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WO2020241814A1 - Agent antiviral ou inhibiteur de formation d'adnccc viral - Google Patents

Agent antiviral ou inhibiteur de formation d'adnccc viral Download PDF

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Publication number
WO2020241814A1
WO2020241814A1 PCT/JP2020/021269 JP2020021269W WO2020241814A1 WO 2020241814 A1 WO2020241814 A1 WO 2020241814A1 JP 2020021269 W JP2020021269 W JP 2020021269W WO 2020241814 A1 WO2020241814 A1 WO 2020241814A1
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Prior art keywords
allicin
antiviral agent
antiviral
formation inhibitor
cccdna formation
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Japanese (ja)
Inventor
恭爾 森屋
和也 奥新
秀剛 藤永
崇介 福原
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University of Tokyo NUC
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University of Tokyo NUC
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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
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • 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
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • 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
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • the present invention relates to an antiviral agent (for example, an anti-hepatitis B virus agent) or a viral cccDNA formation inhibitor.
  • an antiviral agent for example, an anti-hepatitis B virus agent
  • a viral cccDNA formation inhibitor for example, an anti-hepatitis B virus agent
  • Hepatitis B virus causes chronic hepatitis in humans and is involved in the development of liver cirrhosis and liver cancer.
  • HBV Hepatitis B virus
  • Interferon (IFN) and nucleic acid analog preparations are the first-line drugs for the treatment of HBV.
  • HBV forms extremely stable closed circular DNA (cccDNA) in the nucleus of infected hepatocytes.
  • cccDNA extremely stable closed circular DNA
  • Non-Patent Document 1 Non-Patent Document 1
  • the present inventor finds that allicin or a related compound thereof can exert an antiviral effect on HBV, for example, an inhibitory effect on cccDNA formation, and that allicin or a related compound thereof is used in combination with other antiviral agents.
  • the present invention has been completed by finding that the effect is remarkably enhanced.
  • the present invention includes the following embodiments.
  • An anti-hepatitis B virus agent containing a compound selected from the group consisting of allicin, alliin, allyl sulfide, S-allylcysteine, and ajoene.
  • a viral cccDNA formation inhibitor containing a compound selected from the group consisting of allicin, alliin, allyl sulfide, S-allylcysteine, and ajoene.
  • An antiviral agent comprising a compound selected from the group consisting of allicin, alliin, allyl sulfide, S-allylcysteine, and ajoene, which is administered together with one or more other antiviral agents.
  • Agent. A viral cccDNA formation inhibitor comprising a compound selected from the group consisting of allicin, alliin, allyl sulfide, S-allylcysteine, and ajoene, which is administered with one or more other antiviral agents. Viral ccc DNA formation inhibitor. (6) The antiviral agent according to (4) or the viral cccDNA formation inhibitor according to (5), wherein the compound is allicin or alliin.
  • An antiviral agent containing the viral cccDNA formation inhibitor according to (2) as an active ingredient (12) An antiviral composition comprising the antiviral agent according to (11) and one or more other antiviral agents. (13) The antiviral composition according to (12), wherein the other antiviral agent is one or more selected from the group consisting of cytokines, nucleic acid analogs, and statins. (14) The antiviral composition according to (13), wherein the cytokine is interferon- ⁇ , the nucleic acid analog is entecavir, and the statins are fluvastatin. (15) The antiviral composition according to any one of (12) to (14), wherein the virus is hepatitis B virus. This specification includes the disclosure of Japanese Patent Application No. 2019-099892, which is the basis of the priority of the present application.
  • the present invention provides an antiviral agent against viruses such as HBV, for example, a virus cccDNA formation inhibitor.
  • FIG. 1 shows the measurement results of the antiviral activity of allicin or allicin in vitro.
  • ETV shows the effect of entecavir, which is a positive control.
  • Figure 2 shows treatment in A: treatment group A (IFN + entecavir + fluvastatin + alicin 4 drugs), B: treatment group B (IFN + lamivudine + fluvastatin 3 drugs administration), and C: no treatment group C.
  • the measurement result of the amount of HBV cccDNA in the liver on the 56th day after that is shown. * Indicates a significant difference (p ⁇ 0.05) from the untreated group C.
  • FIG. 3 shows the measurement results of serum HBV DNA concentration up to 56 days after the treatment. Each group is similar to the group in FIG.
  • FIG. 4 shows the measurement results of body weight (A) and blood human albumin (hAlb) concentration (B) up to 56 days after the treatment. Each treatment group is similar to the group in FIG. * Indicates a significant difference (p ⁇ 0.05) from the untreated group C.
  • FIG. 5 shows the measurement results of serum AST activity (A) and serum ALT activity (B) up to 56 days after the treatment. Each treatment group is similar to the group in FIG.
  • FIG. 6 shows the result (A) of correcting the amount of HBV cccDNA in the liver 56 days after the treatment with the blood human albumin (hAlb) measurement value, and the HBV-DNA measurement value up to the 56th day after the treatment in the blood.
  • FIG. 7 shows the measurement results of serum HBV DNA concentration up to 56 days after the treatment.
  • A indicates treatment group A (IFN + entecavir + fluvastatin + allicin)
  • C indicates no treatment group C.
  • E indicates the treatment group E (IFN + entecavir + allicin) in which fluvastatin was removed from the treatment group A.
  • FIG. 8 is a Western blot showing the amount of ISG15 in the liver 56 days after treatment detected with an anti-ISG15 antibody.
  • Lanes 1 and 2 are HBV-uninfected untreated groups (Mock), lanes 3 and 4 are HBV-infected untreated groups (HBV), and lanes 5 and 6 are entecavir-treated groups in which HBV-infected individuals are treated with entecavir alone.
  • HBV + ETV Lanes 7 and 8 are HBV-infected individuals treated with flubustatin alone (HBV + Statin)
  • Lanes 9 and 10 are HBV-infected individuals treated with Alicin alone (HBV + Allicin)
  • lanes 11 and 12 show the peginterferon ⁇ treatment group (HBV + PegIFN) in which peginterferon ⁇ was administered as a single agent to HBV-infected individuals.
  • the present invention relates to an anti-hepatitis B virus agent or viral cccDNA formation inhibitor, including alicin or an analog thereof.
  • Allicin is a volatile odor compound derived from plants of the genus Allium such as garlic, and is produced by converting alliin, a substrate component contained in cells, with the enzyme alliinase due to cell destruction. Allicin is known to have strong antibacterial / antifungal activity, antioxidant activity, and immunostimulatory activity.
  • allicin analogs include compounds such as alliin, allyl sulfide, S-allylcysteine, and ajoene.
  • Alliin is a derivative of cysteine, a naturally occurring sulfoxide.
  • allyl sulfide refers to a sulfide having a structure in which at least one allyl group is bonded to a sulfur atom (for example, 1 to 6 sulfur atoms), and examples thereof include diallyl sulfide and diallyl.
  • the anti-hepatitis B virus agent or viral cccDNA formation inhibitor of the present invention may contain only one allicin or an analog thereof, or may contain two or more allicin.
  • Allicin or its analogs can be obtained by methods known to those skilled in the art. For example, a chemically synthesized product may be used, a product extracted and purified from a natural product (for example, garlic) such as a plant of the genus Allium may be used, or a commercially available product may be used. When extracting or purifying from natural products, allicin or its analogs may be fully purified, partially purified, or extracts.
  • the weight% of alicin or its relatives in the purified product or extract is not limited, but is, for example, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, 90% by weight or more, 95% by weight or more. , 98% by weight or more, or 99% by weight or more.
  • anti-hepatitis B virus agent means, by its application, reducing the number of copies of hepatitis B virus (eg, in a subject or tissue thereof) and / or reducing hepatitis B in a subject. Means a drug that can be treated and / or prevented.
  • treatment includes complete or partial cure, alleviation, or prevention of progression of a disease or associated symptoms in a patient who already has symptoms.
  • prevention includes suppressing the onset or reducing the incidence in a patient who may have the disease.
  • cccDNA formation inhibitor means a drug having a cccDNA formation inhibitory action. Therefore, a cccDNA formation inhibitor can be an active ingredient of an antiviral agent against a virus that forms a cccDNA structure.
  • An antiviral agent that suppresses and inhibits the growth of a virus based on such a cccDNA formation inhibitory effect is often referred to as a "viral cccDNA formation inhibitor" in the present specification.
  • cccDNA means covalently closed circular DNA that occurs in the nucleus of a cell during the growth of some viruses and can function as a transcription template for viral RNA.
  • Viruses capable of forming cccDNA or similar circular DNA include, for example, Hepadnaviridae (Hepadnaviridae) or Caulimoviridae (Carimovirus), and Hepadnaviridae viruses include the genus Orthohepadnavirus, eg, Orthohepadnavirus.
  • Hepadnaviridae virus is mentioned, and the virus of Caulimoviridae includes the genus Caulimovirus, for example, Califlower mosaic virus.
  • the anti-hepatitis B virus agent or viral cccDNA formation inhibitor of the present invention comprises or comprises allicin or an analog thereof, or consists or substantially consists of allicin or an analog thereof.
  • the invention relates to an antiviral agent comprising allicin or an analog thereof for concomitant use with one or more other antiviral agents.
  • a viral cccDNA formation inhibitor comprising allicin or an analog thereof relates to a viral cccDNA formation inhibitor that is administered simultaneously with or separately from one or more other antiviral agents.
  • the antiviral agent of the present invention containing allicin or its relatives and one or more other antiviral agents When the antiviral agent of the present invention containing allicin or its relatives and one or more other antiviral agents are administered at the same time, the antiviral agent containing allicin or its relatives and one or more other antiviral agents It may be administered as an antiviral composition containing, or an antiviral agent containing allicin or an analog thereof may be co-administered separately with one or more other antiviral agents.
  • an antiviral agent containing allicin or its relatives and one or more other antiviral agents are administered separately at different times, the order of administration is not limited and the antiviral agent containing allicin or its relatives was administered. Later, one or more other antiviral agents may be administered, or one or more other antiviral agents may be administered before administration of an antiviral agent containing allicin or an analog thereof.
  • the administration of the antiviral agent containing allicin or its relatives and one or more other antiviral agents may be repeated, in which case the antiviral agent containing allicin or its relatives and one or more other antiviral agents.
  • the dosing intervals may be the same or different.
  • the combined use of two or more drugs is aimed at the combined effect of one or more other antiviral agents with an antiviral agent containing allicin or its relatives
  • the following As for the interval until the antiviral agent is administered it is preferable to administer the other antiviral agent before the effect of the previously administered antiviral agent is lost in vivo.
  • it is used in combination as the antiviral composition.
  • virus species to which the antiviral agent or viral cccDNA formation inhibitor of this embodiment can be applied include DNA viruses such as double-stranded DNA virus, single-stranded DNA virus, and double-stranded DNA reverse transcription virus.
  • double-stranded DNA reverse transcription viruses include Hepadnaviridae (Hepadnaviridae) or Caulimoviridae (Caulifloweridae), and Hepadnaviridae viruses include the genus Orthohepadnavirus, such as hepatnaviridae virus.
  • the virus of Caulimoviridae include the genus Caulimovirus, for example, cauliflower mosaic virus.
  • antiviral agents are, but are not limited to, one or more selected from the group consisting of, for example, cytokines, nucleic acid analogs, and statins (cytokines and nucleic acid analogs, cytokines and statins, or nucleic acid analogs and Statins) or all three may be included.
  • cytokines cytokines and nucleic acid analogs, cytokines and statins, or nucleic acid analogs and Statins
  • each antiviral agent may be administered at the same time or at different time points.
  • cytokines examples include type I interferon, such as interferon ⁇ , interferon ⁇ , and interferon ⁇ . Cytokines may be modified such as PEG modification for the purpose of prolonging the half-life in blood, for example. Specific examples include peginterferon (PegIFN) in which interferon is modified with polyethylene glycol.
  • the dose of cytokine is not limited, but is, for example, about 0.1 ⁇ g to 100 mg / 1 kg body weight, about 1 ⁇ g to 10 mg / 1 kg body weight, about 10 ⁇ g to 1 mg / 1 kg body weight, or about 50 ⁇ g to 200 ⁇ g / 1 kg body weight or about 100 ⁇ g / 1 kg body weight. It may be there.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every 2 weeks. It may be once, once a month, etc.
  • nucleic acid analogs examples include entecavir, lamivudine, adefovir, sofosbuvir, tenofovir, zidovudine, and ribavirin.
  • the dose of the nucleic acid analog is not limited, for example, about 0.03 ⁇ g to 300 mg / 1 kg body weight, about 0.3 ⁇ g to 3 mg / 1 kg body weight, about 3 ⁇ g to 300 ⁇ g / 1 kg body weight, or about 15 ⁇ g to 60 ⁇ g / 1 kg body weight or about 30 ⁇ g /. It may weigh 1 kg.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every 2 weeks. It may be once, once a month, etc.
  • statins examples include fluvastatin, atorvastatin, serivastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.
  • the dose of statins is not limited, for example, about 0.01 mg to 100 mg / 1 kg body weight, about 0.1 m to 100 mg / 1 kg body weight, about 1 mg to 100 mg / 1 kg body weight or about 5 mg to 20 mg / 1 kg body weight or about 10 mg / 1 kg. It may be weight.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every 2 weeks. It may be once, once a month, etc.
  • the invention comprises allicin or an analog thereof, and one or more other components (eg, one or more of the above other antiviral agents and / or the other components below). With respect to antiviral compositions or compositions for inhibiting viral cccDNA formation.
  • compositions of the invention include carriers such as, for example, pharmaceutically acceptable carriers, such as sterile water, saline, buffers, excipients, binders, disintegrants, emulsifiers, surfactants. Activators, stabilizers, lubricants, diluents, fluidity promoters, flavoring agents, colorants, fragrances and the like can be mentioned.
  • pharmaceutically acceptable carriers such as sterile water, saline, buffers, excipients, binders, disintegrants, emulsifiers, surfactants.
  • Activators, stabilizers, lubricants, diluents, fluidity promoters, flavoring agents, colorants, fragrances and the like can be mentioned.
  • composition of the present invention can be formulated by a conventional method.
  • formulation for example, the method described in Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.) Can be referred to.
  • the form of administration is not particularly limited and is appropriately selected as necessary, but is generally oral such as tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, and elixirs. It can be administered as an agent or a parenteral agent such as an injection, a drip, a suppository, an inhalant, a transdermal absorbent, a transmucosal absorbent, a patch, and an ointment.
  • a parenteral agent such as an injection, a drip, a suppository, an inhalant, a transdermal absorbent, a transmucosal absorbent, a patch, and an ointment.
  • the route of administration of the antiviral agent or composition of the present invention is not limited, but for example, by inhalation, spray administration, injection, infusion, oral, transdermal, nasal, translocal, transvaginal, transmucosal, or transrectal. It may be administered. Further, the composition of the present invention is formulated for inhalation, spray administration, injection, infusion, oral, transdermal, nasal, translocal, transvaginal, transmucosal, or transrectal. Good. The route of administration can be appropriately selected depending on the applicable disease and / or the subject.
  • Subjects to whom the antiviral agent or composition of the present invention can be administered are, but are not limited to, animals or plants, such as mammals, such as primates such as humans and chimpanzees, and experimental animals such as rats and mice. , Pigs, cows, horses, sheep, and livestock animals such as goats, and pet animals such as dogs and cats, such as humans or mice, preferably humans.
  • the dosage, administration interval, and administration period of the antiviral agent or composition described in the present specification will vary depending on those skilled in the art, such as the sex, weight, age of the subject, and the course and symptoms of the disease. It can be determined as appropriate in consideration of factors.
  • the dose of the antiviral agent or composition is, for example, about 0.1 ⁇ g to 100 mg / 1 kg body weight, about 1 ⁇ g to 10 mg / 1 kg body weight, about 10 ⁇ g to 1 mg / 1 kg body weight or about 50 ⁇ g to 200 ⁇ g / 1 kg body weight or about 100 ⁇ g / It may weigh 1 kg.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every 2 weeks. It may be once, once a month, etc.
  • the administration period may be, but is not limited to, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.
  • the invention comprises administering to a subject an allicin or analog thereof (and optionally one or more other antiviral agents), or compositions described herein. Concerning therapeutic and / or prophylactic methods, or methods of inhibiting the formation of viral cccDNA.
  • the administration subject, dose, number of administrations, administration route, etc. in this embodiment are as described in the present specification.
  • the invention is the allicin or analog thereof (and optionally one or more other antis) described herein for use in viral treatment and / or prophylactic methods, or methods of inhibiting the formation of viral cccDNA.
  • Viral agents or compositions.
  • Example 1 Measurement of antiviral activity in vitro> (Method) Allicin (Cayman Chemical Company) or Alliin (Sigma-Aldrich) was diluted to 20 mM with DMSO to prepare Master Stock. 2x10 5 cells / 24 well HepG2 / NTCP-C4 cells (HBV highly infection-tolerant liver cancer cell line: distributed by Dr. Koichi Watashi, National Institute of Infectious Diseases) at a concentration of 1x10 4 GEq / cell of HBV (HepAD38 cells) Obtained from the supernatant, genotype D) was infected. At the same time, the above Master stock was added and processed so that the final concentration was 10 ⁇ M or 1 ⁇ M. As a positive control, the nucleic acid analog entecavir (ETV) (selleck chemicals) known as an antiviral agent was used at a final concentration of 500 nM.
  • ETV nucleic acid analog entecavir
  • Cells were collected on the 14th day and copies / well were quantified. Specifically, Core-related rcDNA (relaxed circular DNA) in 1 well was extracted and the copy number was calculated by real-time PCR. Specifically, the cell pellet was lysed with a lysis buffer (50 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1% NP-40) at 4 ° C. for 15 minutes. After centrifuging at 15000 rpm for 5 minutes, the supernatant was incubated with 7 mM magnesium acetate, 0.2 mg / ml Dnase I (Roche), and 0.1 mg / ml Rnase A (Sigma) at 37 ° C. for 3 hours.
  • a lysis buffer 50 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1% NP-40
  • the lysate was digested with proteinase K (0.3 mg / ml, Thermo Fisher Scientific) and 2% SDS at 37 ° C. for 12 hours.
  • the extracted HBV DNA was purified with phenol chloroform isoamyl alcohol, precipitated with ethanol, dissolved in pure water, and the number of copies of Core-related rcDNA was calculated by real-time PCR.
  • Real-time PCR was carried out according to a conventional method using primers of SEQ ID NO: 1 (GGAGGGATACATAGAGGTTCCTTGA) and 2 (GTTGCCCGTTTGTCCTCTAATTC) and Cyber Green as a fluorescent reagent.
  • Example 2 Measurement of antiviral activity in vivo> (Method)
  • uPA / SCID mice mouse with severe immunodeficiency liver injury having both uPA transgenic and SCID traits
  • chimeric mice are used. Made. After infecting 15-week-old chimeric mice with 1.0 ⁇ 10 5 copies / animal or 1.0 ⁇ 10 6 copies / animal of HBV (genotype C), the following groups were administered to 22-week-old HBV-infected chimeric mice. did.
  • Treatment group B (3 drugs): PegIFN, lamivudine (Fujifilm Wako Pure Drug), and fluvastatin (Toronto Research Chemicals), PegIFN subcutaneously injected twice a week at 30 ⁇ g / kg, lamivudine 15
  • Body weight measurement and blood human albumin (h-Alb) concentration measurement The body weight of the animal was measured once a day. On the day of blood collection, the body weight of the animals was measured before the blood collection.
  • Serum ALT and AST activity were measured after the collected serum was diluted 3-fold with physiological saline. The measurement is performed by the POP / POD / leuco dye method (the diarylimidazole leuco dye is colored blue by hydrogen peroxide and peroxidase generated by pyruvate oxidase) using Drychem NX500sV (Fujifilm). Measured according to.
  • ISG15 level in liver The expression level of the ubiquitin-like molecule ISG15 (Interferon Stimulated Gene 15) was detected in the liver tissue 56 days after the start of drug administration.
  • Protein is prepared from 50 mg of liver tissue by a conventional method, 20 ng / ⁇ L of protein is decomposed by SDS-PEGE, and then Western blotting is performed by a conventional method using an anti-ISG15 antibody (# 2743, Cell Signaling Techonology). It was.
  • Figure 3 shows the measurement results of HBV DNA concentration in serum. * Indicates a significant difference (p ⁇ 0.05) from the untreated group C.
  • treatment group A 4 drugs of IFN + entecavir + fluvastatin + alicin
  • HBV-DNA was significantly higher than that of treatment group B (3 drugs of IFN + lamivudine + fluvastatin). The amount was reduced. This indicates that allicin can function as an antiviral agent.
  • Fig. 4A The results of body weight measurement are shown in Fig. 4A, and the results of blood human albumin concentration measurement are shown in Fig. 4B.
  • FIG. 4B * indicates a significant difference (p ⁇ 0.05) from untreated group C.
  • the results of serum ALT activity and serum AST activity measurement are shown in FIG.
  • the result of correcting the amount of HBV cccDNA in the liver 56 days after treatment with the blood human albumin measurement value is shown in Fig. 6A, and the HBV-DNA measurement value up to 56 days after treatment is corrected with the blood human albumin measurement value.
  • the results are shown in Fig. 6B.
  • ISG15 is a protein whose expression is induced by interferon. No induction of ISG15 expression was observed when entecavir (lanes 5 and 6) and fluvastatin (lanes 7 and 8) were administered alone, but when allicin (lanes 9 and 10) was administered as a single agent. , ISG15 expression was induced in the same manner as when peginterferon ⁇ (lanes 11 and 12) was administered as a single agent. These results suggest that allicin exerts antiviral effects by a mechanism similar to interferon. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

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Abstract

Dans un mode de réalisation, la présente invention aborde le problème de la production d'un agent antiviral contre le VHB et d'autres virus. Dans un mode de réalisation, la présente invention aborde le problème de la production d'un inhibiteur de formation d'ADNccc viral.　Dans un mode de réalisation, la solution selon la présente invention porte sur un agent antiviral ou un inhibiteur de formation d'ADNccc viral contenant un composé choisi dans le groupe constitué par l'allicine, l'alliine, le sulfure d'allyle, la S-allylcystéine et l'ajoène (et éventuellement un ou plusieurs autres agents antiviraux).
PCT/JP2020/021269 2019-05-29 2020-05-29 Agent antiviral ou inhibiteur de formation d'adnccc viral Ceased WO2020241814A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011526255A (ja) * 2008-07-03 2011-10-06 ヤオ，イン 天然アリシン（Ａｌｌｉｃｉｎ）錠剤及びその調製方法
JP2015531773A (ja) * 2012-08-28 2015-11-05 ヤンセン・サイエンシズ・アイルランド・ユーシー スルファモイル−アリールアミド及びｂ型肝炎の治療のための薬剤としてのその使用
WO2017155082A1 (fr) * 2016-03-11 2017-09-14 国立大学法人鹿児島大学 Agent permettant de lutter contre des virus liés à un hépatome
WO2018193902A1 (fr) * 2017-04-18 2018-10-25 公立大学法人名古屋市立大学 Procédé d'activité antivirale contre le virus de l'hépatite b au moyen de micro-arn

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011526255A (ja) * 2008-07-03 2011-10-06 ヤオ，イン 天然アリシン（Ａｌｌｉｃｉｎ）錠剤及びその調製方法
JP2015531773A (ja) * 2012-08-28 2015-11-05 ヤンセン・サイエンシズ・アイルランド・ユーシー スルファモイル−アリールアミド及びｂ型肝炎の治療のための薬剤としてのその使用
WO2017155082A1 (fr) * 2016-03-11 2017-09-14 国立大学法人鹿児島大学 Agent permettant de lutter contre des virus liés à un hépatome
WO2018193902A1 (fr) * 2017-04-18 2018-10-25 公立大学法人名古屋市立大学 Procédé d'activité antivirale contre le virus de l'hépatite b au moyen de micro-arn

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Title
N. BHAGYALAKSHMI, R. THIMMARAJU, L. VENKATACHALAM, K. N. CHIDAMBARA MURTHY, R. V. SREEDHAR: "Nutraceutical Applications of Garlic and the Intervention of Biotechnology", CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION, vol. 45, no. 7-8, 2005, pages 607 - 621, XP055769366 *
UTE MÜNCHBERG, AWAIS ANWAR, SUSANNE MECKLENBURG, CLAUS JACOB: "Polysulfides as biologically active ingredients of garlic", ORGANIC AND BIOMOLECULAR CHEMISTRY, vol. 5, no. 10, 2007, pages 1505 - 1518, XP055769360 *

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