Anti-hepatitis b virus agent and use thereof

Abstract

To provide a novel anti-hepatitis B virus agent and related technologies.SOLUTION: The anti-hepatitis B virus agent comprises, as an active ingredient, dimethyl fumarate or a salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anti-hepatitis B virus agent and use thereof. [Background technology]

[0002] Hepatitis B virus (HBV) is a virus known to infect many people worldwide, including Japan. Hepatitis B virus causes hepatitis B (e.g., acute hepatitis B and chronic hepatitis B), which may progress to liver cirrhosis or liver cancer in some cases. Therefore, there is a strong demand for the development of anti-hepatitis B virus agents.

[0003] BACKGROUND ART Interferon or nucleic acid analogue preparations have conventionally been used to treat hepatitis B virus infections such as hepatitis B (see, for example, Non-Patent Documents 1 and 2).

[0004] However, while interferon can reduce the amount of hepatitis B virus in the body, it frequently causes side effects in the body, while nucleoside analogues can reduce the amount of hepatitis B virus in the body when taken, but the amount of hepatitis B virus in the body increases when the drug is discontinued, so it must be taken for a long period of time. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] David Durantel et al., "New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus" J. Hepatol., 2016, 64, S117-S131 [Non-patent document 2] Grance LH Wong et al., "How to achieve functional cure of HBV: Stopping NUCs, adding interferon or new drug development?" J. Hepatol., 2022, 76(6), 1249-1262 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, there is room for improvement in the conventional techniques, and there is a need to develop new anti-hepatitis B virus agents.

[0007] An object of one aspect of the present invention is to provide a new anti-hepatitis B virus agent and related technology. [Means for solving the problem]

[0008] The present inventors have discovered a substance having an anti-hepatitis B virus effect, and have completed the present invention. That is, one aspect of the present invention is as follows.

[0009] [1] An anti-hepatitis B virus agent containing dimethyl fumarate or its salt as an active ingredient.

[0010] [2] Use of dimethyl fumarate or a salt thereof for the production of an anti-hepatitis B virus agent.

[0011] [3] A screening method for anti-hepatitis B virus agents, comprising: an administration step of administering a candidate substance for an anti-hepatitis B virus agent to a non-human organism or cells collected from a living organism; a measurement step of measuring the amount of dimethyl fumarate or a salt thereof in the non-human organism or cells collected from the living organism after the administration step; and a determination step of determining whether the candidate substance is an anti-hepatitis B virus agent by comparing the amount of dimethyl fumarate or a salt thereof measured in the measurement step with a control amount. [Effects of the Invention]

[0012] According to one aspect of the present invention, a new anti-hepatitis B virus agent and related technology can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows graphs and microscopic images showing the test results of toxicity evaluation of a compound that is an active ingredient of a drug in an example of the present invention. [Figure 2] 1 is a graph showing the effect of a compound, which is an active ingredient of a drug, on hepatitis B virus in an example of the present invention. [Figure 3] 1 is a graph showing changes in the expression levels of various genes caused by compounds that are active ingredients of drugs in an example of the present invention. [Figure 4] 1 is a graph showing the effect of FAH, an active ingredient of a drug, on hepatitis B virus in an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B."

[0015] 1. Anti-hepatitis B virus agent and method for producing the anti-hepatitis B virus agent An anti-hepatitis B virus agent according to one embodiment of the present invention contains, as an active ingredient, a compound represented by the following formula I or a salt thereof, a fumarylacetoacetate hydrolase (FAH) protein, or an expression vector for fumarylacetoacetate hydrolase:

[0016] [ka]

[0017] (In Formula I, R 1 and R 2 are independently OH, O - or a C1 to C6 alkoxy group).

[0018] As described above, an anti-hepatitis B virus agent according to one embodiment of the present invention may contain, as an active ingredient, a compound represented by formula I or a salt thereof.

[0019] More specifically, the C1-C6 alkoxy group may be a C1-C5 alkoxy group, a C1-C4 alkoxy group, a C1-C3 alkoxy group, a C1-C2 alkoxy group, or a methoxy group. The C1-C6 alkoxy group may be a linear alkoxy group, a branched alkoxy group, or a cyclic alkoxy group. In this specification, the term "CX alkoxy group" refers to an "alkoxy group having X carbon atoms."

[0020] The compound represented by the above formula I can more specifically be fumaric acid, monomethyl fumarate, or dimethyl fumarate.

[0021] The salt of the compound represented by Formula I above may be any pharmaceutically acceptable salt, and is not limited thereto. Examples of the salt include alkali metal salts (potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), ammonium salt, organic base salts (trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), organic acid salts (acetate salt, maleate salt, tartrate salt, methanesulfonate salt, benzenesulfonate salt, formate salt, toluenesulfonate salt, trifluoroacetate salt, etc.), inorganic acid salts (hydrochloride salt, hydrobromide salt, sulfate salt, phosphate salt, etc.), etc.

[0022] As described above, an anti-hepatitis B virus agent according to one embodiment of the present invention may contain, as an active ingredient, a fumarylacetoacetate hydrolase protein or an expression vector for fumarylacetoacetate hydrolase.

[0023] The expression vector is not limited in its specific structure as long as it can express fumarylacetoacetate hydrolase, and may be composed of DNA or RNA.

[0024] The above expression vector can be prepared by inserting any one of the following polynucleotides (1) to (6) into a known expression vector in a manner that allows expression: (1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; (2) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and encodes a polypeptide having anti-hepatitis B virus activity; (3) a polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 1 and encoding a polypeptide having anti-hepatitis B virus activity; (4) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; (5) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and having anti-hepatitis B virus activity; (6) A polynucleotide encoding a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 and having anti-hepatitis B virus activity.

[0025] The polynucleotide shown in SEQ ID NO: 1 encodes human fumarylacetoacetate hydrolase. The base sequence of the polynucleotide is shown below: SEQ ID NO:1:

[0026] The polypeptide shown in SEQ ID NO: 2 corresponds to human fumarylacetoacetate hydrolase. The amino acid sequence of the polypeptide is shown below: SEQ ID NO:2: MSFIPVAEDSDFPIHNLPYGVFSTRGDPRPRIGVAIGDQILDLSIIKHLFTGPVLSKHQDVFNQPTLNSFMGLGQAAWKEARVFLQNLLSVSQARLRDDTELRKCAFISQASATMHLPATIGDYTDFYSSRQHATNVGIMFRDKENALMPNWLHLPVGYHGRASSVVVSGTPIRMGQMKPDDSKPPVYGACKLLDMELEMAFFVGPGN RLGEPIPISKAHEHIFGMVLMNDWSARDIQKWEYVPLGPFLGKSFGTTVSPWVVPMDALMPFAVPNPKQDPRPLPYLCHDEPYTFDINLSVNLKGEGMSQAATICKSNFKYMYWTMLQQLTHHSVNGCNLRPGDLLASGTISGPEPENFGSMLELSWKGTKPIDLGNGQTRKFLLDGDEVIITGYCQGDGYRIGFGQCAGKVLPALLPS.

[0027] Whether or not a desired polypeptide has anti-hepatitis B virus activity can be determined, for example, by expressing the desired polypeptide in cells infected with hepatitis B virus or in a hepatitis B virus-infected model cell, and then comparing the amounts of hepatitis B virus before and after expression of the polypeptide (e.g., the amount of extracellular HBsAg, the amount of extracellular HBV DNA, the amount of intracellular HBV RNA, and / or the amount of intracellular HBV DNA). If the amount of hepatitis B virus after expression of the polypeptide is reduced compared to the amount of hepatitis B virus before expression of the polypeptide, the polypeptide can be determined to have anti-hepatitis B virus activity. The amount of hepatitis B virus can be measured according to the method described in the Examples below, etc.

[0028] With respect to (2) above, "stringent conditions" refers to conditions under which so-called specific hybrids are formed but nonspecific hybrids are not formed. Examples of stringent conditions include conditions under which highly homologous polynucleotides (e.g., polynucleotides having a homology of 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more) hybridize but polynucleotides having lower homology do not hybridize, or conditions under which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization: 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0029] With regard to (3) and (6) above, the sequence identity can be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0030] The determination of sequence identity between two sequences can, for example, be accomplished using a mathematical algorithm. Such mathematical algorithms include, but are not limited to, the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, with modifications as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0031] With regard to (5) above, "substitution, deletion, insertion, and / or addition of one or several amino acids" refers to the substitution, deletion, insertion, and / or addition of a number of amino acids that maintains the original function of the polypeptide (e.g., 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less). The "substitution, deletion, insertion, and / or addition of one or several amino acids" may include artificially induced mutations and naturally occurring mutations.

[0032] The amount of the active ingredient contained in the anti-hepatitis B virus agent according to one embodiment of the present invention is not limited, and may be, for example, 0.00001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.001% by mass to 100% by mass, 0.01% by mass to 100% by mass, 0.1% by mass to 100% by mass, 0.1% by mass to 95% by mass, 0.1% by mass to 90% by mass, 0.1% by mass to 80% by mass, 0.1% by mass to 70% by mass, 0.1% by mass to 60% by mass, 0.1% by mass to 50% by mass, 0.1% by mass to 40% by mass, 0.1% by mass to 30% by mass, 0.1% by mass to 20% by mass, or 0.1% by mass to 10% by mass, when the anti-hepatitis B virus agent is taken as 100% by mass.

[0033] The anti-hepatitis B virus agent according to one embodiment of the present invention may contain ingredients other than the above-mentioned active ingredients.

[0034] The ingredients other than the active ingredient are not particularly limited and may be, for example, a buffering agent, a pH adjusting agent, an isotonicity agent, a preservative, an antioxidant, a high molecular weight polymer, an excipient, a solvent, an antibacterial agent, or the like.

[0035] Examples of the buffering agent include phosphoric acid or phosphate salts, boric acid or borates, citric acid or citrate salts, acetic acid or acetate salts, carbonic acid or carbonate salts, tartaric acid or tartrate salts, ε-aminocaproic acid, and trometamol. Examples of the phosphate salts include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borates include borax, sodium borate, and potassium borate. Examples of the citrate salts include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate salts include sodium acetate and potassium acetate. Examples of the carbonate salts include sodium carbonate and sodium bicarbonate. Examples of the tartrate salts include sodium tartrate and potassium tartrate.

[0036] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0037] Examples of the isotonic agent include ionic isotonic agents (eg, sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and non-ionic isotonic agents (eg, glycerin, propylene glycol, sorbitol, mannitol).

[0038] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0039] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0040] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0041] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0042] Examples of the solvent include water, physiological saline, and alcohol.

[0043] Examples of the antibacterial agents include β-lactam, aminoglycoside, tetracycline, lincomycin, chloramphenicol, macrolide, ketolide, polypeptide, and glycopeptide antibiotics; and pyridonecarboxylic acid (quinolone), new quinolone, oxazolidinone, and sulfonamide synthetic antibacterial agents.

[0044] The amount of ingredients other than the active ingredient contained in the anti-hepatitis B virus agent according to one embodiment of the present invention is not limited, and can be, for example, 0% to 99.99999% by mass, 0% to 99.9999% by mass, 0% to 99.9999% by mass, 0% to 99.999% by mass, 0% to 99.999% by mass, 0% to 99.99% by mass, 0% to 99.99% by mass, 5% to 99.9% by mass, 10% to 99.9% by mass, 20% to 99.9% by mass, 30% to 99.9% by mass, 40% to 99.9% by mass, 50% to 99.9% by mass, 60% to 99.9% by mass, 70% to 99.9% by mass, 80% to 99.9% by mass, or 90% to 99.9% by mass, when the anti-hepatitis B virus agent is taken as 100% by mass.

[0045] The dosage form of the anti-hepatitis B virus agent according to one embodiment of the present invention is not limited, and may be, for example, an oral agent (e.g., powder, granules, pills, tablets, capsules, oral liquid), an external agent (e.g., external liquid, semi-solid, solid), or an injection.

[0046] The administration interval of the anti-hepatitis B virus agent according to one embodiment of the present invention is not limited, and may be, for example, once every hour, once every 6 hours, once every 12 hours, once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, once every 2 weeks, once every 3 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months.

[0047] The subjects to which the anti-hepatitis B virus agent according to one embodiment of the present invention is administered are not limited, and examples thereof include humans and non-human organisms (e.g., livestock, pets, and laboratory animals). Examples of non-human organisms include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0048] The anti-hepatitis B virus agent according to one embodiment of the present invention can also be used as a reagent for various in vitro or in vivo experiments. In this case, examples of subjects to which the anti-hepatitis B virus agent according to one embodiment of the present invention is administered include cultured tissues, cultured cells, and experimental systems that reproduce the life cycle of the virus (e.g., infection, replication, and proliferation).

[0049] The anti-hepatitis B virus agent according to one embodiment of the present invention has an anti-hepatitis B virus effect, and therefore can also be used as a therapeutic or preventive agent for hepatitis B virus infection.

[0050] As used herein, the term "therapeutic agent" refers to a drug that provides a therapeutic effect. The therapeutic effect includes, but is not limited to, the following effects (1) to (3): (1) The effect of reducing the severity of one or more symptoms of the disease compared to when the therapeutic agent is not administered; (2) The effect of preventing an increase in the severity of one or more symptoms of the disease compared to when the therapeutic agent is not administered; (3) The effect of reducing the rate of increase in the severity of one or more symptoms associated with the disease compared to when the therapeutic agent is not administered.

[0051] As used herein, the term "prophylactic agent" refers to a drug that provides a preventive effect. The preventive effect refers to, but is not limited to, the effects (4) to (6) exemplified below: (4) The effect of preventing the onset of one or more symptoms of the disease or reducing the risk of onset compared to when the prophylactic agent is not administered; (5) The effect of preventing the recurrence of one or more symptoms of the disease or reducing the risk of recurrence compared to when the prophylactic agent is not administered; (6) The effect of preventing the onset of one or more symptoms of a disease or reducing the risk of the onset of the symptoms compared to when the prophylactic agent is not administered.

[0052] Specific examples of the hepatitis B virus infection include, but are not limited to, hepatitis B (acute hepatitis B, chronic hepatitis B), liver cirrhosis, and liver cancer.

[0053] One embodiment of the present invention relates to the use of a compound represented by the following formula I or a salt thereof, a fumarylacetoacetate hydrolase protein (FAH) protein, or an expression vector for fumarylacetoacetate hydrolase (FAH) for the manufacture of an anti-hepatitis B virus agent:

[0054] [ka]

[0055] (In Formula I, R 1 and R 2 are independently OH, O - or a C1 to C6 alkoxy group).

[0056] The anti-hepatitis B virus agent, the compound represented by formula I (for example, fumaric acid, monomethyl fumarate, or dimethyl fumarate) or a salt thereof, and the expression vector for the fumarylacetoacetate hydrolase have already been described in detail, and therefore further description thereof will be omitted here. Note that the method for producing the anti-hepatitis B virus agent is not limited and may be any known formulation method.

[0057] 2. Screening method for anti-hepatitis B virus agents A screening method for an anti-hepatitis B virus agent according to one embodiment of the present invention is a screening method for an anti-hepatitis B virus agent, comprising: an administration step of administering a candidate substance for an anti-hepatitis B virus agent to a non-human organism or cells collected from a living organism; a measurement step of measuring the amount of (i) the compound represented by formula I or a salt thereof, or (ii) the expression level of fumarylacetoacetate hydrolase (FAH) in the non-human organism or cells collected from the living organism after the administration step; and a determination step of determining whether the candidate substance is an anti-hepatitis B virus agent by comparing the amount of (i) the compound represented by formula I or a salt thereof, or (ii) the expression level of fumarylacetoacetate hydrolase (FAH) measured in the measurement step with a control amount:

[0058] [ka]

[0059] (In Formula I, R 1 and R 2 are independently OH, O - or a C1 to C6 alkoxy group).

[0060] The compound represented by the above formula I and its salt, and the above fumarylacetoacetate hydrolase are substances having an anti-hepatitis B virus effect. Candidate substances that can increase the amounts of these substances in vivo can function as anti-hepatitis B virus agents through the anti-hepatitis B virus effect of these substances.

[0061] Each step will be explained below, but explanations of the anti-hepatitis B virus agent, the compound represented by formula I above (for example, fumaric acid, monomethyl fumarate, dimethyl fumarate) or a salt thereof, and the expression vector for the fumarylacetoacetate hydrolase, as well as the matters already explained in [1. Anti-hepatitis B virus agent and method for producing anti-hepatitis B virus agent] above, will basically be omitted below.

[0062] 2-1. Administration Step The administration step is a step of administering a candidate substance for an anti-hepatitis B virus agent to a non-human organism or to cells collected from a living organism.

[0063] Examples of the non-human organisms include livestock, pets, and laboratory animals, and more specifically, monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0064] The cells collected from the living body may be, for example, cells collected from a human or non-human organism (e.g., hepatocytes). The cells collected from the living body may be, for example, tissue collected from a human or non-human organism (e.g., liver). The cells collected from the living body may be a cell line (e.g., HepG2 cells) established from cells collected from a human or non-human organism.

[0065] Examples of the candidate substance include inorganic compounds, organic compounds, animal extracts, plant extracts, microbial extracts, and culture supernatants of various cells. The candidate substance may be used as is or dissolved in a solvent. Examples of the solvent include physiological saline, phosphate-buffered saline, and water.

[0066] Methods for administering a candidate substance for an anti-hepatitis B virus agent to a non-human organism or cells collected from a living organism include, but are not limited to, (i) a method in which the candidate substance is administered orally or by injection to a non-human organism, and (ii) a method in which the candidate substance is administered to a medium in which cells or tissue collected from a living organism are cultured.

[0067] [2-2. Measurement process] The measuring step is a step of measuring (i) the amount of the compound represented by formula I or a salt thereof, or (ii) the expression level of fumarylacetoacetate hydrolase in the non-human organism or cells collected from the organism after the administration step.

[0068] The method for measuring the amount of the compound represented by formula I or a salt thereof is not limited, and known methods can be used. For example, the amount of the compound represented by formula I or a salt thereof can be measured according to HPLC (high performance liquid chromatography) or MS (mass spectrometry). Alternatively, the amount of the compound represented by formula I or a salt thereof can be measured using a commercially available measurement kit.

[0069] The method for measuring the expression level of the fumarylacetoacetate hydrolase is not limited, and known methods can be used. For example, the expression level of the fumarylacetoacetate hydrolase can be measured by RT-PCR (reverse transcription-polymerase chain reaction), Northern blotting, or Western blotting. The primers, probes, antibodies, and the like used in these methods may (i) be commercially available, or (ii) may be designed according to known methods based on the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. That is, the expression level of the fumarylacetoacetate hydrolase may be measured based on the amount of fumarylacetoacetate hydrolase protein or the amount of fumarylacetoacetate hydrolase mRNA.

[0070] [2-3. Judgment process] The determination step is a step of determining whether the candidate substance is an anti-hepatitis B virus agent by comparing the amount of (i) the compound represented by formula I or a salt thereof, or (ii) the expression level of fumarylacetoacetate hydrolase (FAH) measured in the measurement step with a control amount.

[0071] As the control amount, for example, two types of control amounts can be selectively used: (i) the control amount α with respect to the amount of the compound represented by formula I or a salt thereof, or (ii) the control amount β with respect to the expression level of fumarylacetoacetate hydrolase (FAH).

[0072] The control amount is not limited, and examples thereof include (i) the amount of the compound represented by formula I or a salt thereof, or (ii) the expression level of fumarylacetoacetate hydrolase (FAH) in the non-human organism before the administration step or in cells collected from a living body.

[0073] Specifically, in the non-human organism before the administration step or in cells collected from a living body, let the amount of the compound represented by formula I or a salt thereof be A, and (ii) let the expression level of fumarylacetoacetate hydrolase be A'. On the other hand, in the non-human organism after the administration step or in cells collected from a living body, let the amount of the compound represented by formula I or a salt thereof be B, and (ii) let the expression level of fumarylacetoacetate hydrolase be B'. At this time, if "A < B" or "A' < B'", it can be determined that the candidate substance can be used as an active ingredient of an anti-hepatitis B virus agent.

[0074] [3. Others] [1] A method for treating hepatitis B virus, comprising a step of administering to a subject (for example, a human or a non-human organism) an anti-hepatitis B virus agent containing, as an active ingredient, a compound represented by the following formula I or a salt thereof, a fumarylacetoacetate hydrolase protein, or an expression vector of fumarylacetoacetate hydrolase:

[0075] [Chemical formula]

[0076] (In formula I, R 1 and R 2 are independently OH, O -or a C1 to C6 alkoxy group).

[0077] <2> A method for treating or preventing hepatitis B virus infection (e.g., hepatitis B (acute hepatitis B, chronic hepatitis B), liver cirrhosis, or liver cancer), comprising the step of administering to a subject (e.g., a human or non-human organism) an anti-hepatitis B virus agent containing, as an active ingredient, a compound represented by the following formula I or a salt thereof, a fumarylacetoacetate hydrolase protein, or an expression vector for fumarylacetoacetate hydrolase:

[0078] [ka]

[0079] (In Formula I, R 1 and R 2 are independently OH, O - or a C1 to C6 alkoxy group).

[0080] This invention may also contribute to achieving Goal 3 of the United Nations' Sustainable Development Goals (SDGs), including "Ensure good health and promote well-being for all." [Example]

[0081] <1. Preparation of Hepatitis B Virus Infected Model Cells> A 1.3-mer hepatitis B virus (HBV) genome (genotype C2, both basal core promoter (BCP) A1762T / G1764A mutation and precore G1896A mutation, accession number AB819615) was inserted into a plasmid carrying the neomycin resistance gene (pBluescript II SK: Agilent Technologies, Santa Clara, USA).

[0082] Using commercially available reagents, the above plasmid was introduced into HepG2 cells (American Type Culture Collection, Manassas, VA, USA), a cell line derived from human hepatoma, to generate HepG2 cells that release hepatitis B virus into the culture medium. Furthermore, the HepG2 cells were cultured in a neomycin-containing medium and selected to obtain the HepG2.D11 clone, which stably releases hepatitis B virus into the culture medium.

[0083] <2. Compound Tests> <2-1. Toxicity evaluation of compounds> <Test 1> HepG2.D11 clones were seeded onto culture plates and cultured for 1 day, after which fumaric acid (FA), monomethyl fumarate (MMF), or dimethyl fumarate (DMF) was added to the medium in which the HepG2.D11 clones were cultured to a final concentration of 0 μM, 3 μM, 10 μM, 30 μM, or 50 μM, and the clones were cultured for an additional 3 days.

[0084] After 3 days of culture, cell viability was measured using a modified 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with Cell Counting kit-8 (Dojindo, Kumamoto, Japan).

[0085] The results of the MTT assay are shown in 101 of Figure 1. As is clear from 101 of Figure 1, there was no difference in the viability of cells treated with the compounds used in the present invention (see the test results for FA, MMF, and DMF at 3 to 50 μM) and the viability of cells not treated with the compounds used in the present invention (see the test results for FA, MMF, and DMF at 0 μM). This indicates that the compounds used in the present invention have low toxicity to cells.

[0086] <Test 2> HepG2.D11 clones were seeded onto culture plates and cultured for 1 day. After this 1-day culture, fumaric acid (FA), monomethyl fumarate (MMF), dimethyl fumarate (DMA), or control dimethyl sulfoxide (DMSO) was added to the culture medium to a final concentration of 30 μM, and the cells were cultured for an additional 3 days. After this 3-day culture, the cell morphology was observed under a microscope.

[0087] The results of microscopic observation are shown in 102 of Figure 1. As is clear from 102 of Figure 1, there was no difference in the morphology of cells treated with the compounds used in the present invention (see the test results for FA, MMF, and DMF) and cells not treated with the compounds used in the present invention (see the test results for DMSO). This indicates that the compounds used in the present invention have low toxicity to cells.

[0088] <2-2. Effect of compounds on hepatitis B virus> HepG2.D11 clones were seeded onto culture plates and cultured for 1 day, after which fumaric acid (FA), monomethyl fumarate (MMF), dimethyl fumarate (DMA), or dimethyl sulfoxide (DMSO) (control) was added to the culture medium to a final concentration of 30 μM, and the clones were cultured for an additional 3 days.

[0089] After the 3-day culture, the medium and cells were collected, and the amount of extracellular hepatitis B virus was measured using the collected medium, and the amount of intracellular hepatitis B virus was measured using the collected cells.

[0090] Specifically, the amount of hepatitis B virus present outside the cells was measured based on the amount of HBsAg (Hepatitis B surface antigen) present outside the cells and the amount of hepatitis B virus genomic DNA (HBV DNA) present outside the cells.

[0091] The amount of extracellular HBsAg was measured using the Lumipulse HBsAg-HQ immunoassay (Fujirebio Inc., Tokyo, Japan) according to the protocol attached to the kit.

[0092] The amount of extracellular HBV DNA was measured using a known method. First, DNA was purified from the collected culture medium using a Smithest EX-R&D (MBL) kit. To measure the amount of HBV DNA, two primer pairs and a probe corresponding to hepatitis B virus genomic DNA were used (see Table 1 below). These primer pairs and probes were purchased from Takara Bio and Applied Biosystems. Nested PCR was performed on the DNA purified from the culture medium. Furthermore, the amount of HBV DNA was measured by real-time PCR using the StepOne Plus system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Applied Biosystems) and transferrin receptor (TFRC; Sigma-Aldrich) were used as endogenous controls (see Table 2 below).

[0093] Specifically, the amount of hepatitis B virus present in the cells was measured based on the amount of hepatitis B virus genomic DNA (HBV DNA) present in the cells and the amount of hepatitis B virus RNA (HBV RNA) present in the cells.

[0094] The amount of HBV RNA present in the cells was measured using a known method. First, total RNA was extracted from the harvested cells using TRIzol reagent (Thermo Fisher Scientific). Next, cDNA was synthesized using the total RNA and a High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). The amount of HBV RNA was then measured by real-time PCR using the StepOne Plus system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Applied Biosystems) and transferrin receptor (TFRC; Sigma-Aldrich) were used as endogenous controls (see Table 2 below).

[0095] The amount of HBV DNA present in the cells was measured using a known method. First, DNA was purified from the recovered cells using a Smithest EX-R&D (MBL) kit. To measure the amount of HBV DNA, two primer pairs and a probe corresponding to the genomic DNA of hepatitis B virus were used (see Table 1 below). These primer pairs and probes were purchased from Takara Bio and Applied Biosystems. Nested PCR was performed on DNA purified from the culture medium. Furthermore, the amount of HBV DNA was measured by real-time PCR using the StepOne Plus system (Applied Biosystems). In this measurement, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Applied Biosystems) and transferrin receptor (TFRC; Sigma-Aldrich) were used as endogenous controls (see Table 2 below).

[0096] [Table 1]

[0097] [Table 2]

[0098] The test results are shown in 201 and 202 of Figure 2. 201 of Figure 2 shows the measurement results of the amount of HBsAg present outside the cells and the amount of HBV DNA present outside the cells. Meanwhile, 202 of Figure 2 shows the measurement results of the amount of HBV RNA present inside the cells and the amount of HBV RNA present inside the cells. As is clear from 201 and 202 of Figure 2, the amount of hepatitis B virus present inside and outside the cells treated with the compounds used in the present invention (see the test results for FA, MMF, and DMF) was found to be reduced compared to cells not treated with the compounds used in the present invention (see the test results for DMSO). This indicates that the compounds used in the present invention have an anti-hepatitis B virus effect.

[0099] <2-3. Search for genes whose expression levels change depending on compounds> HepG2.D11 clones were seeded onto culture plates and cultured for 1 day, after which fumaric acid (FA), monomethyl fumarate (MMF), dimethyl fumarate (DMA), or dimethyl sulfoxide (DMSO) (control) was added to the culture medium to a final concentration of 30 μM, and the clones were cultured for an additional 3 days.

[0100] After 3 days of culture, the cells were harvested and the expression levels of specific genes (NRF2, p62 / SQSTM1, ATG5, ATG7, APOBEC3A, APOBEC3B, APOBEC3G, IRF3, IRF7, IRF9, STAT1, STAT2, HMOX-1, etc.) were measured.

[0101] Specifically, gene expression was analyzed using a custom TaqMan Array plate and a TaqMan® Array 96-well plate, fast (Thermo Fisher Scientific). The target genes were determined using a StepOne Plus real-time PCR system (Applied Biosystems). The primers used for gene expression analysis are listed in Table 3 below. The expression levels of the target genes were analyzed using the ΔΔCt comparative threshold method. The GAPDH gene was used as an endogenous control.

[0102] [Table 3]

[0103] The test results are shown in Figure 3. As shown in Figure 3, the compounds used in the present invention were found to alter the expression levels of specific genes (such as NRF2, p62 / SQSTM1, ATG5, ATG7, APOBEC3A, APOBEC3B, APOBEC3G, IRF3, IRF7, IRF9, STAT1, STAT2, and HMOX-1). These genes included genes classified as fumaric acid-related genes, autophagy-related genes, or anti-HBV genes.

[0104] <3. Genetic testing> <3-1. Construction of expression vector> The cytomegalovirus vector used to express fumarylacetoacetate hydrolase (FAH) (pRP[Exp]-CMV>hFAH(NM_001374380.1)) was constructed using VectorBuilder (for details, see VectorBuilder ID VB230117-1091yve on the VectorBuilder website).

[0105] The nucleotide sequence of the FAH cDNA inserted into the above cytomegalovirus vector was the nucleotide sequence of SEQ ID NO:1, and the amino acid sequence of the polypeptide encoded by the cDNA was the amino acid sequence of SEQ ID NO:2.

[0106] <3-2. Forced expression of FAH and its effect on hepatitis B virus> HepG2.D11 clones were seeded onto culture plates and cultured for 1 day. After this 1-day culture, the cytomegalovirus vector prepared in <3-1> above was introduced into the HepG2.D11 clones using Lipofectamine® LTX Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the HepG2.D11 clones were cultured for another 1 day.

[0107] After culturing for one day, the medium was replaced with fresh medium, and the HepG2.D11 clones were cultured for an additional two days. After the two-day culture, the HepG2.D11 clones and the medium were collected and used in the following tests.

[0108] First, the expression of FAH in the recovered HepG2.D11 clones was confirmed by qRT-pCR (mRNA expression) and Western blotting (protein expression). The specific procedures for qRT-pCR and Western blotting were performed according to known methods.

[0109] The test results are shown in 401 and 402 in Figure 4. 401 in Figure 4 shows the test results for qRT-pCR, and 402 in Figure 4 shows the test results for Western blot. As is clear from 401 and 402 in Figure 4, it was confirmed that FAH could be forcibly expressed in the HepG2.D11 clone using the cytomegalovirus vector prepared in <3-1> above.

[0110] Next, using the recovered HepG2.D11 clones and culture medium, the amount of extracellular HBsAg, the amount of extracellular HBV DNA, the amount of intracellular HBV RNA, and the amount of intracellular HBV DNA were measured according to the method described above in <2-2>.

[0111] The test results are shown in 403 of Figure 4. As is clear from 403 of Figure 4, the amount of hepatitis B virus present extracellularly and intracellularly in cells expressing FAH (see the test results for the FAH vector) was found to be reduced compared to cells not expressing FAH (see the test results for the empty vector). This indicates that FAH has an anti-hepatitis B virus effect.

[0112] Statistical analyses in Figures 1 to 4 were performed using GraphPad Prism software (version 10; GraphPad Software Inc., San Diego, CA, USA). Statistical significance was determined using Mann-Whitney U tests. p values < 0.05 were considered statistically significant. [Industrial Applicability]

[0113] The present invention can be used for treating or preventing infection with hepatitis B virus. The present invention can be used for treating and preventing agents for hepatitis B virus infection, and for the development of such agents.

Claims

1. An anti-hepatitis B virus agent containing dimethyl fumarate or a salt thereof as an active ingredient.

2. Use of dimethyl fumarate or a salt thereof for the manufacture of an anti-hepatitis B virus agent.

3. an administration step of administering a candidate substance for an anti-hepatitis B virus agent to a non-human organism or cells collected from a living organism; a measuring step of measuring the amount of dimethyl fumarate or a salt thereof in the non-human organism or cells collected from the organism after the administering step; a determination step of determining whether the candidate substance is an anti-hepatitis B virus agent by comparing the amount of dimethyl fumarate or its salt measured in the measurement step with a control amount.

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