JP7845648B2 - Inhibitor of HBV-derived cccDNA, method of inhibition, medical composition for HBV infection, and method for treating HBV infection - Google Patents

Description

This invention relates to an inhibitor of HBV-derived cccDNA, a method for inhibition, a medical composition for HBV infection, and a method for treating HBV infection.

Hepatitis B virus (HBV) infection is known to cause acute or chronic hepatitis, and if left untreated for a long period, it can lead to cirrhosis and hepatocellular carcinoma.

HBV normally exists in the form of mature DNA (relaxed circular DNA: rcDNA). When HBV infects a cell and enters the nucleus from the cytoplasm, enzymes in the host cell convert it into a replication intermediate, covalently closed circular DNA (cccDNA). This is then transcribed into RNA, which is released from the nucleus into the cytoplasm. The virus's reverse transcriptase then transcribes this RNA back into rcDNA, and the mature HBV is released extracellularly.

Current treatments for HBV infection generally involve inhibitors of reverse transcriptase, thereby inhibiting the re-transcription of RNA to DNA (transcription to rcDNA) and suppressing the release of replicated HBV outside the cell. However, these treatments cannot remove cccDNA present in the nucleus. Therefore, if administration is discontinued, transcription from cccDNA to RNA occurs in the nucleus, and subsequently, reverse transcription from RNA to rcDNA resumes in the cytoplasm. For this reason, there is a need for the development of treatments that act upstream of the reverse transcriptase process, specifically, treatments that reduce nuclear cccDNA by inhibiting cccDNA synthesis.

Therefore, the present invention aims to provide a novel drug that suppresses cccDNA present in the nucleus.

To achieve the above objective, the HBV-derived cccDNA inhibitor of the present invention is characterized by comprising a ferritin light chain inhibitor that suppresses the ferritin light chain.

The pharmaceutical composition for HBV infection of the present invention is characterized by containing the HBV-derived cccDNA inhibitor of the present invention.

The present invention provides a method for suppressing HBV-derived cccDNA, characterized by including a suppression step that suppresses the ferritin light chain.

The present invention relates to a method for treating HBV infection, characterized by including an inhibitory step that suppresses ferritin light chains.

As a result of diligent research, the inventors have discovered that the amount of cccDNA produced from HBV infecting cells can be reduced by inhibiting ferritin light chains. According to the present invention, since the reduction of cccDNA, a replication intermediate upstream of transcription to rcDNA by reverse transcriptase inhibitors as described above, viral replication can be suppressed more effectively. Therefore, the present invention is extremely useful, for example, as a new treatment method for HBV infection.

Figure 1 is a graph showing the suppression of cccDNA by hFTL-specific siRNA in the cell line in Example 1. Figure 2 is a graph showing the suppression of hFTL expression by various siRNAs in the cell line in Example 2. Figure 3 is a graph showing the reduction in HBV cccDNA and rcDNA in cultured cells when hFTL siRNA and entecavir are used in Example 3. Figure 4 shows the results of Example 4, demonstrating the suppression of cccDNA in cultured cells by hFTL siRNA, hFTH siRNA, and hTFRC siRNA.

Unless otherwise specified, terms used herein may be used in the sense commonly used in the art.

The HBVcccDNA inhibitor of the present invention is, for example, an expression inhibitor that suppresses the expression of the ferritin light chain or a function inhibitor that suppresses the function of the ferritin light chain.

The HBVcccDNA inhibitor of the present invention is, for example, at least one selected from the group consisting of a substance that suppresses transcription from the gene encoding the ferritin light chain, a substance that degrades the transcribed transcript, and a substance that suppresses the translation of the protein from the transcript.

The HBVcccDNA inhibitor of the present invention is, for example, one nucleic acid substance selected from the group consisting of miRNA, siRNA, antisense, and ribozyme.

The HBVcccDNA inhibitor of the present invention is, for example, an expression vector that expresses the nucleic acid substance, where the expression inhibitor is the expression inhibitor.

The HBVcccDNA inhibitor of the present invention, for example, is a substance that inhibits or neutralizes the activity of the ferritin light chain.

The HBVcccDNA inhibitor of the present invention, for example, has an active neutralized product that is an antibody or antigen-binding fragment against the ferritin light chain.

The HBVcccDNA inhibitor of the present invention is, for example, an expression vector in which the functional inhibitor expresses a substance that neutralizes the activity of the ferritin light chain.

The present invention's method for suppressing HBVcccDNA involves, for example, suppressing ferritin light chain expression in the aforementioned suppression step.

The present invention provides a method for treating HBV infection, for example, by suppressing the expression of ferritin light chains in the suppression step.

In this invention, "treatment" has a broad meaning and includes, for example, treatment in a narrow sense such as suppression of progression, improvement (alleviation), and complete cure, as well as prevention such as prevention of infection and prevention of recurrence. In this invention, for example, one of these purposes may be used, or two or more may be used.

In this invention, HBV infection refers to a disease caused by HBV infection, such as hepatitis B (including acute or chronic hepatitis), cirrhosis, and liver cancer.

(1) Inhibitor of HBV-derived cccDNA The inhibitor of HBV-derived cccDNA of the present invention is characterized by containing a ferritin light chain inhibitor that inhibits ferritin light chains. In the present invention, HBV-derived cccDNA will also be referred to as "HBV cccDNA" or "cccDNA", ferritin light chain will also be referred to as "FTL", ferritin light chain inhibitor will be referred to as "FTL inhibitor", and the inhibitor of HBV-derived cccDNA will also be referred to as "HBV cccDNA inhibitor" or "cccDNA inhibitor".

When HBV infects hepatocytes, it follows the following replication process: First, (1) HBV binds to receptors on the surface of hepatocytes and enters the cell. (2) In the cytoplasm, the membrane structure on the surface of HBV breaks down, and the mature viral genome (rcDNA) migrates into the nucleus. (3) In the nucleus, the rcDNA is converted to cccDNA by host cell enzymes, and four types of RNA are transcribed. (4) Of the transcribed RNAs, Pregenetic RNA is released outside the nucleus and reverse-transcribed into rcDNA (mature form) by viral reverse transcriptase in the cytoplasm. (5) Finally, in the cytoplasm, the rcDNA is covered with a capsid and released outside the cell. This invention is an inhibitor that suppresses cccDNA in step (3) above.

In this invention, the suppression of HBV-derived cccDNA refers, for example, to the suppression of cccDNA production. This production suppression may involve, for example, the suppression of cccDNA synthesis itself, or the degradation of synthesized cccDNA; preferably, the suppression of cccDNA synthesis itself. This production suppression means, for example, a relative decrease in the amount of cccDNA in cells compared to the case without the addition of the FTL inhibitor.

The HBV cccDNA inhibitor of the present invention is characterized by containing the FLT inhibitor, and other components and conditions are not particularly limited. Furthermore, the FTL inhibitor of the present invention can be described by reference to the method for inhibiting HBV-derived cccDNA of the present invention, as described later.

In the present invention, the FTL inhibitor may be, for example, an expression inhibitor that suppresses FTL expression, or a function inhibitor that suppresses FTL function. The HBVcccDNA inhibitor of the present invention may, for example, contain only the expression inhibitor, only the function inhibitor, or both as the FTL inhibitor.

The type of FTL inhibitor is not particularly limited and includes, for example, low-molecular-weight compounds such as nucleic acid substances, proteins such as antibodies, and peptides such as antigen-binding fragments.

The aforementioned expression repressor may, for example, repress either the transcriptional or translational process in the expression of FTL from a gene encoding FTL (hereinafter also referred to as the FTL gene), and is not particularly limited. Examples of transcriptional repression include inhibition of transcription from DNA to mRNA precursor, inhibition of RNA processing that forms mature mRNA from mRNA precursor, and degradation of mRNA precursor or mature mRNA. Examples of translational repression include inhibition of translation from mature mRNA and inhibition of modification of translation products.

The expression inhibitor is, for example, a nucleic acid substance (hereinafter also referred to as a nucleic acid-type inhibitor), and may be in a form that inhibits expression directly (first form), or in a precursor form (second form) that inhibits expression under in vivo or in vitro conditions.

Examples of the first form of expression repressor include antigens, antisense (antisense oligonucleotides), RNA interference (RNAi) substances, and ribozymes. Examples of RNAi substances include siRNA, miRNA, and artificial mimic miRNA. Antigenes inhibit mRNA transcription, antisense and miRNA inhibit translation from mRNA, and siRNA and ribozymes degrade mRNA. An example of the artificial mimic miRNA is the structure described in WO2015/099122. These expression repressors may target either the entire or partial region of the FTL gene. Specifically, antisense and miRNA can be designed to bind to the 3'UTR region of mRNA transcribed from the FTL gene, and siRNA and ribozymes can be designed to bind to a specific region of mRNA transcribed from the FTL gene in a completely complementary manner.

The gene and protein sequences of human FTLs are registered in the database (NCBI), for example, under accession numbers NM_000146.4 and NM_000137.2 (Sequence IDs 1 and 2).

The first embodiment of the expression repressor preferably has, for example, an antisense sequence (also called a guide sequence) that is complementary to the target sequence of the FTL gene. The target sequence of the FTL gene is, for example, a series of 19 to 31 or 19 to 25 sequences within the region enclosed by the rectangle in Table 1, i.e., within the region of positions 533 to 563 (ftl_#1), 599 to 629 (ftl_#2), or 822 to 852 (ftl_#3) of Sequence ID No. 1, as shown below, with the underlined part being an example of a 19-base target sequence.
ftl_#1
ttggat cttcatgccctgggttctg cccgca (Sequence ID 3)
cttcatgccctgggttctg (Sequence ID 4)
ftl_#2
ttccta gatgaggaagtgaagctta tcaaga (Sequence ID 5)
gatgaggaagtgaagctta (SEQ ID NO: 6)
ftl_#3
aactat cctaacaagccttggacca aatgga (Sequence ID 7)
cctaacaagccttggacca (SEQ ID NO: 8)

When the expression repressant is siRNA, it is preferable that it is composed of a double helix consisting of an antisense strand and a sense strand, and that the antisense strand (also called the guide strand) has the antisense sequence. It is preferable that both the antisense strand and the sense strand have an overhang of several bases (for example, 1 to 3 bases) added to their 3' ends.

Table 1 shows specific examples of siRNAs for the aforementioned FTL gene. These siRNAs were designed for the 19-base target sequence mentioned above. In the sequences in Table 2, lowercase letters indicate overhangs. Note that the present invention is not limited to these examples.

The first form of gene expression inhibitor can be obtained, for example, by a screening method utilizing an FTL gene expression system, or it can be designed from the sequence of the FTL gene.

The first form of the expression inhibitor may be single-stranded or double-stranded, for example. The constituent units of the expression inhibitor are not particularly limited and may include, for example, a deoxyribonucleotide skeleton or a ribonucleotide skeleton containing a sugar, a base such as purine or pyrimidine, and a phosphate group. Other examples include non-nucleotide skeletons containing bases such as pyrrolidine or piperidine. These skeletons may be modified or unmodified. Furthermore, the constituent units may be, for example, natural or artificial non-natural. The expression inhibitor may be formed from the same constituent unit, or from two or more different constituent units.

The second form of the expression inhibitor is, as described above, the precursor, and a specific example is a precursor that expresses the first form of the expression inhibitor. By administering the precursor to a target, the first form of the expression inhibitor can be expressed and made functional, for example, in vivo or in vitro.

The precursor may, for example, include a form comprising the first form of expression inhibitor and a linker. A specific example is a form in which both strands of siRNA are linked by the linker. With such a precursor, for example, under in vivo or in vitro conditions, the precursor is cleaved, removing the linker and generating (expressing) double-stranded siRNA. A specific example of the precursor is, for example, shRNA that generates siRNA upon cleavage.

Furthermore, the precursor may be, for example, an expression vector into which the coding sequence of the first form of the expression repressor has been inserted. The expression vector allows for the expression of the first form of the expression repressor, for example, in vivo or in vitro. The expression vector may also contain, for example, the coding sequence of the aforementioned shRNA or other precursor. The type of expression vector is not particularly limited; examples include plasmid vectors and viral vectors, and examples of viral vectors include adenovirus vectors and Sendai virus vectors.

Examples of the function-inhibiting substances include activity inhibitors that inhibit the activity of the FTL, and activity-neutralizing substances that neutralize the activity of the FTL. The activity inhibitors are not particularly limited and include low-molecular-weight compounds, etc.

The aforementioned active neutralizing substance may be, for example, an antibody or antigen-binding fragment (antigen-binding peptide) against the FTL (Functional Threshold Light). The aforementioned antibody-type suppressor is also called a neutralizing antibody or neutralizing antigen-binding fragment because, for example, it can suppress the function of the FTL by binding to it. The aforementioned antibody-type suppressor can also be obtained, for example, by a screening method as described later.

The antibody may be, for example, a monoclonal antibody or a polyclonal antibody, and its isotype is not particularly limited; examples include IgG, IgM, IgA, etc. When administered to humans, the antibody is preferably, for example, a fully human antibody, a humanized antibody, or a chimeric antibody.

The antigen-binding fragment can, for example, recognize and bind to the target site of the FTL, and may be a fragment possessing the complementarity-determining region (CDR) of the antibody. Specific examples of the antigen-binding fragment include fragments such as Fab, Fab', and F(ab').

The functional inhibitor may be, for example, a first form that inhibits the function of the FTL as is, or a second form of precursor that inhibits the function of the FTL under in vivo or in vitro conditions. The first form of the functional inhibitor is, for example, an antibody-type inhibitor as described above. The second form of precursor may be, for example, an expression vector into which a coding sequence of a protein or peptide that inhibits the function of the FTL is inserted. The type of expression vector is not particularly limited, and examples include plasmid vectors, viral vectors, etc., as described above.

Furthermore, the function-inhibiting substance may be, for example, a substance that inhibits the state in which the FTL is active and has not lost its function, but is capable of functioning. Specifically, it may be a substance that reduces other substances necessary for the function of the FTL, or alters those other substances. The reduction of these other substances may, for example, be through the inhibition of their formation or through their decomposition.

The cccDNA inhibitor of the present invention may, for example, contain only the FTL inhibitor as an active ingredient, or it may contain the FTL inhibitor and other agents against HBV (HBV agents). Examples of HBV agents include replication inhibitors of viruses such as HBV. Examples of replication inhibitors include nucleoside reverse transcriptase inhibitors (NRTIs). NRTIs are modified nucleosides lacking the 3' hydroxyl group of a pentose sugar, and in cells, a phosphate group is added to the 3' by phosphorylation enzymes to become the active nucleotide form. In the process of viral DNA elongation by reverse transcriptase using viral RNA as a template, when the nucleotide form is incorporated into the DNA chain during elongation as a substrate, the lack of the hydroxyl group of the pentose sugar prevents the next nucleotide from binding, thus halting viral DNA elongation.

Specific examples of the aforementioned NRTIs include, for example, entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, lamivudine, adefovir, and interferon preparations (alpha-interferon, beta-interferon, polyethylene glycol-modified alpha-interferon). Other examples of the aforementioned NRTIs include, for example, zidovudine (AZT, or ZDV), didanosine (ddI), stavudine (d4T), abacavir (ABC), and emtricitabine (FTC). Furthermore, examples of combination drugs that can be used in combination with the aforementioned FTL inhibitors include epzicom (EPZ) (lamivudine + abacavir [3TC + ABC]), truvada (TVD) (tenofovir + emtricitabine [TDF + FTC]), combivir (CBV) (zidovudine + lamivudine [AZT + 3TC]), and descovy (DVY) (tenofovir alafenamide + emtricitabine [TAF + FTC]).

Furthermore, non-nucleoside reverse transcriptase inhibitors that can be used in combination include, for example, nevirapine (NVP), efavirenz (EFV), delavirdine (DLV), etravirine (ETR), and rilpivirine (RPV). Combination drugs include, for example, complera (CMP) (rilpivirine + tenofovir + emtricitabine [RPV + TDF + FTC]).

The cccDNA inhibitor of the present invention may, for example, contain only the active ingredient described above, or it may also contain other additive components. The additive components are not particularly limited, and examples include the following components, preferably pharmacologically acceptable components. The additive components can be appropriately determined, for example, depending on the method of administration, target recipient, and dosage form of the cccDNA inhibitor.

The aforementioned additive components include, for example, excipients. Examples of excipients include liquid media such as aqueous solvents, alcohol solvents, polyalcohol solvents, oily solvents, and mixed solvents thereof (e.g., emulsifying solvents), lactose, and starch. Examples of aqueous solvents include water, physiological saline, and isotonic solutions such as sodium chloride, while examples of oily solvents include soybean oil. Other examples of additive components include binders such as starch paste; disintegrants such as starch and carbonate; and lubricants such as talc and wax. Furthermore, the additive components may include, for example, a DDS (drug delivery system) agent for delivering the active ingredient to the target site.

In this invention, the cells targeted for cccDNA suppression are not particularly limited and include, for example, cells infected with HBV, cells potentially infected with HBV, and cells that should not be infected with HBV. The type of cell is, for example, liver cells.

The method of using the cccDNA inhibitor of the present invention is not particularly limited, and for example, it may be added to a target to which the production of HBV cccDNA is to be suppressed. The method of addition is not particularly limited, and for example, it may be done in vivo or in vitro. The target to which the cccDNA inhibitor of the present invention is added is, for example, cells, tissues, or living organisms. The type of cells and tissues, and the part (organ) of the living organism are not particularly limited, and for example, the liver. The cells and tissues may be, for example, isolated from living organisms, or from cell lines or cultures thereof. The cells and tissues to which the inhibitor is added may be, for example, derived from humans or from non-human animals, and the living organism to which the inhibitor is added may be, for example, a human or a non-human animal. Examples of non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cattle, and camels. When the target of the additive is derived from a non-human animal or is a non-human animal, the FTL inhibitor in the cccDNA inhibitor is preferably an inhibitor that specifically corresponds to the FTL or FTL gene of that particular non-human animal. When the target of the additive is derived from a human or is human, the FTL inhibitor is preferably an inhibitor that specifically corresponds to the FTL or FTL gene of human origin.

(2) Method for suppressing HBV-derived cccDNA The method for suppressing HBV-cccDNA of the present invention is characterized by including a step of suppressing FTL (also referred to as the first suppression method of the present invention), as described above. The key point of the present invention is the discovery that HBV-cccDNA can be suppressed by suppressing FTL, and the method of suppressing FTL, other steps and conditions are not limited in any way. In the first suppression method of the present invention, FTL suppression can be performed, for example, by adding the HBV-cccDNA inhibitor of the present invention.

The method for suppressing HBV-derived cccDNA according to the present invention is characterized by adding the HBV-derived cccDNA inhibitor of the present invention, as described above (also referred to as the second suppression method of the present invention). The second suppression method of the present invention can suppress HBV-derived cccDNA by adding the active ingredient of the HBV cccDNA inhibitor of the present invention, i.e., the FTL inhibitor. The key point of the second suppression method of the present invention is the use of the HBV cccDNA inhibitor of the present invention; other steps and conditions are not limited in any way.

The HBVcccDNA suppression method of the present invention can, for example, be based on the description of the HBVcccDNA inhibitor of the present invention.

When the target of the HBV cccDNA inhibitor is cells, for example, the production of HV-derived cccDNA can be suppressed by adding the HBV cccDNA inhibitor in the presence of a culture medium and incubating the cells. The incubation conditions are not particularly limited; for example, the culture medium, temperature, time, humidity, etc., can be set according to the type of cell.

When the target of the additive is tissue, for example, by adding the HBVcccDNA inhibitor in the presence of a culture medium and incubating, the production of HBVcccDNA in the cells constituting the tissue can be suppressed. The incubation conditions are not particularly limited; for example, the culture medium, temperature, time, humidity, etc., can be set according to the type and size of the tissue.

When the target of the additive (the target of administration) is a living organism, the method of administration of the HBVcccDNA inhibitor is not particularly limited and may include parenteral administration, oral administration, intravenous administration, etc. The administration conditions are not particularly limited and can be appropriately determined, for example, depending on the type of organism.

In the case of parenteral administration, the administration site may be, for example, the target organ, i.e., the liver, or a site from which the active ingredient can be delivered to the target organ. For example, if the target organ is the liver, the administration site may be the liver itself, or a site from which the active ingredient can be delivered to the liver. Methods of parenteral administration include, for example, local injection, intravenous injection, subcutaneous injection, intradermal injection, intravenous infusion, and transdermal administration. The form of the HBVcccDNA inhibitor is not particularly limited and can be appropriately determined according to the administration method, etc., as described above, and the above description can be applied accordingly.

In the case of parenteral administration, the dosage form is not particularly limited and can be appropriately determined depending on the administration method. For example, it may be a liquid, cream, or gel, and can be prepared by mixing a medium with the active ingredient. Among the mediums, the aqueous solvent may be, for example, physiological saline or isotonic solution; the oily solvent may be, for example, soybean oil; and the emulsifying solvent may be, for example, a mixture thereof. The parenteral administration agent may further contain, for example, alcohol, polyalcohol, or surfactant. Furthermore, the parenteral administration agent may contain a DDS agent for effectively delivering the active ingredient from an organ other than the target organ to the target organ. Also, when effectively delivering the active ingredient to, for example, HBV-infected cells within the target organ, the parenteral administration agent may contain, for example, a DDS agent that specifically recognizes the HBV-infected cells.

In the case of oral administration, the dosage form of the oral preparation is not particularly limited and may include, for example, tablets, pills, granules, powders, capsules, syrups, etc. The oral preparation may also contain, for example, diluents, excipients, carriers, etc. Furthermore, the oral preparation may also contain, for example, a DDS agent for effectively delivering the active ingredient to the target site. In addition, when effectively delivering the active ingredient to, for example, HBV-infected cells within the target organ, the oral preparation may also contain, for example, a DDS agent that specifically recognizes the HBV-infected cells.

In administering the HBVcccDNA inhibitor to a living organism, the administration conditions are not particularly limited and can be appropriately determined according to, for example, age, weight, type of organ to be administered, sex, etc. The living organism may, for example, be a subject infected with HBV from a therapeutic standpoint, or a subject not infected with HBV, or a subject whose infection status is unknown, from a preventative standpoint.

As a specific example, if the FTL inhibitor is a nucleic acid-type inhibitor such as siRNA, the administration conditions may include, for example, a dose of 0.1 to 0.5 mg/kg/day of the FTL inhibitor, an administration interval of once every 1 to 3 weeks, and administration over a period of 70 minutes or more (for example, 1 ml/min for the first 15 minutes of administration, and then 3 ml/min thereafter). Furthermore, if an infection reaction (acute fluid reaction) is expected, it is desirable to premedicate with, for example, the following drugs prior to the administration of the FTL inhibitor.
- Corticosteroid (dexamethasone 10 mg or equivalent) (intravenous administration)
Acetaminophen (500 mg) (oral administration)
• H1 antagonist (chlorpheniramine maleate 5 mg or equivalent) (intravenous administration)
• H2 antagonist (famotidine 20 mg or equivalent) (intravenous administration)

In the present invention, for example, when the FTL inhibitor and the replication inhibitor are used in combination as described above, they may be administered simultaneously or at different timings using the same administration method, or at different timings using the same administration method, or simultaneously or at different timings using different administration methods. In the present invention, for example, it is preferable to reduce the amount of HBV by administering the reverse transcriptase inhibitor and/or an expression inhibitor such as siRNA against HBV DNA (e.g., rcDNA), and then further suppress cccDNA by treating with the FTL inhibitor.

(3) Pharmaceutical composition for HBV infection The pharmaceutical composition for HBV infection of the present invention is characterized by containing the HBVccc DNA inhibitor of the present invention, as described above. The pharmaceutical composition for HBV infection of the present invention is characterized by containing the active ingredient of the HBVccc DNA inhibitor of the present invention, i.e., the FLT inhibitor, and other components and conditions are not particularly limited. Furthermore, the pharmaceutical composition for HBV infection of the present invention can be made by reference to the description of the HBVccc DNA inhibitor and the method for inhibiting HBVccc DNA of the present invention.

As described above, the HBV infection pharmaceutical composition of the present invention may further contain, in addition to the FTL inhibitor that suppresses the production of HBVccc DNA, a replication inhibitor such as a nucleoside reverse transcriptase inhibitor (NRTI) as an active ingredient. The FTL inhibitor and the NRTI may be administered by the same method, or by different methods. In the latter case, the HBV infection pharmaceutical composition of the present invention may be a composition in which the FTL inhibitor and the NRTI inhibitor are administered separately.

(4) Method for treating HBV infection The method for treating HBV infection of the present invention is characterized by including a step of suppressing FTL in the target organ of the patient, as described above (also referred to as the first method for treating the present invention). The key point of the present invention is that it has been found that the production of HBVccc DNA can be suppressed by suppressing FTL, and the method of suppressing FTL, other steps and conditions are not limited in any way. In the first method for treating the present invention, the suppression of FTL can be carried out, for example, by adding the HBVccc DNA inhibitor of the present invention (or the pharmaceutical composition for HBV infection of the present invention).

The present invention provides a method for treating HBV infection, characterized by administering the HBV-derived cccDNA inhibitor (or the HBV infection pharmaceutical composition of the present invention) to a patient (also referred to as the second treatment method of the present invention). The second treatment method of the present invention treats HBV infection by suppressing the production of HBV-derived cccDNA through the addition of the active ingredient of the HBV cccDNA inhibitor of the present invention, i.e., the FTL inhibitor. The key point of the second treatment method of the present invention is the use of the HBV cccDNA inhibitor (or the HBV infection pharmaceutical composition of the present invention), and other steps and conditions are not limited in any way.

The present invention's method for treating HBV infection, for example, the description of the HBVcccDNA suppression method of the present invention, can be applied.

(5) Uses The present invention is an FTL inhibitor for the treatment of HBV infection, which suppresses the FTL. The FTL inhibitor can be described by reference to the above description.

The present invention will be described in detail below with reference to examples, etc., but the present invention is not limited to these examples.

(Example 1)
We confirmed that siRNA against human FTL suppresses HBV cccDNA synthesis in the cell nucleus.

For the examples, hFTL_#3 shown in Table 1 was synthesized as the hFTL siRNA (SEQ ID NOs: 6 and 7). For the comparative examples, the following siRNA targeting HBV genomic DNA (hereinafter referred to as "genomic siRNA") and the following siRNA not targeting human or viral genes (hereinafter referred to as "negative control (NC)") were used.

As human hepatocytes, we used the cell line Hep38.7-Tet cells. These cells were developed by incorporating the HBV genome into the nucleus of the human liver cancer-derived cell line HepAD38, thereby increasing the expression efficiency of HBVcccDNA. Transcription of the HBV genome is stopped by the addition of tetracycline. Hep38.7-Tet cells are known to have a copy number of 1 copy of HBVcccDNA per cell and a copy number of 2 copies of the RNaseP gene per cell (Kurusu et al., Proceedings of the Research Institute for Mathematical Sciences, 2017, Vol. 2043, pp. 95-101, Publisher: Research Institute for Mathematical Sciences, Kyoto University).

In a 96-well plate, 2.4 μL of the 500 nmol/L siRNA and 7.6 μL of OPTI-MEN® were added, followed by 10 μL of lipofectamine solution. The plate was incubated at room temperature for 20 minutes. The lipofectamine solution consisted of 0.2 μL of lipofectamine RNAiMAX® and 9.8 μL of OPTI-MEM® per well.

Hep38.7-tet cells were collected and prepared in Dulbecco's modified Eagle medium (D-MEM Tet(+)) supplemented with tetracycline (0.3 μg/ml) to a concentration of 3 × ^10⁴ cells/100 μL. 100 μL of this medium was added to each well of a 96-well plate and incubated at 37°C and 5% _CO₂ for 20 hours. The medium was removed and replaced with 200 μL of Dulbecco's modified Eagle medium (D-MEM Tet(-)) without tetracycline, and incubated under the same conditions for another 72 hours. After observing the cells, the entire cell supernatant (approximately 200 μL) was collected, and DNA was extracted from 50 μL of the supernatant and from all cells in the wells using a DNA extraction reagent (product name SMITEST EX-R&D, MBL Life Sciences). Then, the rcDNA was quantified from the supernatant DNA extract, and the cccDNA was quantified from the cell-derived DNA extract by digital PCR.

The quantification of cccDNA was performed by the following method. Specifically, without treating the DNA sample derived from the cells with exogenous DNase, a reaction solution with the following composition was prepared. This reaction solution was then distributed into a 20,000-well plate and subjected to digital PCR, and the fluorescence signal was detected. Digital PCR was performed using a commercially available instrument (product name QuantStudio® 3D Digital PCR System, Applied Biolad Systems), and the operating conditions followed the instructions in its manual.

The HBVcccDNA sequence is registered in the database (GenBank) under accession no. D12980, and the sequence of the region from position 1541 to 1920 is shown in SEQ ID NO: 22. The forward primer was the sequence from position 1550 to 1570 (SEQ ID NO: 19), the reverse primer was the complementary sequence for position 1882 to 1863 (SEQ ID NO: 20), and the probe was the sequence from position 1781 to 1805 (SEQ ID NO: 21).
Forward primer 1 (SEQ ID NO: 19) (approximately 1550-1570)
5'-cgtctgtgccttctcatctgc-3'
Reverse primer 1 (SEQ ID NO: 20) (1882-1863):
5'-gcacagcttggaggcttgaa-3'
Probe 1 (Sequence No. 21) (Ranks 1781-1805)
5'- FAM/CT GTA GGC A/ZEN/T AAA TTG GTC TGT TCA /TAMSp -3'

The relative values of each fluorescence signal were calculated, with the fluorescence signal of the NC DNA sample set as a relative value of 1. These results are shown in Figure 1. When siRNA targeting the HBV genome was added, HBV DNA (rcDNA) in the cell culture supernatant fraction decreased compared to the negative control (not shown). However, as shown in Figure 1, no decrease in cccDNA in the cell nucleus was observed compared to the negative control. In contrast, a significant decrease in cccDNA was observed in the DNA sample obtained by introducing hFTL_#3 (a decrease of approximately 20%). From these results, it was found that an expression inhibitor designed to target FTL can reduce the production of cccDNA in the nucleus of HBV-infected cells. While reverse transcription of rdDNA from pregenetic RNA in the cytoplasm by reverse transcriptase can only be suppressed while a reverse transcriptase inhibitor is supplied, according to the present invention, the production of cccDNA in the nucleus upstream of pregenetic RNA can be suppressed, thus effectively inhibiting HBV replication and suppressing infection.

Furthermore, cell culture and introduction of the siRNA from the example were performed using a similar method, and the changes in cccDNA over time were confirmed. Specifically, the introduction of siRNA was designated as Day 0, and cccDNA was measured 24 hours later on Day 1, Day 3, and Day 5. As a comparative example, the changes in cccDNA over time were also confirmed in parallel and in the same manner for the system with the siRNA from the NC example introduced (NC) and the system without siRNA (NT).

The fluorescence signals of the DNA samples from Day 1, Day 3, and Day 5 of the NC (Nutrition Control) system were set to a relative value of 1, and the relative values of the fluorescence signals for each day were calculated. As a result, in the system without added siRNA (NT), no decrease in cccDNA was observed compared to NC even after Day 3. In contrast, in the system with the hFTL siRNA introduced in the example, a decrease in cccDNA was observed compared to NC from Day 3, and a significant decrease in cccDNA was confirmed on Day 5 (relative value 0.78).

(Example 2)
In Example 1, it was confirmed that siRNA against human FTL could suppress HBV cccDNA production in the cell nucleus.

Therefore, we confirmed the inhibitory effect on hFTL expression of the hFTL-specific siRNA (hFTL_#3) used in Example 1 and the other hFTL-specific siRNAs (hFTL_#1, #2).

Specifically, in the same manner as in Example 1, each siRNA was introduced into Hep38.7-Tet cells, the cell supernatant was collected, and a DNA sample was prepared. The expression level of the hFTL gene in the DNA sample derived from the cell supernatant was then measured as a fluorescence signal by real-time PCR. The control (NT) was processed and measured in the same manner, except that no siRNA was added.

Then, the relative values for each group were calculated, with the control (NT) measurement result set as a relative value of 1. These results are shown in Figure 2. As shown in Figure 2, in all cases where hFTL-targeting siRNAs were introduced, the expression level of the hFTL gene was significantly reduced compared to the control. As shown in Example 1, it has been confirmed that the introduction of hFTL_#3, which targets the hFTL gene, suppresses the production of cccDNA in the nucleus of HBV-infected cells. Therefore, it is clear that the production of cccDNA will similarly be suppressed by other siRNAs (#1 or #2) that target hFTL and suppress the expression of the hFTL gene.

(Example 3)
In Example 1, it was confirmed that siRNA targeting FTL can suppress the production of cccDNA in the nucleus of HVB-infected cells. On the other hand, regarding the suppression of HBV replication in HBV infection, the reverse transcriptase inhibitor entecavir, as described above, is known as a mechanism different from that of the present invention. In HBV-infected cells, pregenetic RNA transcribed from cccDNA in the nucleus is released into the cytoplasm and reverse transcribed into rcDNA. Entecavir inhibits this reverse transcription process. Since the cccDNA inhibitor of the present invention can suppress the production of cccDNA in the nucleus, which is upstream, it becomes possible to suppress HBV replication stepwise by using it in combination with a reverse transcriptase inhibitor that suppresses a different process. Therefore, in this example, the suppression of cccDNA production and the suppression of HBV rcDNA production were confirmed by using the hFTL-targeting siRNA (hFTL_#3) used in Example 1 in combination with entecavir.

Human primary cultured hepatocytes (PhoenixBio) were used as human stem cells. Commercial well plates (PhoenixBio) with the aforementioned cells (PXB cells) seeded at 4 × ^10⁵ cells/well (dHCGM medium) were purchased, and the medium in each well was replaced with dHCGM medium containing HBV DNA (rcDNA) at 6 × ^10⁶ Geq (Genome Equivalent)/ml (Day 0), and further culture was performed. The HBV DNA (rcDNA) used was derived from the serum of hepatitis B patients. Then, on Day 3, Day 8, Day 13, and Day 18, the medium was replaced with fresh dHCGM medium, and each time it was replaced, siRNA was added to the medium to a final concentration of 10 nmol/l (final concentration) and entecavir was added to 1 ng/ml (final concentration). On Day 23, cells and cell supernatant were collected, and DNA samples were prepared. For the cell-derived DNA samples, cccDNA was measured by digital PCR in the same manner as in Example 1. For the cell supernatant-derived DNA samples, real-time PCR was performed using a primer set and probe for rcDNA, and the expression level of HBV DNA was measured as a fluorescence signal. Controls consisted of a system with only siRNA of NC added (NC) and a system with only entecavir added, and culture and measurement were performed in the same manner.

These results are shown in Figure 3. In Figure 3, the vertical axis of the left bar represents the amount of cccDNA (copies/cell) and the vertical axis of the right bar represents the amount of HBV DNA (copies/ml) for each system. Since the cell supernatant does not contain cccDNA, the amount of HBV DNA in the DNA sample of the cell supernatant is theoretically almost entirely HBV rcDNA. As shown in Figure 3, compared to the system in which only NC siRNA was introduced, the system in which only entecavir was added showed a significant reduction in HBV rcDNA, but no reduction in cccDNA. In contrast, the system in which hFTL siRNA and entecavir were used in combination showed a significant reduction in both cccDNA and HBV rcDNA.

(Example 4)
Ferritin (FT) is one of the nuclear proteins involved in iron metabolism and is a heterooligomer composed of two types of subunits: L-type (Light; light chain) and H-type (Heavy; heavy chain). These subunits polymerize to retain iron in the nucleus. In Example 1, suppression of cccDNA production was confirmed by targeting FTL, the L-type subunit of FT, with siRNA. Therefore, suppression of cccDNA production was confirmed when targeting the H-type subunit of FT (hereinafter referred to as FTH) and transferrin, a protein that retains iron similarly to FT.

Except for using hFTL_#3, human FTH-targeting siRNA (hFTH1_#1, #2, #3), and human transferrin receptor-targeting siRNA (TFRC_#1, #2, #3) as in Example 1, cccDNA was measured in the same manner as in Example 1. Specifically, cccDNA in a single cell was measured. Since two copies of the RNaseP molecule exist in a single cell, the value of cccDNA in a single cell was calculated by dividing the "detected cccDNA value" by "RNaseP detection value / 2," which is derived from the number of cells.

These results are shown in Figure 4. In Figure 4, the vertical axis represents the amount of cccDNA in one cell (copies/cell). As shown in Figure 4, a decrease in cccDNA was observed only in hFTL-mediated siRNA. This result suggests that suppression of FTL expression is involved in the suppression of cccDNA production in the nucleus.

The present invention has been described above with reference to embodiments and examples, but the present invention is not limited to the above embodiments. Various modifications to the configuration and details of the present invention are possible, as can be understood by those skilled in the art within the scope of the present invention.

According to the present invention, instead of reducing the transcription of rcDNA by reverse transcriptase inhibitors as described above, the replication intermediate cccDNA upstream can be reduced, thus more effectively suppressing viral replication. Therefore, the present invention is extremely useful, for example, as a new treatment method for HBV infection.

Claims (5)

As a ferritin light chain inhibitor that suppresses the ferritin light chain,
The expression inhibitor suppresses the expression of the FTL gene encoding the ferritin light chain, or the function inhibitor suppresses the function of the ferritin light chain ,
The expression inhibitor is at least one selected from the group consisting of RNA interferants, antisenses, antigens, ribozymes, and expression vectors expressing any of these.
An inhibitor of HBV-derived cccDNA, characterized in that the function inhibitor is at least one selected from the group consisting of an antibody against the ferritin light chain, an antigen-binding fragment thereof, and an expression vector expressing any of these . The RNA interfering substance is at least one nucleic acid substance selected from the group consisting of miRNA, siRNA, and their precursors .
The inhibitor according to claim 1 , wherein the precursor is shRNA or an expression vector for the shRNA . The inhibitor according to claim 2, wherein the antisense strand of the siRNA is any of the following:
hFTL_#1
CAGAACCCAGGGCAUGAAGtt (Sequence No. 10)
hFTL_#2
UAAGCUUCACUUCCUCAUCtt (Sequence ID 12)
hFTL_#3
UGGUCCAAGGCUUGUUAGGtt (Sequence ID 14) A pharmaceutical composition for HBV infection, characterized by comprising an HBV-derived cccDNA inhibitor according to any one of claims 1 to 3 . The process includes an addition step of adding an HBV-derived cccDNA inhibitor according to any one of claims 1 to 3 ,
A method for suppressing HBV-derived cccDNA, characterized in that the addition step is performed in vitro or on a non-human animal .