present invention will be explained more specifically by the following examples, but the present invention is not limited by the examples.
　（ＢｓａＩ（－）ベクターの作製）
　ｐＵＣ１９のアンピシリン耐性遺伝子内にある制限酵素ＢｓａＩサイト（ｇｇｔｃｔｃ）をＱｕｉｃｋＣｈａｎｇｅ　ｍｕｔａｇｅｎｅｓｉｓ（Ｓｔｒａｔａｇｅｎｅ社製）を用いて、“ｇＡｔｃｔｃ”になるよう遺伝子導入を行い、ＢｓａＩ欠損プラスミドｐＵＣ１９ｂを構築した。 (Preparation of BsaI (-) vector)
The restriction enzyme BsaI site (ggtctc) in the ampicillin resistant gene of pUC19 was transfected into "gAtctc" using QuickChange mutagenesis (manufactured by Stratagene) to construct a BsaI-deficient plasmid pUC19b.
　ｐＳＭＡＲＴ　ＢＡＣのｓｏｐＢ遺伝子内にある制限酵素ＢｓａＩサイト（ｇｇｔｃｔｃ）をＱｕｉｃｋＣｈａｎｇｅ　ｍｕｔａｇｅｎｅｓｉｓを用いて、“ｇＡｔｃｔｃ”になるよう遺伝子導入を行い、ＢｓａＩ欠損プラスミドｐＳＭＡＲＴ　ＢＡＣｂを構築した。 Using QuickChange mutagenesis, the restriction enzyme BsaI site (ggtctc) in the sopB gene of pSMART BAC was transfected into "gAtctc" to construct the BsaI-deficient plasmid pSMART BACb.
　（人工遺伝子の設計）
　図１（Ａ）はＳＡＲＳ－ＣｏＶ－２のゲノムの構成を示す。ＳＡＲＳ－ＣｏＶ－２のゲノムは、非構造蛋白遺伝子１ａ（ＯＲＦ１ａ）及び非構造蛋白遺伝子１ｂ（ＯＲＦ１ｂ）を含む非構造領域と、Ｓ遺伝子、ＯＲＦ３ａ、Ｅ遺伝子、Ｍ遺伝子、ＯＲＦ６、ＯＲＦ７ａ、ＯＲＦ７ｂ、ＯＲＦ８、Ｎ遺伝子及びＯＲＦ１０を含む構造領域と、を有する。ＳＡＲＳ－ＣｏＶ－２のゲノムの５’末端及び３’末端には、それぞれ非翻訳領域（５’ＵＴＲ及び３’ＵＴＲ）がある。ＳＡＲＳ－ＣｏＶ－２のゲノムの塩基配列（ＮＣＢＩ　Ｒｅｆｅｒｅｎｃｅ　Ｓｅｑｕｅｎｃｅ：ＮＣ＿０４５５１２）は配列番号１に例示される。 (Design of artificial gene)
FIG. 1(A) shows the organization of the genome of SARS-CoV-2. The genome of SARS-CoV-2 consists of nonstructural regions including nonstructural protein gene 1a (ORF1a) and nonstructural protein gene 1b (ORF1b), S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF7b, and a structural region containing ORF8, the N gene and ORF10. At the 5' and 3' ends of the genome of SARS-CoV-2 are untranslated regions (5'UTR and 3'UTR), respectively. The genome sequence of SARS-CoV-2 (NCBI Reference Sequence: NC_045512) is exemplified in SEQ ID NO:1.
　本実施例に係るレプリコンＤＮＡ（以下、“ＳＣ２Ｒ”とする）は、ＳＡＲＳ－ＣｏＶ－２のゲノムの塩基配列に基づいて構築されるＤＮＡ構築物である。ＳＣ２Ｒの塩基配列は配列番号２に示される。一般にウイルスのレプリコンは、非構造領域を維持し、構造領域を欠くため、感染性を有していないが自律増殖する。ＳＣ２Ｒの構成を図１（Ｂ）に示す。ＳＣ２Ｒは、ＳＡＲＳ－ＣｏＶ－２の非構造領域を有し、Ｎ遺伝子を除く構造領域に含まれる遺伝子を有していない。詳細には、ＳＣ２Ｒは、５’末端側からサイトメガロウイルス（ＣＭＶ）プロモーター、ＯＲＦ１ａ、ＯＲＦ１ｂ、分泌型ルシフェラーゼ遺伝子（ｓＮＬｕｃ）、Ｎ遺伝子、ポリＡ領域（ｐＡ）、肝炎デルタウィルスのリボザイム遺伝子（Ｒｚ）及びウシ成長ホルモンポリＡシグナル（ＢＧＨ）を有する。 The replicon DNA (hereinafter referred to as "SC2R") according to this example is a DNA construct constructed based on the nucleotide sequence of the genome of SARS-CoV-2. The nucleotide sequence of SC2R is shown in SEQ ID NO:2. In general, a viral replicon maintains a nonstructural region and lacks a structural region, so that it does not have infectivity but propagates autonomously. The structure of SC2R is shown in FIG. 1(B). SC2R has the non-structural region of SARS-CoV-2 and has no genes contained in the structural region except the N gene. Specifically, SC2R consists of cytomegalovirus (CMV) promoter, ORF1a, ORF1b, secretory luciferase gene (sNLuc), N gene, poly A region (pA), hepatitis delta virus ribozyme gene (Rz ) and bovine growth hormone poly A signal (BGH).
　Ｎ遺伝子はゲノムＲＮＡの複製レベルを向上させる機能を有する。ＳＣ２Ｒは、細胞内でＲＮＡ（レプリコンＲＮＡ）に転写されたうえで、翻訳され、複製される。ｓＮＬｕｃの活性を介して複製されたＳＣ２Ｒを定量することができる。Ｎｅｏは、マーカー遺伝子としての薬剤耐性遺伝子である。ｐＡは転写産物の安定性に寄与する。Ｒｚは複製後の自己切断による切り出しに必要である。ＢＧＨは転写を終結させる。 The N gene has the function of improving the replication level of genomic RNA. SC2R is transcribed into RNA (replicon RNA) in cells, translated, and replicated. Duplicated SC2R can be quantified through the activity of sNLuc. Neo is a drug resistance gene as a marker gene. pA contributes to transcript stability. Rz is required for excision by self-cleavage after replication. BGH terminates transcription.
　ＳＣ２Ｒを、天然ウイルスを鋳型として使用しない人工合成により、タイプＩＩＳ型制限酵素を用いるゴールデンゲート法で以下のように合成した。 SC2R was synthesized by artificial synthesis without using a natural virus as a template, by the golden gate method using type IIS restriction enzyme, as follows.
　ＳＣ２Ｒの非構造領域では、配列番号１に示す塩基配列の５’末端から１７９７２番目から１７９７７番目までのＢｓａＩサイト（ｇａｇａｃｃ）に相当する塩基配列を同義置換するために、“ｇＧｇａｃｃ”とした。 In the non-structural region of SC2R, "gGgacc" was used to synonymously replace the base sequence corresponding to the BsaI site (gagacc) from 17972nd to 17977th from the 5' end of the base sequence shown in SEQ ID NO: 1.
　ＢｓａＩサイトを欠失させたＳＣ２Ｒ（２５５６２ｂｐ）を図１（Ｃ）に示すように２～３ｋｂの１０個のフラグメントＦ１～Ｆ１０に分けて人工遺伝子を合成した。詳細には、配列番号２に示すＳＣ２Ｒの塩基配列の５’末端から１～２７３５番目の２７３５ｂｐをＦ１、２７３２～５３６０番目の２８２９ｂｐをＦ２、５３５７～８１５１番目の２７９５ｂｐをＦ３、８１４８～１０５９３番目の２４４６ｂｐをＦ４、１０５９０～１３３７９番目の２７９０ｂｐをＦ５、１３３７６～１５８７７番目の２５０２ｂｐをＦ６、１５８７４～１８７０８番目の２８３５ｂｐをＦ７、１８７０５～２１１１４番目の２４１０ｂｐをＦ８、２１１１１～２３２８８番目の２１７８ｂｐをＦ９、２３２８５～２５５６２番目の２２７８ｂｐをＦ１０とした。各フラグメントの両端にＢｓａＩサイトの塩基配列を付加して、シームレスにＳＣ２ＲをＢＡＣベクターに組込むことができるように設計した。 An artificial gene was synthesized by dividing SC2R (25562 bp) with the BsaI site deleted into 10 fragments F1 to F10 of 2 to 3 kb as shown in Fig. 1 (C). Specifically, from the 5′ end of the SC2R nucleotide sequence shown in SEQ ID NO: 2, 2735 bp from 1 to 2735 is F1, 2829 bp from 2732 to 5360 is F2, 2795 bp from 5357 to 8151 is F3, and 2795 bp from 8148 to 10593 is F3. 2446 bp F4, 10590 to 13379 2790 bp F5, 13376 to 15877 2502 bp F6, 15874 to 18708 2835 bp F7, 18705 to 21114 2410 bp F8, 21111 to 23288 2178 bp to F9, 23285 2278 bp at position ~25562 was designated as F10. It was designed so that the nucleotide sequence of the BsaI site was added to both ends of each fragment so that SC2R could be seamlessly incorporated into the BAC vector.
　（人工遺伝子の合成）
　１０個のフラグメントそれぞれの５’末端と３’末端にＢｓａＩサイトを導入した。Ｆ１～４とＦ６～１０はｐＵＣ１９ｂに導入した（ｐＵＣ１９ｂ／ＳＣ２Ｒ－Ｆ１，Ｆ２，Ｆ３，Ｆ４，Ｆ６，Ｆ７，Ｆ８，Ｆ９，Ｆ１０）。Ｆ５はｐＣＣ１－４ｋベクターに導入した（ｐＣＣ１－４ｋ／ＳＣ２Ｒ－Ｆ５）。 (Synthesis of artificial genes)
A BsaI site was introduced at the 5' and 3' ends of each of the 10 fragments. F1-4 and F6-10 were introduced into pUC19b (pUC19b/SC2R-F1, F2, F3, F4, F6, F7, F8, F9, F10). F5 was introduced into the pCC1-4k vector (pCC1-4k/SC2R-F5).
　（クローニングベクターｐＳＭＡＲＴ　ＢＡＣｂｃの作製）
　人工的に合成したマルチクローニングサイトが導入されたｐＵＣ５７　ＮｏｔＩ－ＢｓａＩｃ－ＢｓａＩ－ＥｃｏＲＩ－ＨｉｎｄＩＩＩ－ＢａｍＨＩ－ＸｈｏＩ－ＡｖｒＩＩを制限酵素ＮｏｔＩとＡｖｒＩＩで切断し、配列番号３に示す９５５ｂｐの人工遺伝子を得た。ｐＳＭＡＲＴ　ＢＡＣｂのＮｏｔＩ－ＡｖｒＩＩサイトに人工遺伝子をライゲーションして、ｐＳＭＡＲＴ　ＢＡＣｂｃを作製した。 (Creation of cloning vector pSMART BACbc)
pUC57 NotI-BsaIc-BsaI-EcoRI-HindIII-BamHI-XhoI-AvrII into which an artificially synthesized multicloning site was introduced was cleaved with restriction enzymes NotI and AvrII to obtain an artificial gene of 955 bp shown in SEQ ID NO: 3. . The artificial gene was ligated into the NotI-AvrII sites of pSMART BACb to create pSMART BACbc.
　（クローニングベクターｐＨＳＧ２９８ｃ及びｐＣＣ１－４ｋｃの作製）
　ｐＨＳＧ２９８ベクターにＢｓａＩサイトを導入するため、ＥｃｏＲＩとＢａｍＨＩの内側にＢｓａＩを含むＰＣＲフラグメントＡ及びＢを作製した。ＰＣＲフラグメントＡの作製では、配列番号４に示すＨＲＰ遺伝子を鋳型として、フォワードプライマー（配列番号５）及びリバースプライマー（配列番号６）を用いてＰＣＲを行った。ＰＣＲフラグメントＡの塩基配列を配列番号７に示す。ＰＣＲフラグメントＢの作製では、配列番号４に示すＨＲＰ遺伝子を鋳型として、フォワードプライマー（配列番号８）及びリバースプライマー（配列番号９）を用いてＰＣＲを行った。ＰＣＲフラグメントＢの塩基配列を配列番号１０に示す。 (Creation of cloning vector pHSG298c and pCC1-4kc)
To introduce a BsaI site into the pHSG298 vector, PCR fragments A and B containing BsaI inside EcoRI and BamHI were prepared. In the preparation of PCR fragment A, PCR was performed using the HRP gene shown in SEQ ID NO: 4 as a template and a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6). The nucleotide sequence of PCR fragment A is shown in SEQ ID NO:7. In the preparation of PCR fragment B, PCR was performed using the HRP gene shown in SEQ ID NO: 4 as a template and a forward primer (SEQ ID NO: 8) and a reverse primer (SEQ ID NO: 9). The nucleotide sequence of PCR fragment B is shown in SEQ ID NO:10.
　また、ｐＣＣ１－４ｋベクターにＢｓａＩサイトを導入するため、ＨｉｎｄＩＩＩとＢａｍＨＩの内側にＢｓａＩを含むＰＣＲフラグメントＣを作製した。ＰＣＲフラグメントＣの作製では、配列番号４に示すＨＲＰ遺伝子を鋳型として、フォワードプライマー（配列番号１１）及びリバースプライマー（配列番号１２）を用いてＰＣＲを行った。ＰＣＲフラグメントＣの塩基配列を配列番号１３に示す。 In addition, in order to introduce a BsaI site into the pCC1-4k vector, a PCR fragment C containing BsaI inside HindIII and BamHI was prepared. In the preparation of PCR fragment C, PCR was performed using the HRP gene shown in SEQ ID NO: 4 as a template and a forward primer (SEQ ID NO: 11) and a reverse primer (SEQ ID NO: 12). The nucleotide sequence of PCR fragment C is shown in SEQ ID NO:13.
　ｐＨＳＧ２９８をＥｃｏＲＩとＢａｍＨＩで切断し、ＰＣＲフラグメントＡをライゲーションして、ｐＨＳＧ２９８ｃベクターＡを構築した。また、ｐＨＳＧ２９８をＥｃｏＲＩとＢａｍＨＩで切断し、ＰＣＲフラグメントＢをライゲーションして、ｐＨＳＧ２９８ｃベクターＢを構築した。ｐＣＣ１－４ｋをＨｉｎｄＩＩＩとＢａｍＨＩで切断し、ＰＣＲフラグメントＣをライゲーションして、ｐＣＣ１－４ｋｃベクターを構築した。 pHSG298 was cleaved with EcoRI and BamHI, and PCR fragment A was ligated to construct pHSG298c vector A. Further, pHSG298 was cleaved with EcoRI and BamHI, and PCR fragment B was ligated to construct pHSG298c vector B. pCC1-4k was cleaved with HindIII and BamHI and PCR fragment C was ligated to construct pCC1-4kc vector.
　（中間フラグメントｐＨＳＧ２９８ｃ／Ｆ１２３、ｐＣＣ１－４ｋｃ／Ｆ４５６及びｐＨＳＧ２９８ｃ／Ｆ７－１０の作製）
　ｐＵＣ１９ｂ／Ｆ１，Ｆ２，Ｆ３，Ｆ４，Ｆ６，Ｆ７，Ｆ８，Ｆ９，Ｆ１０及びｐＣＣ１－４ｋ／Ｆ５をそれぞれＢｓａＩで切断した。Ｆ１、Ｆ２及びＦ３をｐＨＳＧ２９８ｃベクターＡに導入し、ｐＨＳＧ２９８ｃ／Ｆ１２３とした。Ｆ４、Ｆ５及びＦ６をｐＣＣ１－４ｋｃベクターに導入し、ｐＣＣ１－４ｋｃ／Ｆ４５６とした。Ｆ７、Ｆ８、Ｆ９及びＦ１０をｐＨＳＧ２９８ｃベクターＢに導入し、ｐＨＳＧ２９８ｃ／Ｆ７－１０とした。 (Generation of intermediate fragments pHSG298c/F123, pCC1-4kc/F456 and pHSG298c/F7-10)
pUC19b/F1, F2, F3, F4, F6, F7, F8, F9, F10 and pCC1-4k/F5 were each cut with BsaI. F1, F2 and F3 were introduced into pHSG298c vector A, resulting in pHSG298c/F123. F4, F5 and F6 were introduced into the pCC1-4kc vector, resulting in pCC1-4kc/F456. F7, F8, F9 and F10 were introduced into pHSG298c vector B, resulting in pHSG298c/F7-10.
　（ＳＣ２Ｒベクターの作製）
　ｐＨＳＧ２９８ｃ／Ｆ１２３、ｐＣＣ１－４ｋｃ／Ｆ４５６及びｐＨＳＧ２９８ｃ／Ｆ７－１０をＢｓａＩで切断し、各フラグメントをｐＳＭＡＲＴ　ＢＡＣｂｃのＢｓａＩ－ＢｓａＩサイトにライゲーションした。ライゲーション後、エレクトロポレーション法にてＢＡＣ－Ｏｐｔｉｍｉｚｅｄ　Ｒｅｐｌｉｃａｔｏｒ　ｖ２．０　Ｅｌｅｃｔｒｏｃｏｍｐｅｔｅｎｔ　Ｃｅｌｌｓ（Ｌｕｃｉｇｅｎ社製）に導入しクローニングを行った。以上により、ｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒを得た。 (Preparation of SC2R vector)
pHSG298c/F123, pCC1-4kc/F456 and pHSG298c/F7-10 were cleaved with BsaI and each fragment was ligated to the BsaI-BsaI site of pSMART BACbc. After ligation, cloning was performed by introducing into BAC-Optimized Replicator v2.0 Electrocompetent Cells (manufactured by Lucigen) by electroporation. As described above, pSMART BACbc/SC2R was obtained.
　（ｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒ　ＳＡＡベクターの作製）
　Ｆ７に存在するＲＮＡ依存性ＲＮＡポリメラーゼ（ＲｄＲＰ）の活性部位はモチーフである“ＳＤＤ”（ｔｃｔ　ｇａｃ　ｇａｔ）を有している。ＱｕｉｃｋＣｈａｎｇｅ　ｍｕｔａｇｅｎｅｓｉｓを用いて、“ＳＤＤ”から“ＳＡＡ”（ｔｃｔ　ｇＣｃ　ｇＣｔ）になるよう置換を行い、ＲｄＲＰ活性を欠損させたｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒ　ＳＡＡベクターをｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒと同様に構築した。 (Preparation of pSMART BACbc/SC2R SAA vector)
The active site of the RNA-dependent RNA polymerase (RdRP) present in F7 has the motif "SDD" (tct gac gat). Using QuickChange mutagenesis, "SDD" was replaced with "SAA" (tct gCc gCt) to construct pSMART BACbc/SC2R SAA vector lacking RdRP activity in the same manner as pSMART BACbc/SC2R.
　（ｐＣＸ４ｂｓｒ／ＳＡＲＳ－ＣｏＶ－２－Ｎベクターの作製）
　ＳＡＲＳ－ＣｏＶ－２のＮ遺伝子を増幅するためにｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒを鋳型としてＰＣＲを行なった。当該ＰＣＲに使用したフォワードプライマー及びリバースプライマーの塩基配列を、それぞれ配列番号１４及び１５に示す。５’末端と３’末端にＭｌｕＩとＮｏｔＩサイトを導入したＰＣＲフラグメントを作製し、ＭｌｕＩとＮｏｔＩで切断したｐＣＸ４ｂｓｒベクターにＰＣＲフラグメントをライゲーションして、ｐＣＸ４ｂｓｒ／ＳＡＲＳ－ＣｏＶ－２－Ｎベクターを構築した。 (Preparation of pCX4bsr/SARS-CoV-2-N vector)
PCR was performed using pSMART BACbc/SC2R as a template to amplify the N gene of SARS-CoV-2. The nucleotide sequences of the forward primer and reverse primer used for the PCR are shown in SEQ ID NOs: 14 and 15, respectively. A PCR fragment was prepared by introducing MluI and NotI sites at the 5' end and 3' end, and the PCR fragment was ligated to the pCX4bsr vector cleaved with MluI and NotI to construct the pCX4bsr/SARS-CoV-2-N vector. .
　（ＨｕＨ－７．６ｃ細胞の樹立）
　レポーター全長ＨＣＶ　ＲＮＡをエレクトロポレーションによりＨｕＨ－７細胞に導入後Ｇ４１８を添加するとＨＣＶ　ＲＮＡ複製レベルの高い細胞が選択されてコロニーを形成する。コロニーの中で最もＨＣＶ　ＲＮＡ複製レベルの高いクローン化細胞を細胞株として樹立したものがＯＲ６細胞株となる。次に、ＯＲ６細胞にＩＦＮを添加することでＨＣＶ　ＲＮＡを排除して治癒細胞株（ＨｕＨ－７．６ｃ細胞株）を樹立した。治癒細胞株は親株のＨｕＨ－７細胞株よりもＨＣＶ　ＲＮＡの複製レベルが高いことが知られている（Ｍａｓａｎｏｒｉ　Ｉｋｅｄａ、外５名、「Ｅｆｆｉｃｉｅｎｔ　ｒｅｐｌｉｃａｔｉｏｎ　ｏｆ　ａ　ｆｕｌｌ－ｌｅｎｇｔｈ　ｈｅｐａｔｉｔｉｓ　Ｃ　ｖｉｒｕｓ　ｇｅｎｏｍｅ，　ｓｔｒａｉｎ　Ｏ，ｉｎ　ｃｅｌｌ　ｃｕｌｔｕｒｅ，ａｎｄ　ｄｅｖｅｌｏｐｍｅｎｔ　ｏｆ　ａ　ｌｕｃｉｆｅｒａｓｅ　ｒｅｐｏｒｔｅｒ　ｓｙｓｔｅｍ」、Ｂｉｏｃｈｅｍ　Ｂｉｏｐｈｙｓ　Ｒｅｓ　Ｃｏｍｍｕｎ．、２００５年、３２９（４）：１３５０－９）。 (Establishment of HuH-7.6c cells)
When the full-length reporter HCV RNA was introduced into HuH-7 cells by electroporation and then G418 was added, cells with high HCV RNA replication levels were selected to form colonies. A cloned cell with the highest HCV RNA replication level among the colonies was established as a cell line to become the OR6 cell line. Next, a curative cell line (HuH-7.6c cell line) was established by eliminating HCV RNA by adding IFN to OR6 cells. It is known that the cured cell line has a higher level of HCV RNA replication than the parent HuH-7 cell line (Masanori Ikeda, et al., ``Efficient replication of a full-length hepatitis C virus genome, strain O,''). in cell culture, and development of a luciferase reporter system,” Biochem Biophys Res Commun., 2005, 329(4):1350-9).
　（ＳＣ２Ｒアッセイによる評価）
　ＨｕＨ－７．６ｃ細胞を６ウェルプレートに１×１０５細胞／ウェルになるように播種し、翌日ｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒ　２μｇをＦｕｇｅｎｅ　ＨＤ試薬を用いてトランスフェクションし細胞に導入した。２４時間後、トリプシンを用いて細胞を剥離し、２４ウェルプレートに３×１０４細胞／ウェルになるように播種した。さらに２４時間後、化合物を所定の濃度になるように添加した。４８時間培養後、培養上清を採取し、Ｎａｎｏ－Ｇｌｏ　Ｌｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ　Ｓｙｓｔｅｍを用いてルシフェラーゼ活性を測定した。化合物としてレムデシビルを添加した場合、濃度依存的にルシフェラーゼ活性が低下したため、レムデシビルによってＳＣ２Ｒの複製が抑制された。すなわち、ＳＣ２Ｒアッセイで抗ＳＡＲＳ－ＣｏＶ－２活性を有する化合物を同定できることを確認した。 (Evaluation by SC2R assay)
HuH-7.6c cells were seeded in a 6-well plate at 1×10 5 cells/well, and the next day, 2 μg of pSMART BACbc/SC2R was transfected using Fugene HD reagent and introduced into the cells. After 24 hours, cells were detached using trypsin and seeded at 3 x 104 cells/well in 24-well plates. After an additional 24 hours, compounds were added to the indicated concentrations. After culturing for 48 hours, the culture supernatant was collected and luciferase activity was measured using the Nano-Glo Luciferase Assay System. When remdesivir was added as a compound, the luciferase activity decreased in a concentration-dependent manner, and SC2R replication was suppressed by remdesivir. That is, it was confirmed that compounds having anti-SARS-CoV-2 activity could be identified by the SC2R assay.
　（細胞毒性の評価）
　ＨｕＨ－７．６ｃ細胞を、９６ウェルプレートに５×１０３個になるように播種した。培養開始から２４時間後に、化合物を、所定の濃度になるように細胞に添加した。４８時間培養した後、Ｐｒｅｍｉｘ　ＷＳＴ－１　Ｃｅｌｌ　Ｐｒｏｌｉｆｅｒａｔｉｏｎ　Ａｓｓａｙ　Ｓｙｓｔｅｍ（タカラバイオ社製）１０μｌを培地に添加して、３７℃で２時間培養後、マイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定した。 (Evaluation of cytotoxicity)
HuH-7.6c cells were seeded in 96-well plates at 5×10 3 cells. Twenty-four hours after the initiation of culture, compounds were added to the cells at the given concentrations. After culturing for 48 hours, 10 μl of Premix WST-1 Cell Proliferation Assay System (manufactured by Takara Bio Inc.) was added to the medium, cultured at 37° C. for 2 hours, and absorbance at 450 nm was measured using a microplate reader.
　（ウエスタンブロット解析）
　ＳＣ２Ｒアッセイと同様に６ウェルプレートで培養したＨｕＨ－７．６ｃにＳＤＳゲルローディングバッファーを加えて、超音波法にて粗タンパク質分画を回収した。これをＳＤＳ－ポリアクリルアミドゲル電気泳動法により分離した。タンパク質を分離する際に使用したＳＤＳ－ポリアクリルアミドゲルは１０％の濃度で使用した。分離したタンパク質をセミドライブロッティング法にてＰＶＤＦメンブレンに転写した。転写後、メンブレンをブロッキングバッファーにて室温で１時間ブロッキングした後、目的のタンパク質に対する特異的な抗体で、４℃で一晩反応させた。抗体には、ＳＡＲＳ－ＣｏＶ－２　Ｎｕｃｌｅｏｃａｐｓｉｄ　Ｐｒｏｔｅｉｎ（Ｅ８Ｒ１Ｌ）　Ｍｏｕｓｅ　ｍＡｂ（１／１０００、Ｃｅｌｌ　ｓｉｇｎａｌｉｎｇ社製）と抗β－アクチン抗体（１／４０００、ＡＣ－１５、Ｓｉｇｍａ社製）を用いた。 (Western blot analysis)
SDS gel loading buffer was added to HuH-7.6c cultured in a 6-well plate in the same manner as in the SC2R assay, and a crude protein fraction was collected by ultrasonication. This was separated by SDS-polyacrylamide gel electrophoresis. The SDS-polyacrylamide gel used for protein separation was used at a concentration of 10%. Separated proteins were transferred to a PVDF membrane by a semi-dry blotting method. After transfer, the membrane was blocked with a blocking buffer at room temperature for 1 hour, and then reacted overnight at 4°C with a specific antibody against the protein of interest. SARS-CoV-2 Nucleocapsid Protein (E8R1L) Mouse mAb (1/1000, Cell Signaling) and anti-β-actin antibody (1/4000, AC-15, Sigma) were used as antibodies.
　１次抗体を一晩反応させた後、２次抗体ＨＲＰ－ａｎｔｉ－ｍｏｕｓｅと、室温で２５分間反応させた。２次抗体反応後、Ｗｅｓｔｅｒｎ　Ｌｉｇｈｔｉｎｇ　Ｐｌｕｓ－ＥＣＬ　Ｅｎｈａｎｃｅｄ　Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅ　Ｓｕｂｓｔｒａｔｅ（Ｐｅｒｋｉｎ　Ｅｉｍｅｒ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅ社製）を用いて発光させ、ＢｉｏＭａｘ　Ｌｉｇｈｔ　Ｆｉｌｍ（ＫＯＤＡＫ社製）に感光した。現像液にはＲｅｎｄｏｌ（富士フイルム社製）、定着液にはＳＵＰＥＲＦＵＪＩＦＩＸ－Ｌ（富士フイルム社製）を使用して、フイルムを現像した。なお、陽性対照及び陰性対照として、ｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒに代えて、それぞれｐＣＸ４ｂｓｒ／ＳＡＲＳ－ＣｏＶ－２－Ｎベクター及びｐＳＭＡＲＴ　ＢＡＣｂｃ／ＳＣ２Ｒ　ＳＡＡベクターを使用した。 After allowing the primary antibody to react overnight, it was allowed to react with the secondary antibody HRP-anti-mouse at room temperature for 25 minutes. After the secondary antibody reaction, Western Lighting Plus-ECL Enhanced Chemiluminescence Substrate (manufactured by Perkin Eimer Life Science) was used to emit light and exposure to BioMax Light Film (manufactured by KODAK). The film was developed using Rendol (manufactured by Fuji Film Co., Ltd.) as a developer and SUPERFUJIFIX-L (manufactured by Fuji Film Co., Ltd.) as a fixer. As positive and negative controls, pCX4bsr/SARS-CoV-2-N vector and pSMART BACbc/SC2R SAA vector were used instead of pSMART BACbc/SC2R, respectively.
　（結果）
　化合物として食事療法関連化合物ライブラリ（Ｌ６８００、Ｓｅｌｌｅｃｋ社製）の３７３種類の化合物（１０μＭ）をＳＣ２Ｒアッセイで評価した。また、各化合物（１０μＭ）について細胞毒性を評価した。図２にその一部の結果を示す。陽性対照として１００ｎＭレムデジビルを使用した。（２０Ｒ）－プロトパナキサジオールのみが５０％以上のルシフェラーゼ活性抑制を示した。 (result)
As compounds, 373 kinds of compounds (10 μM) in a diet-related compound library (L6800, Selleck) were evaluated by the SC2R assay. Cytotoxicity was also evaluated for each compound (10 μM). FIG. 2 shows some of the results. 100 nM Remdesivir was used as a positive control. Only (20R)-protopanaxadiol showed more than 50% inhibition of luciferase activity.
　図３は、（２０Ｒ）－プロトパナキサジオール及び（２０Ｓ）－プロトパナキサジオールのＳＣ２Ｒアッセイの結果を示す。（２０Ｒ）－プロトパナキサジオールは、濃度依存的にルシフェラーゼ活性を抑制し、ＥＣ５０は５．４６μＭであった。（２０Ｓ）－プロトパナキサジオールにもルシフェラーゼ活性の抑制を認めた。図４は、各濃度の（２０Ｒ）－プロトパナキサジオールでの細胞生存率を示す。（２０Ｒ）－プロトパナキサジオールのＣＣ５０は２６．７μＭであった。 FIG. 3 shows the SC2R assay results for (20R)-protopanaxadiol and (20S)-protopanaxadiol. (20R)-protopanaxadiol inhibited luciferase activity in a concentration-dependent manner with an EC50 of 5.46 μM. (20S)-protopanaxadiol was also found to inhibit luciferase activity. FIG. 4 shows cell viability at each concentration of (20R)-protopanaxadiol. The CC 50 of (20R)-protopanaxadiol was 26.7 μM.
　図５は、ウエスタンブロット解析の結果とルシフェラーゼ活性の相対値を示す。（２０Ｒ）－プロトパナキサジオール及び（２０Ｓ）－プロトパナキサジオールによって、Ｎタンパク質の発現が抑制されることを確認した。 Fig. 5 shows the results of Western blot analysis and the relative values of luciferase activity. It was confirmed that (20R)-protopanaxadiol and (20S)-protopanaxadiol suppress the expression of N protein.
　上述した実施の形態は、本発明を説明するためのものであり、本発明の範囲を限定するものではない。すなわち、本発明の範囲は、実施の形態ではなく、特許請求の範囲によって示される。そして、特許請求の範囲内及びそれと同等の発明の意義の範囲内で施される様々な変形が、本発明の範囲内とみなされる。 The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.
　本出願は、２０２２年２月３日に出願された、日本国特許出願２０２２－１５４９２号に基づく。本明細書中に日本国特許出願２０２２－１５４９２号の明細書、特許請求の範囲、図面全体を参照として取り込むものとする。 This application is based on Japanese Patent Application No. 2022-15492 filed on February 3, 2022. The entire specification, claims, and drawings of Japanese Patent Application No. 2022-15492 are incorporated herein by reference.
　本発明はＣＯＶＩＤ－１９の治療に有用である。 The present invention is useful for treating COVID-19.

Claims (4)

  1. 　プロトパナキサジオール又は薬理学的に許容されるその塩を含む、
    　ＣＯＶＩＤ－１９治療薬。     Protopanaxadiol or a pharmacologically acceptable salt thereof,
    COVID-19 treatment.
  2. 　前記プロトパナキサジオールは、
    　（２０Ｒ）－プロトパナキサジオールである、
    　請求項１に記載のＣＯＶＩＤ－１９治療薬。     The protopanaxadiol is
    is (20R)-protopanaxadiol;
    The COVID-19 therapeutic of claim 1.
  3. 　プロトパナキサジオール又は薬理学的に許容されるその塩を含む、
    　ＣＯＶＩＤ－１９治療用経口組成物。     Protopanaxadiol or a pharmacologically acceptable salt thereof,
    Oral compositions for treating COVID-19.
  4. 　ＣＯＶＩＤ－１９治療薬の製造のためのプロトパナキサジオール又は薬理学的に許容されるその塩の使用。 　Use of protopanaxadiol or a pharmacologically acceptable salt thereof for the manufacture of a COVID-19 therapeutic agent.
PCT/JP2023/001263 2022-02-03 2023-01-18 Covid-19 therapeutic, oral composition for treating covid-19, and use of compounds for manufacturing covid-19 therapeutic WO2023149204A1 (en)

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