WO2020075836A1 - B型肝炎ウイルス複製阻害剤及びそれを含むb型肝炎治療用医薬組成物 - Google Patents
B型肝炎ウイルス複製阻害剤及びそれを含むb型肝炎治療用医薬組成物 Download PDFInfo
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- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
Definitions
- the present invention relates to a hepatitis B virus replication inhibitor and a pharmaceutical composition for treating hepatitis B containing the same as an active ingredient.
- Hepatitis B is viral hepatitis caused by infection with hepatitis B virus (hepatitis B virus: often referred to as “HBV” in this specification). Since hepatitis B is transmitted through the blood and body fluids of HBV-infected persons, it causes vertical transmission (mother-to-child transmission) of the HBV-infected mother through the blood of the mother of the HBV-infected mother and sexual contact. Horizontal infection due to misuse of syringes and accidental needle sticks in tattoos, blood transfusions, and mass vaccinations is known as the main infection route (Non-Patent Document 1).
- HBV infection is roughly classified into transient infection and persistent infection. Infections at age 5 and older are often transient because of the well-developed immune system. 70-80% of these are subclinical infections and the remaining 20-30% develop acute hepatitis B. However, in most cases, HBs antibody is induced, so that lifelong immunity is not acquired and persistent infection does not occur. On the other hand, if HBV infection occurs due to mother-infant infection or when the immune system of the child is immature at the age of 3 or younger due to medical treatment or family infection, a persistent infection can be established. The majority of HBV persistently infected patients progress as "HBV carriers" who maintain normal liver function, of which 85-90% undergo seroconversion and become asymptomatic carriers. However, the remaining 10 to 15% develop chronic hepatitis and progress to cirrhosis and hepatocellular carcinoma. The number of HBV persistent infections is estimated to be 1.5 million in Japan and 300 to 400 million worldwide (Non-Patent Document 2).
- nucleic acid analog preparations such as Lamivudine and Entecavir are widely used as therapeutic agents for chronic hepatitis B. These therapeutic agents can reduce the amount of HBV in blood by competitive antagonism against HBV DNA polymerase and HBV DNA elongation-terminating action, thereby delaying the onset and progression of cirrhosis and hepatocellular carcinoma.
- HBV DNA in hepatocytes cannot be eliminated, if the administration of the drug is stopped, blood HBV DNA will rise again and hepatitis will relapse. Therefore, long-term administration of therapeutic agents is required. Further, relapse during long-term treatment using the nucleic acid analog preparation is accompanied by the emergence of drug resistant virus.
- Non-patent Documents 3 and 4 That makes the treatment of chronic hepatitis B more difficult.
- Non-patent Documents 3 and 4 it is desired to develop a novel therapeutic agent for hepatitis B having a mechanism of action different from that of conventional nucleic acid analog preparations.
- the present invention is to develop and provide a novel therapeutic agent for hepatitis B, which has a different mechanism of action or target object from the conventional therapeutic agents for hepatitis B.
- the present inventors have attempted to develop a novel hepatitis B therapeutic agent that inhibits HBV replication.
- HBV is a DNA virus, and its genome has a single-stranded structure as shown in Fig. 1 in which the positive strand ((+) strand) is shorter than the negative strand ((-)). It consists of approximately 3.2 Kb of circular incomplete double-stranded DNA (relaxed circular DNA: rcDNA).
- rcDNA circular incomplete double-stranded DNA
- the ORF (open reading frame) (C-ORF) of the C gene is the main component of the nucleocapsid of HBV, encodes the core protein (HBc) and HBe antigen that are essential for HBV replication, and the ORF of the P gene.
- P-ORF encodes reverse transcriptase (HBV-Pol).
- the ORF of the S gene (S-ORF) encodes three types of S protein regions (preS1, preS2, and S) that compose the envelope, and the ORF of the X gene (X-ORF) is a transcriptional regulator. , Encodes an X protein (HBx) that is considered to be important for the establishment of hepatocellular carcinoma.
- HBV infection and replication The mechanism of HBV infection and replication is that HBV first enters the host hepatocytes via an unknown HBV-specific receptor and is infected. After infection, the single-stranded portion is repaired in the nucleus of the host cell by the endogenous DNA polymerase derived from the host cell, resulting in complete double-stranded DNA (covalently closed circular DNA: cccDNA). Next, using the (-) strand of this cccDNA as a template, RNA polymerase II (RNApol II) from the host cell was used to generate four types of mRNA (3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb) with different lengths. Is synthesized.
- RNApol II RNA polymerase II
- HBc is a major component of the nucleocapsid that forms the diaphyseal part of virus particles, and at the same time, is an extremely important protein that plays an essential role in genome replication.
- the present inventors focused on HBc as a target protein of a novel hepatitis B therapeutic agent that inhibits HBV replication, and attempted to create a mutant HBc that can competitively inhibit HBV replication.
- the infection efficiency is very low, and replication is possible using cell lines (HepG2.2.15, HepAD38, etc.) in which the HBV genome has been inserted. It took 7 to 12 days for the culture to be detected. Therefore, the conventional HBV replication evaluation system has a big problem that it lacks throughput.
- the present inventors in WO2018 / 030534, without using infectious HBV to solve the above problems, using general cells, inexpensive, safe and quickly HBV genomic replication in a short time
- the present inventors used the HBV replication activity evaluation system described in WO2018 / 030534 to verify the effects of various deletion mutant HBc on HBV replication, and act antagonistically on HBc to replicate HBV.
- the present invention is based on the research results, and provides the following.
- An HBV replication inhibitor comprising any of the following (I) to (III).
- (I) Peptide fragment constituting a spike region in HBV core protein (II) Peptide fragment in which an arbitrary amino acid sequence different from that of the core protein is added to the N-terminal and / or C-terminal of the spike region (III)
- the peptide fragment constituting the spike region is the following (a) to ( The HBV replication inhibitor according to (1), which consists of the amino acid sequence of any one of c).
- nucleic acid comprises any of the following base sequences (i) to (iv) The HBV replication inhibitor according to (1).
- any of the base sequences shown in SEQ ID NOs: 8 to 14 (ii) any of the base sequences shown in SEQ ID NOs: 8 to 14 with one or more bases added, deleted, or replaced (Iii) a nucleotide sequence having a base identity of 80% or more with any of the nucleotide sequences shown in SEQ ID NOs: 8 to 14 (iv) a nucleotide sequence complementary to any of the nucleotide sequences shown in SEQ ID NOs: 8 to 14
- the phenylalanine (F) residue at position 23 is an alanine (A) residue
- the leucine (L) residue at the position is an alanine (A) residue, or in the amino acid sequence of SEQ ID NO: 7, the phenylalanine (F) residue at the 35th position is an alanine (A) residue, and
- An HBV nucleocapsid formation inhibitor comprising any of the following (I) to (III).
- An expression vector comprising a nucleic acid encoding the peptide fragment described in (I) or (II) and capable of expressing the peptide fragment in a cell
- a pharmaceutical composition for treating hepatitis B which comprises the HBV replication inhibitor, and a carrier and / or a solvent.
- the pharmaceutical composition for treating hepatitis B according to (6) which further comprises an anti-HBV agent.
- (9) (I) A peptide fragment that constitutes a spike region in the HBV core protein, (II) A peptide fragment in which an arbitrary amino acid sequence different from that of the core protein is added to the N-terminal and / or C-terminal of the spike region Or a method for inhibiting HBV replication, which comprises the step of introducing into a host an expression vector containing the nucleic acid encoding the peptide fragment described in (I) or (II) above and capable of expressing the peptide fragment in cells.
- the present specification includes the disclosure content of Japanese Patent Application No. 2018-193812, which is the basis of priority of the present application.
- the HBV replication inhibitor of the present invention can be a novel anti-HBV agent.
- hepatitis B of the present invention by using the HBV replication inhibitor of the present invention as an active ingredient, hepatitis B having a different mechanism of action or target target from conventional hepatitis B therapeutic agents, A therapeutic pharmaceutical composition can be provided.
- the thick black line in the center is an incomplete circular double-stranded genomic DNA (rcDNA) of HBV consisting of about 3.2 Kb.
- the thick gray line is the ORF (open reading frame) of the four genes encoded in the HBV genomic DNA, where C is the C gene, P is the P gene, S is the S gene, and X is the position of the X gene.
- the thin black line at the outer edge is pregenomic RNA (pregenomic RNA), which is the longest 3.5 kb mRNA among mRNA synthesized using the (-) strand of HBV genomic DNA as a template: often referred to as "pgRNA" in this specification. ) Is shown.
- HBc HBV core protein
- HBc consists of a total length of 183 amino acids, and consists of an N-terminal assembly domain (1-144 residues) and a C-terminal RNA / DNA binding domain (145-183 residues).
- the assembly domain contains five ⁇ -helices ( ⁇ 1- ⁇ 3, ⁇ 4a, ⁇ 4b, and ⁇ 5.
- B Schematic diagram of HBc conformation (modified from Wynne et al., 1999).
- FIG. 2 is an alignment diagram of amino acid sequences constituting each genotype of HBc. An asterisk at the bottom of the sequence indicates that the amino acid at the corresponding position in all genotypes is identical, and a colon indicates that the amino acid at the corresponding position in all genotypes is similar.
- FIG. 3 is a conceptual diagram showing an example of various expression vectors constituting the HBV replication activity evaluation system described in WO2018 / 030534 used in Examples.
- FIG. 3 is a conceptual diagram of pCI-HBc, which is an expression vector of a C gene encoding C: HBc.
- FIG. 3 is a conceptual diagram of pCI-HBx, which is an expression vector of an X gene encoding D: HBx.
- A It is a conceptual diagram showing an example of a nucleic acid for evaluating HBV replication activity of the present invention.
- FIG. 2 is a conceptual diagram of a reporter pgRNA when an HBV replication activity evaluation vector, in which the HBV replication activity evaluation nucleic acid represented by B: A is incorporated into an expression vector, is introduced into cells and expressed.
- FIG. 2 is a conceptual diagram of reporter minus-strand DNA (reporter ( ⁇ ) DNA) synthesized by the reverse transcription activity of HBV-Pol using a reporter pgRNA represented by C: B as a template.
- HBV replication activity evaluation nucleic acid and reporter minus strand DNA can be distinguished by the presence or absence of an intron. It is a figure which showed the inhibitory effect to HBV replication of (DELTA) HBc.
- a to C are the results of evaluation by the HBV replication activity evaluation system of the effects of 11 types of ⁇ HBc constructed in the example on HBV replication.
- FIG. 8 is a graph showing the quantitative effect of HBV replication inhibition by ⁇ HBc ( ⁇ 1-4b) corresponding to the spiked region of HBc in which the HBV replication inhibition effect was observed in FIG. 7.
- the ⁇ HBc ( ⁇ 1-4b) expression vector was introduced into HeLa cells together with the HBV replication activity evaluation system.
- ⁇ HBc ( ⁇ 1-4b) is a quantitative ratio with the full-length wild-type HBc expression vector contained in the HBV replication activity evaluation system, and ⁇ HBc ( ⁇ 1-4b) / WT-HBc is 1/9, 3/9, 9/9. , And 26/9.
- FIG. 3 is a diagram showing the HBV replication inhibitory effect of ⁇ HBc ( ⁇ 1-4b) derived from each genotype.
- FIG. 3 is a diagram showing the effects of HBV nucleocapsid formation inhibition and HBV replication inhibition by HBc amino acid point mutation.
- WT indicates full length wild type HBc
- F23A indicates full length HBc-F23A
- L42A indicates full length HBc-L42A.
- B Results of particle blotting.
- C It is a figure showing the HBV replication activity when the HBc expression vector contained in the HBV replication activity evaluation system is replaced with the F23A or L42A mutant HBc expression vector.
- FIG. 6 is a view showing an inhibitory effect on HBV replication by ⁇ HBc ( ⁇ 1-4b) introduced with an F23A or L42A mutation.
- A Inhibition of HBV replication of ⁇ HBc ( ⁇ 1-4b) introduced with a point mutation using the HBV replication activity evaluation system.
- B It is a figure which shows the result of the dose-dependent effect of HBV replication inhibition.
- FIG. 3 is a graph showing the inhibitory effect of ⁇ HBc ( ⁇ 1-4b) or ⁇ HBc ( ⁇ 1-4b) -F23A on HBc multimer formation and the inhibitory effect of HBc on nucleocapsid formation.
- A The effect of the expression of ⁇ HBc ( ⁇ 1-4b) or ⁇ HBc ( ⁇ 1-4b) -F23A added with a PA tag on dimerization and multimerization of HBc was measured using anti-HBc monoclonal antibody # 511. It is the result of analysis by Western blotting.
- Mlt is a multimer
- T is a multimer
- D is a dimer
- M is a monomer band position.
- FIG. 2 shows the quantitative effect of HBV replication inhibition by the nucleocapsid formation inhibitor GLS4.
- HBc-WT wild-type HBc
- HBc-T33N HBc introduced with a T33N mutation in place of wild-type HBc was used.
- the vertical axis of the figure shows the inhibition efficiency of HBV replication.
- the inhibitory efficiency of HBV replication in the presence of a specific concentration of GLS4 is based on the measurement value of HBV replication activity at that concentration, ⁇ 0% inhibition '' of HBV replication activity when GLS4 concentration is 0 ⁇ M, and HBV replication activity is 0%. The value is calculated as "100% inhibition" in the case of.
- FIG. 6 shows the quantitative effect of HBV replication inhibition by ⁇ HBc ( ⁇ 1-4b).
- HBc included in the HBV replication activity evaluation system wild-type HBc (HBc-WT) or HBc-T33N was used instead of HBc-WT.
- the ⁇ HBc ( ⁇ 1-4b) expression vector was introduced into HeLa cells together with the HBV replication activity evaluation system.
- ⁇ HBc ( ⁇ 1-4b) is the amount ratio with HBc-WT or HBc-T33N contained in the HBV replication activity evaluation system, ⁇ HBc ( ⁇ 1-4b) / HBc-WT or ⁇ HBc ( ⁇ 1-4b) / HBc-T33N
- the vertical axis of the figure shows the relative HBV replication activity when the replication activity when ⁇ HBc ( ⁇ 1-4b) was not introduced is 1.
- HBV replication inhibitor Hepatitis B virus replication inhibitor 1-1.
- the first aspect of the present invention is an HBV replication inhibitor.
- the HBV replication inhibitor of the present invention is a peptide fragment constituting a spike region of a core protein (HBc), or a peptide fragment in which an arbitrary amino acid sequence different from the core protein is added to the N-terminal and / or C-terminal of the spike region. Or an expression vector capable of expressing the spike region in cells.
- the HBV replication inhibitor of the present invention can be an active ingredient of a pharmaceutical composition for treating hepatitis B, which has a high inhibitory effect on HBV proliferation in hepatocytes.
- Hepatitis B virus is a DNA virus belonging to the genus Orthohepadnavirus of the family Hepadnaviridae and is a causative virus of hepatitis B.
- HBV is known to have eight genotypes (genotypes A, B, C, D, E, F, G, and H) depending on the difference in gene sequence. These genotypes differ in terms of regional distribution and pathological conditions. For example, in Japan, conventional genotype C (often referred to as “HBV / C” in this specification; the same applies to other genotypes) occupy the majority, followed by HBV / B.
- HBV / A is known to transfer to chronic hepatitis in about 20 to 30% after acute hepatitis, but HBV / B and HBV / C have a low chronicity rate after acute hepatitis.
- the “core protein” is a protein constituting a nucleocapsid essential for HBV replication. As shown in FIG. 2A, it is composed of an “assembly domain” on the N-terminal side and a subsequent “RNA / DNA binding domain” on the C-terminal side. The assembly domain is further composed of an N-terminal side "spike region” and a C-terminal side "hand region (HR)” (Wynne SA, et al., 1999). , Mol Cell., 3: 771-780). HBc expressed in virus-infected cells forms a dimer in the assembly domain (Fig.
- the assembly domain consists of five ⁇ -helices as shown in Fig. 2B, the spike region is four ⁇ -helices on the N-terminal side, and the hand region is the C-terminal side. Containing one ⁇ -helix.
- FIG. 3 eight types of genotypes corresponding to HBV / A to HBV / H are also known for HBc. In the present specification, each genotype of HBc is represented as “HBc / A” for HBc of HBV / A.
- HBc / A to HBc / F and HBc / H are all 183 amino acid residues in length, of which the assembly domain is 144 amino acid residues from 1 to 144, and the RNA / DNA binding domain is 145 position. It is composed of 39 amino acid residues at positions 183. Only HBc / G contains an additional sequence consisting of 12 amino acid residues at the N-terminal side, so it consists of 195 amino acid residues in total length, the assembly domain is 156 amino acid residues from 1 to 156, and RNA / DNA The binding domain is composed of 39 amino acid residues at positions 157-195.
- the full-length amino acid sequence of each genotype of HBc is HBc / A in SEQ ID NO: 15, HBc / B and HBc / C in which the amino acid sequences are completely the same are SEQ ID NO: 16, HBc / D is SEQ ID NO: 17, HBc / E. Is SEQ ID NO: 18, HBc / F is SEQ ID NO: 19, HBc / H is SEQ ID NO: 20, and HBc / G having a different total length from other genotypes is SEQ ID NO: 21.
- the “C gene” is a gene encoding a core protein, and is one of the four types of genes encoded in the HBV genome as described above.
- the “expression vector” refers to a vector containing a gene or gene fragment (hereinafter referred to as “gene etc.”) in an expressible state and including an expression unit capable of controlling the expression of the gene or the like.
- the "expressible state” means that a gene or the like to be expressed is arranged in the downstream region of the promoter under the control of the promoter.
- a plasmid vector, a virus vector and the like are known, and any vector can be used. Usually, a plasmid vector that can be easily subjected to gene recombination is sufficient.
- the expression vector a commercially available expression vector for mammalian cells may be used. For example, pCI vector and pSI vector manufactured by Promega are listed. Further, the expression vector may be a shuttle vector capable of replicating between mammalian cells and bacteria such as Escherichia coli.
- the “promoter” is a gene expression control region capable of controlling the expression of a gene or the like located downstream (3 ′ end side) in a cell into which an expression vector has been introduced. Promoters can be classified into ubiquitous promoters (systemic promoters) and site-specific promoters based on the location where a gene under expression control is expressed.
- the ubiquitous promoter is a promoter that controls the expression of a target gene or the like (target gene or the like) in all cells, that is, the entire host individual.
- a site-specific promoter is a promoter that controls the expression of a target gene or the like only in specific cells or tissues.
- promoters are classified into constitutively active promoters, expression-inducible promoters or time-specific active promoters based on the timing of expression.
- the constitutively active promoter can constitutively express the gene of interest in the cell.
- the expression-inducible promoter can induce the expression of the target gene or the like in the cell at any time.
- the stage-specific active promoter can induce expression of a target gene or the like in cells only at a specific stage of the developmental stage. Any promoter can be considered as an overexpressing promoter because it can cause overexpression of the gene of interest in the host cell.
- inhibiting HBV nucleocapsid formation inhibits dimerization of HBc and multimerization of dimer as a unit, and normal uptake of pgRNA into HBc multimer. Thereby inhibiting the formation of functional nucleocapsids.
- the “anti-HBV agent” refers to a drug having an action of suppressing or inhibiting HBV replication or proliferation.
- the HBV replication inhibitor of the present invention is also included in the anti-HBV agent.
- treatment refers to alleviation or elimination of symptoms associated with the onset of a disease, and / or inhibition or suppression of progression of a disease, and cure of a disease.
- disease as used herein means hepatitis B unless otherwise specified.
- the HBV replication inhibitor of the present invention comprises a peptide fragment or an expression vector. Each configuration will be specifically described below.
- the "peptide fragment" constituting the HBV replication inhibitor of the present invention has a spike region of HBV or an arbitrary amino acid sequence different from HBc added to the N-terminal and / or C-terminal of the spike region.
- the resulting peptide fragment is a component contained on the N-terminal side of the HBc assembly domain, as described above.
- HBc / A to HBc / F and HBc / H spike regions consist of 111 amino acid residues consisting of HBc positions 1 to 111, and HBc / G spike regions consist of HBc positions 1 to 123 123. Corresponds to an amino acid residue.
- Amino acids in the spike region between each genotype are highly conserved, and between HBc / A to HBc / F and HBc / H, amino acid similarity of 95% or more and amino acid identity of 84% or more. Have.
- amino acid sequences of the peptide fragments constituting the HBV replication inhibitor of the present invention include (a) any of the amino acid sequences shown in SEQ ID NOs: 1 to 7 and (b) any of the amino acid sequences shown in SEQ ID NOs: 1 to 7. 82% or more, 84% or more, 86% or more, with the amino acid sequence of 1 or more amino acids added, deleted, or substituted in the amino acid sequence of Examples include amino acid sequences having amino acid identities of% or more, 88% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more.
- SEQ ID NO: 1 is a spike region derived from HBc / A
- SEQ ID NO: 2 is derived from HBc / B and HBc / C
- SEQ ID NO: 3 is derived from HBc / D
- SEQ ID NO: 4 is derived from HBc / E
- SEQ ID NO: 5 shows the amino acid sequence derived from HBc / F
- SEQ ID NO: 6 shows the amino acid sequence derived from HBc / H
- SEQ ID NO: 7 shows the amino acid sequence derived from HBc / G.
- “plurality” means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.
- the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid residue.
- the "conservative amino acid substitution” refers to a substitution between amino acids having similar properties such as charge, side chain, polarity and aromaticity.
- Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), apolar.
- Amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. it can.
- amino acid substitution in the amino acid sequence constituting the peptide fragment is, but not limited to, point mutation.
- point mutation in the amino acid sequence of any of the spike regions shown in SEQ ID NOs: 1 to 6, the phenylalanine (F) residue at position 23 is replaced with an alanine (A) residue (in this specification, this point mutation is referred to as “F23A”).
- F23A this point mutation
- L42A a point mutation in which the leucine (L) residue at position 42 is replaced with an alanine (A) residue
- the phenylalanine (F) residue at position 35 is an alanine (A) residue (F35A) and / or the leucine (L) residue at position 54 is an alanine ( A)
- a point mutation that substitutes the residue (L54A) can be mentioned.
- amino acid identity means that when two amino acid sequences are aligned (aligned) and a gap is introduced as necessary so that the degree of amino acid identity between the two amino acid sequences is the highest. The ratio (%) of the same amino acid residue between the two amino acid sequences to the total amino acid residues of the amino acid sequence. Amino acid identity can be calculated using a protein search system based on BLAST or FASTA.
- An arbitrary amino acid sequence different from HBc added to the N-terminal and / or C-terminal of the spike region is not particularly limited. Examples include ubiquitinated sequences, nuclear localization signals, tag sequences and the like.
- the peptide fragment can include one or more amino acid sequences of the spike region at either the N-terminus or the C-terminus, or both. The number of amino acids added is not limited, for example, 20, 19, 18, 17, 17, 16, 15, 14, 13, 12, 11, 10, 9, respectively. , 8, 7, 6, 5, 4, 3, 2, or 1.
- the "expression vector" constituting the HBV replication inhibitor of the present invention is an expression vector containing a nucleic acid encoding the peptide fragment and a promoter and capable of expressing the spike region in cells.
- the expression vector may contain components such as a marker gene (selection marker), an enhancer, a terminator, an origin of replication, and a poly A signal, if necessary, in addition to the nucleic acid and the promoter which are the above components.
- selection marker selection marker
- an enhancer an enhancer
- a terminator an origin of replication
- poly A signal if necessary, in addition to the nucleic acid and the promoter which are the above components.
- nucleic acid encoding a peptide fragment that constitutes the spike region of HBc may be any nucleic acid that includes a nucleic acid encoding any spike region of each genotype.
- the base sequence of such nucleic acid is not limited.
- the nucleotide sequences of genes of each genotype encoded on the genome can be mentioned.
- the nucleotide sequence of the nucleic acid encoding the HBc / A spike region of Accession No. AY707087.1 the nucleotide sequence of the nucleic acid encoding the HBc / B spike region of Accession No.
- GU357842.1 the access sequence Base sequence of nucleic acid encoding spike region of HBc / C of session No.AB033556.1, base sequence of nucleic acid encoding spike region of HBc / D of accession No.GU357846.1, accession No.X75664.1 Nucleotide sequence encoding the HBc / E spike region of HBc / E, nucleotide sequence encoding the HBc / F spike region of Accession No. JN792913.1, HBc / G spike of Accession No.
- nucleotide sequence of the nucleic acid encoding the spike region of HBc / A shown in SEQ ID NO: 8 the nucleotide sequence of the nucleic acid encoding the spike region of HBc / B or HBc / C shown in SEQ ID NO: 9, the sequence The nucleotide sequence of the nucleic acid encoding the spike region of HBc / D shown in No. 10
- nucleotide sequence of the nucleic acid encoding the HBc / H spike region shown in SEQ ID NO: 13 or the nucleotide sequence of the nucleic acid encoding the HBc / G spike region shown in SEQ ID NO: 14.
- a termination codon TGA is added to each 3 ′ end in order to express only the spike region.
- one or more bases are added, deleted, or substituted, or any of the above base sequences, 80% or more, 82% or more, 85% or more, 88 % Or more, 90% or more, 93% or more, 95% or more, 98% or more, 99% or more of base sequence having a base identity, or a base sequence complementary to any of the above base sequences and highly stringent conditions Examples include base sequences that hybridize below.
- nucleotide sequences constituting the nucleic acid examples include, but are not limited to, degenerate mutations, gene polymorphisms such as SNIPs, splice mutations, point mutations, and the like.
- point mutations include point mutations in which any of the nucleotide sequences shown in SEQ ID NOs: 8 to 13 has TTC at positions 67 to 69 replaced with GCC, and / or CTG at positions 124 to 126 replaced with GCC. .
- nucleotide sequence shown in SEQ ID NO: 14 there may be mentioned point mutations in which the TTC at positions 106 to 108 are replaced with GCC and / or the CTG at positions 160 to 162 are replaced with GCC.
- base identity means that when two base sequences are aligned (aligned) and a gap is introduced as necessary so that the base coincidence between the two becomes highest. It refers to the ratio (%) of the same base between the two base sequences to all the bases of the base sequence represented by SEQ ID NO.
- hybridize under the condition of high stringency means that hybridization and washing are performed under conditions of low salt concentration and / or high temperature.
- the salt concentration in the washing solution is lowered to 0.1 ⁇ SSC, and the temperature is raised to 68 ° C., and washing is performed until no background signal is detected.
- the promoter is a promoter capable of inducing expression of a nucleic acid encoding the peptide fragment in cells. Since the target cell to which the HBV replication inhibitor of the present invention is applied, that is, the expression vector is introduced, is a mammalian cell, in particular, a cell derived from human or chimpanzee in principle, so that a gene downstream thereof can be expressed in those cells. Any promoter will do. Examples include CMV promoter (CMV-IE promoter), SV40 early promoter, RSV promoter, EF1 ⁇ promoter, Ub promoter and the like.
- a "marker gene” is a gene that encodes a marker protein also called a selectable marker or reporter protein.
- the “labeled protein” refers to a peptide that can determine the presence or absence of expression of the labeled gene based on its activity. The activity may be detected by directly detecting the activity of the labeled protein itself or indirectly by detecting a metabolite generated by the activity of the labeled protein such as a dye. Good.
- Detection includes biological detection (including detection by binding of peptides or nucleic acids such as antibodies and aptamers), chemical detection (including enzymatic reaction detection), physical detection (including behavioral analysis detection), or detection It may be any of sensory detection (including detection by sight, touch, smell, hearing, and taste) of a person.
- labeled protein encoded by the labeled gene is not particularly limited as long as its activity can be detected by a method known in the art. Labeled proteins that are less invasive to transformants upon detection are preferred. Examples include tag peptides, drug resistant proteins, dye proteins, fluorescent proteins, luminescent proteins and the like.
- Tag peptide is a short peptide consisting of dozens of amino acids to several tens of amino acids that can label proteins, and is used for protein detection and purification. Usually, the nucleotide sequence encoding the tag peptide is ligated to the 5'-terminal side or the 3'-terminal side of the gene encoding the protein to be labeled, and the gene is expressed as a fusion protein with the tag peptide for labeling. Although various types of tag peptides have been developed in the art, any tag peptide may be used. Specific examples of the tag peptide include FLAG, HA, His, myc and the like.
- Drug-resistant protein is a protein that imparts resistance to cells such as antibiotics added to the medium such as antibiotics, and most of them are enzymes. Examples include ⁇ -lactamase that confers resistance to ampicillin, aminoglycoside 3'phosphotransferase that confers resistance to kanamycin, tetracycline efflux transporter that confers resistance to tetracycline, and chloramphenicol. Examples thereof include CAT (chloramphenicol acetyltransferase) which imparts resistance.
- “Dye protein” is a protein involved in dye biosynthesis, or a protein that enables chemical detection of a transformant by a dye by adding a substrate, and is usually an enzyme.
- the term “dye” as used herein refers to a low molecular weight compound or peptide capable of imparting a dye to a transformant, and its type is not limited.
- ⁇ -galactosidase (LacZ) ⁇ -glucuronidase (GUS), melanin pigment synthetic protein, omochrome pigment, or pteridine pigment may be mentioned.
- Fluorescent protein refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either a natural type or a non-natural type. Moreover, the excitation wavelength and the fluorescence wavelength are not particularly limited. Specific examples include CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP), and the like. To be
- Luminescent protein refers to a substrate protein that can emit light without the need for excitation light or an enzyme that catalyzes the light emission of the substrate protein.
- luciferin or aequorin as a substrate protein and luciferase as an enzyme can be mentioned.
- the “enhancer” is not particularly limited as long as it can enhance the expression efficiency of the gene or the fragment thereof in the vector.
- the “terminator” is a sequence capable of terminating the transcription of the expressed gene or the like by the activity of the promoter.
- the type of terminator is not particularly limited. Preferred is a terminator derived from the same species as the promoter. A terminator paired with the promoter on the genome in the one-gene expression control system is particularly preferable.
- the expression vector of the present invention only needs to be capable of transiently expressing the spike region of HBc in mammalian cells. Therefore, no origin of replication for mammalian cells is required.
- the shuttle vector when expressed in bacteria such as Escherichia coli, its origin of replication is essential.
- a known sequence can be used as the origin of replication. For example, if the origin of replication is E. coli, f1 origin or the like may be used.
- HBV nucleocapsid formation inhibitor HBV nucleocapsid formation inhibitor
- HBV nucleocapsid formation inhibitors target HBc.
- HBc is a protein essential for HBV genome replication, and at the same time, it is also a main component of the nucleocapsid that forms the diaphysis of HBV particles.
- the inhibitory effect on HBV nucleocapsid formation simultaneously brings about an inhibitory effect on HBV replication.
- the HBV replication inhibitor of the present invention may also function as an HBV nucleocapsid formation inhibitor.
- the composition of the HBV nucleocapsid formation inhibitor may be the same as the composition described in HBV replication inhibitor.
- the second aspect of the present invention is a pharmaceutical composition for treating hepatitis B.
- the pharmaceutical composition for treating hepatitis B of the present invention comprises the HBV replication inhibitor of the first aspect as an essential active ingredient, inhibits the replication of HBV after HBV infection, and suppresses the growth of HBV. Hepatitis can be treated.
- composition 2-2-1 Constituents
- the constituents of the pharmaceutical composition for treating hepatitis B of the present invention will be described.
- the pharmaceutical composition for treating hepatitis B of the present invention contains one or more active ingredients as essential constituents, a solvent and / or a carrier, and further drug delivery system (DDS) particles.
- DDS drug delivery system
- the pharmaceutical composition for treating hepatitis B of the present invention includes the HBV replication inhibitor according to the first aspect as an essential active ingredient.
- one or more anti-hepatitis B virus agents may be included, if necessary.
- the structure of the HBV replication inhibitor, which is an essential active ingredient, has been described in detail in the first aspect, and thus a detailed description thereof is omitted here.
- the pharmaceutical composition for treating hepatitis B of the present invention can contain one or more HBV replication inhibitors.
- the pharmaceutical composition for treating hepatitis B of the present invention may contain only an essential HBV replication inhibitor as an active ingredient, but a combination composition further containing one or more other anti-HBV agents in combination of two or more. Good as a thing.
- Such other anti-HBV agents include, but are not limited to, known anti-HBV agents such as hepatitis B therapeutic nucleic acid analogs and hepatitis B virus polymerase activity inhibitors (HBV-Pol activity inhibitors). It
- Nucleic acid analogs for treating hepatitis B include, for example, Entecavir (ETV), Lamivudine (LAM), Adefovir (Adefovir), Tenofovir (Tenofovir), Terbivudine, Clevudine, etc.
- ETV Entecavir
- LAM Lamivudine
- Adefovir Adefovir
- Tenofovir Teenofovir
- Terbivudine Clevudine, etc.
- pharmaceutical compositions for treating hepatitis B which inhibit the reverse transcriptase activity of HBV.
- the “HBV-Pol activity inhibitor” includes a phosphorylation inhibitor that inhibits phosphorylation of the activation site in HBV-Pol described in WO2018 / 030534 developed by the present inventor.
- MAPK kinase inhibitors are relevant. More specific examples of the MAPK kinase inhibitor include the following hypothemycin represented by Formula 1, trametinib represented by Formula 2, PD98059 represented by Formula 3, PD184352 represented by Formula 4, and Formula 5 below. U-126 and the like are shown.
- Nucleic acid analogs for treating hepatitis B have competitive antagonism against HBV DNA polymerase and HBV DNA elongation-terminating action. Therefore, the HBV replication inhibitor described in the first aspect acts on the HBV replication pathway. Mechanism is different.
- HBV-Pol activity inhibitors act on the HBV replication pathway in the same manner as HBV replication inhibitors, but use TxY motif as the target site, and proteins containing such motifs, such as MAPK kinase, are the target molecule. Therefore, the target target is different from the HBV replication inhibitor described in the first aspect, which uses HBcDNA as the target molecule.
- HBV replication inhibitor according to the first aspect with another anti-HBV agent such as a nucleic acid analog for treating hepatitis B and / or an HBV-Pol activity inhibitor, a synergistic effect on anti-HBV inhibition is obtained. Can be obtained.
- another anti-HBV agent such as a nucleic acid analog for treating hepatitis B and / or an HBV-Pol activity inhibitor
- the content of the active ingredient contained in the pharmaceutical composition for treating hepatitis B of the present invention is not particularly limited. Generally, the content varies depending on the type of active ingredient, the dosage form, and the types of solvents and carriers which are other constituents described later. Therefore, it may be appropriately determined in consideration of each condition. It suffices that a single application amount of the pharmaceutical composition for treating hepatitis B contains an effective amount of the active ingredient. However, when it is necessary to administer a large amount of the pharmaceutical composition for treating hepatitis B to the subject in order to obtain the pharmacological effect of the active ingredient, it is administered in several divided doses to reduce the burden on the subject. You can also In this case, the amount of the active ingredient may include the effective amount in the total amount.
- Effective amount means an amount necessary for exhibiting the function as an active ingredient and giving little or no harmful side effects to the subject to which it is applied. This effective amount can vary depending on various conditions such as the subject's information, the route of application, and the number of applications. Therefore, when the pharmaceutical composition for treating hepatitis B is used as a medicine, the content of the active ingredient is finally determined by the judgment of a doctor or pharmacist.
- the “subject” refers to an object to which the HBV replication inhibitor according to the first aspect or the pharmaceutical composition for treating hepatitis B according to the present aspect is applied.
- cells including cultured cells
- tissues including organs, or individuals.
- subject In the case of an individual, it is preferably a human individual, in which case it is particularly referred to as “subject”.
- Subjects infected with HBV, ie patients with hepatitis B, are particularly preferred.
- the “subject information” is various information regarding the characteristics and condition of the subject.
- examples include age, weight, sex, general health status, presence / absence of disease, disease progression / severity, drug sensitivity, presence / absence of concomitant drug, resistance to treatment, and the like. .
- the pharmaceutical composition for treating hepatitis B of the present invention may contain a pharmaceutically acceptable solvent, if necessary.
- a pharmaceutically acceptable solvent refers to a solvent usually used in the technical field of formulation.
- water or an aqueous solution, or an organic solvent can be used.
- Aqueous solutions include, for example, saline, isotonic solutions containing glucose or other auxiliaries, phosphate buffers, sodium acetate buffers.
- the auxiliary agent include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.
- the organic solvent include ethanol.
- composition for treating hepatitis B of the present invention can contain a pharmaceutically acceptable carrier, if necessary.
- “Pharmaceutically acceptable carrier” refers to an additive usually used in the technical field of formulation. Examples thereof include excipients, binders, disintegrants, fillers, emulsifiers, flow control agents, lubricants, human serum albumin and the like.
- Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronics, kaolin, silicic acid, or combinations thereof.
- sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronics, kaolin, silicic acid, or combinations thereof.
- the binder for example, starch paste using vegetable starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone or a combination thereof. Can be mentioned.
- the disintegrant for example, the starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearin.
- examples thereof include acid monoglyceride and salts thereof.
- Examples of the filler include petrolatum, the above sugar and / or calcium phosphate.
- emulsifiers examples include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, and propylene glycol fatty acid ester.
- fluid addition modifiers and lubricants examples include silicates, talc, stearates or polyethylene glycols.
- solubilizers in addition to the above, if necessary, solubilizers, suspensions, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption enhancers, bulking agents, etc. that are commonly used in pharmaceutical compositions and the like.
- Moisturizers moisturizers, humectants, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, preservatives, antioxidants, fragrances, flavors, sweeteners, buffers, isotonic agents.
- An agent and the like can also be included as appropriate.
- the carrier is used for avoiding or suppressing the decomposition of the active ingredient by an enzyme or the like in the subject, facilitating the formulation and administration method, and maintaining the dosage form and the drug effect, and is appropriately used as necessary. You can use it.
- DDS particles Drug delivery system particles
- the pharmaceutical composition for treating hepatitis B of the present invention may optionally contain DDS particles.
- DDS particles include an active ingredient and other carriers in the interior thereof, etc. to deliver the contents to the target site, particularly the active ingredient without degrading it, and to temporally distribute the drug in the living body. It refers to particles that can be quantitatively controlled. Since the active ingredient of the pharmaceutical composition for treating hepatitis B of the present invention is a peptide or a nucleic acid, the use of DDS particles is also suitable for protecting from degradation by protease or nuclease in vivo after administration.
- the type of DDS particles does not matter. Examples thereof include liposomes, polymer micelles, virus particles and the like.
- the dosage form of the pharmaceutical composition for treating hepatitis B of the present invention is not particularly limited.
- the form may be such that the active ingredient is delivered to the intended site without inactivating the active ingredient in the subject.
- the specific dosage form depends on the application method described below. The method of application can be roughly divided into parenteral administration and oral administration, and thus the dosage form may be adapted to each administration method.
- the preferred dosage form is a liquid formulation that can be directly administered to the target site or systemically administered via the circulatory system.
- An injection is mentioned as a preferable example of the liquid preparation.
- injectables are appropriately combined with the above-mentioned excipients, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc. in addition to solvents, and mixed in the unit dosage form generally required for pharmaceutical practice. By doing so, it can be formulated.
- preferable dosage forms include solid preparations (including tablets, capsules, drops, and troches), granules, powders, powders, solutions (internal water preparations, emulsions, syrups) ) Is mentioned. If it is a solid formulation, it may be formed into a coated form known in the art, for example, a sugar-coated tablet, a gelatin-coated tablet, an enteric coated tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary. be able to.
- the specific shapes and sizes of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art for each dosage form.
- the method for producing the pharmaceutical composition for treating hepatitis B of the present invention may be formulated according to a conventional method in the art.
- the method of applying the pharmaceutical composition for treating hepatitis B of the present invention may be oral administration or parenteral administration.
- the oral administration method is systemic administration, but the parenteral administration method can be further subdivided into systemic administration and local administration.
- Local administration includes, for example, intramuscular administration, subcutaneous administration, tissue administration, and organ administration, and systemic administration of parenteral administration includes intracardiac administration, such as intravenous administration (intravenous injection) and arterial administration. Internal administration and intralymphatic administration are included.
- the pharmaceutical composition for treating hepatitis B of the present invention When the pharmaceutical composition for treating hepatitis B of the present invention is locally administered, it may be directly administered to the liver by injection or the like. Further, in the case of systemic administration, it may be administered into the circulatory organ such as intravenous injection.
- the dose may be an amount effective for the active ingredient to respond effectively. The effective amount is appropriately selected depending on the subject information as described above.
- two or more other known anti-HBV agents can be separately used in combination.
- a third aspect of the present invention is a method for inhibiting HBV replication.
- the method for inhibiting HBV replication of the present invention comprises: (I) a peptide fragment constituting a spike region in a core protein of hepatitis B virus in an HBV-infected cell or a cell that may be infected with the HBV-infected cell; Or a peptide fragment having an arbitrary amino acid sequence different from the core protein added to the C-terminus, or (III) containing the nucleic acid encoding the peptide fragment described in (I) or (II), and the peptide in a cell
- an expression vector capable of expressing the fragment HBV replication in cells can be inhibited, thereby inhibiting HBV replication and suppressing proliferation.
- this method is administered to a patient suffering from hepatitis B or a person at risk of suffering from it, it can be a method for treating hepatit
- the method for inhibiting HBV replication in this embodiment includes an introduction step as an essential step.
- the "introduction step” is (I) a peptide fragment constituting the spike region of HBc, (II) a peptide fragment in which an arbitrary amino acid sequence different from the core protein is added to the N-terminal and / or C-terminal of the spike region.
- an expression vector comprising the nucleic acid encoding the peptide fragment described in (I) or (II) above and capable of expressing the peptide fragment in a cell, that is, the HBV replication inhibitor according to the first aspect. Is a step of introducing into the host.
- the “host” refers to a cell, tissue or individual into which an HBV replication inhibitor can be introduced.
- the cell may be one or more mammalian cells. Preferred are cells derived from human or chimpanzee, which is the host of HBV. The type of origin cell does not matter. In addition to hepatocytes, which are the target cells for HBV infection, cells derived from various organs and tissues can be targeted.
- the host may be either a cell line system or a primary culture cell line.
- the host is preferably, but not limited to, a cell infected with HBV or a cell that may be infected with HBV. When the host is an individual, the above-mentioned subject may be used.
- the method of introducing each expression vector into the host is not particularly limited.
- the host is a cell or tissue
- lipofectin method PNAS, 1989, 86: 6077; PNAS, 1987, 84: 7413
- electroporation method calcium phosphate method (Virology, 1973, 52: 456-467), DEAE-Dextran method and the like can be mentioned.
- the host is an individual, for example, a subject
- the HBV replication inhibitor or a medicament for treating hepatitis B which comprises the subject as an active ingredient, according to the method described in "2-3.
- the composition may be administered.
- the method for inhibiting HBV replication of this embodiment can be a method for treating hepatitis B.
- Example 1 Inhibitory effect of mutant HBc on HBV replication> (Purpose) Mutant HBc in which HBc is deleted in various forms is prepared, and its inhibitory effect on HBV replication is verified.
- HBc # 1 is an RNA / DNA binding domain (RDBD) deletion type and is composed only of the assembly domain consisting of positions 1-144 of SEQ ID NO: 16. In the embodiment, it is written as "AD”.
- ⁇ HBc # 2 consists of positions 1-111 of SEQ ID NO: 16.
- This ⁇ 1-4b corresponds to the spike region of HBc in which the hand region (HR) is deleted from AD.
- ⁇ HBc # 3 consists of positions 1 to 91 of SEQ ID NO: 16.
- (4) ⁇ HBc # 4 consists of positions 1 to 78 of SEQ ID NO: 16.
- a structure in which ⁇ 4a to ⁇ 5 are deleted from ⁇ HBc # 1 and includes ⁇ 1 to ⁇ 3. In the examples, it is described as " ⁇ 1-3”.
- ⁇ HBc # 5 consists of positions 1 to 49 of SEQ ID NO: 16.
- ⁇ HBc # 6 consists of positions 1 to 26 of SEQ ID NO: 16.
- ⁇ 2 to ⁇ 5 are deleted from ⁇ HBc # 1 and includes only ⁇ 1.
- ⁇ HBc # 7 consists of the 1st position (starting methionine) and 18th to 111st positions of SEQ ID NO: 16.
- ⁇ 1 and ⁇ 5 are deleted from ⁇ HBc # 1 and includes ⁇ 2 to ⁇ 4b helices. In the examples, it is described as “ ⁇ 2-4b”.
- ⁇ HBc # 8 consists of position 1 (starting methionine) and positions 44 to 111 of SEQ ID NO: 16.
- ⁇ HBc # 9 consists of position 1 (starting methionine) and positions 74 to 111 of SEQ ID NO: 16.
- a structure in which ⁇ 1 to ⁇ 3 and ⁇ 5 are deleted from ⁇ HBc # 1 and includes ⁇ 4a and ⁇ 4b. In the examples, it is described as “ ⁇ 4ab”.
- ⁇ HBc # 10 consists of positions 1 (starting methionine) and 111-144 of SEQ ID NO: 16. This is a structure in which ⁇ 1 to ⁇ 4b are deleted from ⁇ HBc # 1 and includes only ⁇ 5. In the examples, it is written as "HR”. This HR corresponds to the hand region (HR) of HBc in which the spike region has been deleted from AD. (11) ⁇ HBc # 11 consists of position 1 (starting methionine) and positions 111 to 183 of SEQ ID NO: 16. It is a structure in which ⁇ 1 to ⁇ 4b is deleted from HBc, and contains ⁇ 5 corresponding to HR and an RNA / DNA binding domain (RDBD). In the examples, it is described as "HR-RDBD”.
- HBc / C gene represented by SEQ ID NO: 22 in which the codon of the DNA sequence encoding full length HBc / C is optimized for human cells
- the region encoding each ⁇ HBc is excised and the mammal shown in FIG.
- pCI Promega
- the expression vector thus obtained is ⁇ HBc expression vector (pCI- ⁇ HBc), for example, ⁇ HBc expression vector incorporating ⁇ 1-4b of ⁇ HBc # 2 is ⁇ HBc ( ⁇ 1-4b) expression vector (pCI- ⁇ HBc ( ⁇ 1-4b)).
- an HBV replication activity evaluation vector (pBB-intron) encoding the reporter pgRNA shown in FIG. 5A, an HBV-P expression vector (pCI-HBV-Pol) shown in FIG. 5B, and an HBc expression vector shown in FIG. 5C.
- PCI-HBc the HBV replication activity evaluation system consisting of the HBx expression vector (pCI-HBx) shown in FIG. 5D were introduced into HeLa cells together with each pCI- ⁇ HBc.
- ⁇ HBc is about 3 times (2.89 times) the amount of wild-type HBc that acts on HBV replication.
- empty vector pCI
- the electroporation method was used for gene transfer into HeLa cells. Using an electroporator Nepa21 (Neppagene), 10 ⁇ g of DNA was introduced into about 1 ⁇ 10 6 cells under the condition of 125 V / 2.5 ms plus length. The HeLa cells after gene transfer were cultured in 2 mL of 10% FBS-added DMEM in the presence of 5% CO 2 at 37 ° C. for 24 hours.
- RNA-pol II of HeLa cells synthesizes the pre-mRNA shown in FIG. 6A, and then pBB-in the HeLa cells by pre-mRNA splicing.
- the intron contained in the intron reporter sequence is immediately removed.
- the mature mRNA with the intron spliced out becomes the reporter pgRNA shown in FIG. 6B.
- the reporter pgRNA is reverse transcribed by the action of HBV-Pol, HBc, and HBx expressed from the HBV-P expression vector, the HBc expression vector, and the HBx expression vector, respectively, and is shown in FIG. 6A as shown in FIG. 6C.
- the amount of this reporter ( ⁇ ) DNA reflects the replication activity and replication amount of HBV. Therefore, by extracting the DNA from the cells introduced with the HBV replication activity evaluation system and pCI- ⁇ HBc, and quantifying the amount with a primer set specific to the reporter (-) DNA, by comparing with the amount of the positive control, The effect of pCI- ⁇ HBc on HBV replication can be evaluated.
- PrimerF forward primer
- PrimerR reverse primer
- PrimerF is designed so that the two bases at the 3'end match the two bases at the 5'end in the downstream exon, but not the two bases at the 5'end of the intron. Therefore, it functions as a primer only when a reporter (-) DNA having a reporter sequence with an intron removed is present, and a 131-base DNA fragment is amplified.
- Example 2 Quantitative effect of HBV replication inhibition by spike region> (Purpose) It is verified whether the spiked region inhibits HBV replication in a dose-dependent manner.
- FIG. 8 shows the results. As shown in this figure, replication of HBV was suppressed in a dose-dependent manner of pCI- ⁇ HBc ( ⁇ 1-4b) to be introduced, and about 3 times the amount (26/9 amount) of ⁇ HBc ( ⁇ 1- It was revealed that when 4b) was introduced, the replication activity of HBV was almost lost. From this result, it was shown that the spike region of HBc inhibits HBV replication depending on its expression level.
- Example 3 Effect of HBV replication inhibitory activity of spike region derived from each HBc genotype> (Purpose) There are eight genotypes (HBc / A to HBc / H) in HBc. It is verified that the effect of HBV replication inhibitory activity by the spiked region of HBc / C obtained in Example 1 can be obtained also by spiked regions of other genotypes.
- the wild type nucleotide sequence information of each genotype of the HBc gene is the nucleotide sequence shown in SEQ ID NO: 25, the HBc / D gene Is based on the nucleotide sequence represented by SEQ ID NO: 26, the HBc / E gene is represented by SEQ ID NO: 27, and the HBc / F gene is represented by SEQ ID NO: 28, using pCI- ⁇ HBc ( ⁇ 1-4b) as a template.
- Sitedirected mutagenesis (PrimeSTAR Mutagenesis BasalKit, TaKaRa) was used to introduce amino acid substitutions to generate ⁇ HBc ⁇ 1-4b expression plasmids derived from genotypes A, D, E and F.
- FIG. 9 shows the results. As shown in this figure, a remarkable HBV replication inhibitory activity was observed in all genotype spike regions. This result revealed that the spiked region of HBc has HBV inhibitory activity regardless of genotype.
- Example 4 Verification of HBV nucleocapsid formation inhibition and HBV replication inhibition by amino acid point mutation of HBc> (Purpose) From the structural analysis of HBc, phenylalanine (F) at the 23rd position (35th position in HBc / G) or leucine (L) at the 42nd position (54th position in HBc / G) in the spike region ( ⁇ 1-4b) was replaced with alanine (L). It has been reported that the substitution of A) can inhibit the nucleocapsid formation of HBc (Alexander CG, et al., 2013, PNAS, 110 (30): E2782-E2791). Therefore, it is examined whether or not these point mutations have an inhibitory effect on nucleocapsid formation and HBV replication.
- Anti-HBc monoclonal antibody for HBc detection by Western blotting or the like was prepared. Synthesized a peptide represented by SEQ ID NO: 29 (PAYRPPNAPILSTLP) corresponding to positions 130 to 144 in the HBc / C hand region, and immunized BALB / c mice (8 weeks old, female) with the synthetic peptide. Then, a mouse anti-human HBc monoclonal antibody (# 511) was prepared by a hybridoma method using spleen cells of a mouse immunized by a conventional method.
- HBV replication activity was based on the method described in Example 1. However, in this example, the HBc expression vector (pCI-HBc) contained in the HBV replication evaluation system described in Example 1 was replaced with HBc-F23A or HBc-L42A.
- FIG. A is the result of Western blotting under non-reducing conditions.
- M monomer band
- Mlt multimer band
- D dimer
- C is a result of examining the inhibitory effect of HBV replication by introducing HBc-F23A or HBc-L42A into cells in place of HBc-WT in the HBV replication activity evaluation system. From the results of A, it was revealed that HBc-F23A has almost completely lost the HBV replication activity, although there was no significant difference in the ability to form multimers from HBc-WT. Although HBc-L42A retains the replication activity, it was revealed to be significantly attenuated as compared with that of HBc-WT. From the above results, it was clarified that the introduction of the F23A or L42A point mutation into the full-length wild type HBc affects the HBV replication inhibitory activity.
- ⁇ Example 5 Verification of HBV replication inhibitory activity of known amino acid mutations that inhibit HBc nucleocapsid formation> (Purpose) It is examined how the introduction of the F23A or L42A mutation into ⁇ HBc ( ⁇ 1-4b) affects the HBV replication inhibitory activity of ⁇ HBc ( ⁇ 1-4b).
- ⁇ HBc ( ⁇ 1-4b) Expression Vector Introduced with F24A or L42A
- genotype C ⁇ HBc ( ⁇ 1-4b) expression vector (pCI- ⁇ HBc ( ⁇ 1-4b)) used in Example 1 The 23rd F residue or the 42nd L residue was replaced with an A residue by a site-directed mutagenesis method using PrimeSTAR Mutagenesis Basal Kit (TaKaRa) (represented as F23A and L42A, respectively).
- ⁇ HBc ( ⁇ 1-4b) introduced with F23A or L42A is designated as ⁇ HBc ( ⁇ 1-4b) -F23A and ⁇ HBc ( ⁇ 1-4b) -L42A, respectively.
- HBV replication activity of HBc having a point mutation introduced was based on the method described in Example 1.
- wild-type pCI- ⁇ HBc ( ⁇ 1-4b), point mutant pCI- ⁇ HBc ( ⁇ 1-4b) -F23A or pCI- ⁇ HBc ( ⁇ 1- 4b) -L42A was introduced into HeLa cells.
- pCI- ⁇ HBc ( ⁇ 1-4b) and pCI- ⁇ HBc ( ⁇ 1-4b) -F23A the plasmid to be introduced was serially diluted with an empty vector (pCI) in the same manner as in Example 2 to obtain the respective HBV. The dose-dependent effect of replication inhibition was verified.
- FIG. A shows the result of HBV replication activity evaluation. From this figure, pCI- ⁇ HBc ( ⁇ 1-4b) -F23A, and pCI- ⁇ HBc ( ⁇ 1-4b) -L42A was introduced, the inhibitory effect of HBV replication activity was maintained, pCI- ⁇ HBc ( In the cells into which ⁇ 1-4b) -L42A had been introduced, the HBV replication inhibitory activity was weaker than that of wild-type pCI- ⁇ HBc ( ⁇ 1-4b).
- HeLa cells were introduced with pCI- ⁇ HBc ( ⁇ 1-4b) -PA alone or pCI- ⁇ HBc ( ⁇ 1-4b) -PA and pCI- ⁇ HBc ( ⁇ 1-4b) -F23A-PA at a ratio of 1: 1. .
- the anti-HBc monoclonal antibody # 511 and the anti-PA monoclonal antibody NZ-1 (Fujifilm Wako Pure Chemical Industries) shown in Example 4 were used.
- Anti-HBc monoclonal antibody # 511 recognizes the hand region of HBc, and therefore only recognizes full-length HBc here, and the hand region is deleted ⁇ HBc ( ⁇ 1-4b) -PA and ⁇ HBc ( ⁇ 1-4b) -F23A- PA does not recognize.
- the anti-PA monoclonal antibody NZ-1 recognizes the PA tag, it recognizes only ⁇ HBc ( ⁇ 1-4b) -PA and ⁇ HBc ( ⁇ 1-4b) -F23A-PA, and recognizes full-length HBc without PA tag. do not do.
- Particle blotting The basic operation was in accordance with the method described in Example 4.
- the introduction ratio of pCI- ⁇ HBc ( ⁇ 1-4b) -PA and pCI- ⁇ HBc ( ⁇ 1-4b) -F23A-PA was 1: 3.
- HeLa cells were lysed with 300 ⁇ L of TNE buffer (10 mM Tris pH8.0, 100 mM NaCl, 1 mM EDTA).
- 100 ⁇ L of 4 x PNE buffer (26% PEG 8000, 1.4 M NaCl, 40 mM EDTA) was added to the sample, incubated on ice for 2 hours, and then centrifuged at 15,000 rpm for 15 minutes at 4 ° C., and then in Example 4.
- Particle blotting was performed by the method described.
- FIG. A The effect of the expression of ⁇ HBc ( ⁇ 1-4b) or ⁇ HBc ( ⁇ 1-4b) -F23A added with a PA tag on dimerization and multimerization of HBc was measured using anti-HBc monoclonal antibody # 511. It is the result of analysis by Western blotting. From these results, in cells expressing only full-length HBc (lanes 1 to 3), full-length HBc exists as a multimer (Mlt) of dimer (D) or more, and monomer (M) Hardly detected.
- Mlt multimer of dimer
- M monomer
- HBc-T33N HBc with a T33N mutation
- HBV replication inhibition efficiency The measurement of HBV replication activity by HBc-WT or HBc-T33N was carried out by using HBc-WT or HBc-T33N as HBc contained in the HBV replication evaluation system described in Example 1, It was performed according to the method described in Example 1 in the presence of GLS4 at a concentration of 0 ⁇ M, 0.08 ⁇ M, 0.16 ⁇ M, 0.31 ⁇ M, 0.63 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, or 5 ⁇ M. The HBV replication inhibitory efficiency (%) was calculated based on the obtained measurement value of HBV replication activity (amount of reverse-transcribed HBV DNA).
- HBV replication inhibition efficiency (%) in the presence of a specific concentration of GLS4 is “0% inhibition” of reverse transcribed HBV DNA amount when GLS4 concentration is 0 ⁇ M (GLS4 is not added), and reverse transcribed HBV DNA amount. Is a value calculated as “100% inhibition” when 0 is 0 (when reverse transcription does not occur at all).
- FIG. 13 shows the results of examining the inhibitory effect of HBV replication in the presence of various concentrations of GLS4 by transfecting HBc-WT or HBc-T33N in place of HBc-WT in a cell using an HBV replication activity evaluation system.
- GLS4 inhibited HBV replication by HBc-WT in a concentration-dependent manner, while showing no inhibitory effect on HBV replication by HBc-T33N, indicating that HBc-T33N is resistant to GLS4. It was From this result, it was shown that by using HBc-T33N in place of HBc-WT in the HBV replication activity evaluation system, replication of nucleocapsid formation inhibitor resistant virus is reproduced.
- ⁇ Example 8 Examination of inhibitory effect of spike region ⁇ HBc ( ⁇ 1-4b) on HBV replication by HBc-T33N> (Purpose) It is verified whether the spiked region ⁇ HBc ( ⁇ 1-4b) inhibits HBV replication by nucleocapsid formation inhibitor resistant mutant HBc (HBc-T33N).
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Abstract
Description
(I)HBVのコアタンパク質におけるスパイク領域を構成するペプチド断片
(II)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片
(III)前記(I)又は(II)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクター
(2)前記スパイク領域を構成するペプチド断片が以下の(a)~(c)のいずれかのアミノ酸配列からなる、(1)に記載のHBV複製阻害剤。
(a)配列番号1~7で示すいずれかのアミノ酸配列
(b)配列番号1~7で示すいずれかのアミノ酸配列において、1又は複数個のアミノ酸が付加、欠失、又は置換されたアミノ酸配列
(c)配列番号1~7で示すいずれかのアミノ酸配列と82%以上のアミノ酸同一性を有するアミノ酸配列
(3)前記核酸が以下の(i)~(iv)のいずれかの塩基配列からなる、(1)に記載のHBV複製阻害剤。
(i)配列番号8~14で示すいずれかの塩基配列
(ii)配列番号8~14で示すいずれかの塩基配列において、1又は複数個の塩基が付加、欠失、又は置換された塩基配列
(iii)配列番号8~14で示すいずれかの塩基配列と80%以上の塩基同一性を有する塩基配列
(iv)配列番号8~14で示すいずれかの塩基配列に相補的な塩基配列と高ストリンジェントな条件下でハイブリダイズする塩基配列
(4)配列番号1~6で示すいずれかのアミノ酸配列において、23位のフェニルアラニン(F)残基がアラニン(A)残基に、及び/又は42位のロイシン(L)残基がアラニン(A)残基に、又は配列番号7で示すアミノ酸配列において、35位のフェニルアラニン(F)残基がアラニン(A)残基に、及び/又は54位のロイシン(L)残基がアラニン(A)残基に、置換された、(2)に記載のHBV複製阻害剤。
(5)以下の(I)~(III)のいずれかからなるHBVヌクレオカプシド形成阻害剤。
(I)HBVのコアタンパク質におけるスパイク領域を構成するペプチド断片
(II)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片
(III)前記(I)又は(II)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクター
(6)有効成分としての(1)~(4)のいずれかに記載のHBV複製阻害剤、及び担体及び/又は溶媒を含むB型肝炎治療用医薬組成物。
(7)抗HBV剤をさらに含む、(6)に記載のB型肝炎治療用医薬組成物。
(8)抗HBV剤が核酸アナログ及び/又はHBV-Pol活性阻害剤である、(7)に記載のB型肝炎治療用医薬組成物。
(9)(I)HBVのコアタンパク質におけるスパイク領域を構成するペプチド断片、(II)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片、又は前記(I)又は(II)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクターを宿主内に導入する工程を含むHBV複製阻害方法。
本明細書は本願の優先権の基礎となる日本国特許出願番号2018-193812号の開示内容を包含する。
1-1.概要
本発明の第1の態様はHBV複製阻害剤である。本発明のHBV複製阻害剤は、コアタンパク質(HBc)のスパイク領域を構成するペプチド断片、スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片、又は細胞内でスパイク領域を発現可能な発現ベクターからなる。本発明のHBV複製阻害剤は、肝細胞におけるHBVの増殖抑制効果の高いB型肝炎治療用医薬組成物の有効成分となり得る。
本明細書で使用する用語について、以下で定義する。
「B型肝炎ウイルス(hepatitis B virus:HBV)」とは、ヘパドナウイルス科オルソヘパドナウイルス属に属するDNAウイルスで、B型肝炎の原因ウイルスである。HBVは、遺伝子配列の違いにより8種類の遺伝子型(ジェノタイプA、B、C、D、E、F、G、及びH)が知られている。これらの遺伝子型には、地域分布や病態面で差異が見られる。例えば、日本では、従来ジェノタイプC(本明細書では、しばしば「HBV/C」と表記する。他のジェノタイプについても同様とする。)感染者が大半を占め、次いでHBV/B感染者が多くみられていたが、近年ではHBV/A感染者が増加している。一方、欧米ではHBV/AやHBV/Dの感染者が多くみられる。HBV/Aでは、急性肝炎罹患後の約20~30%が慢性肝炎に移行することが知られているが、HBV/BやHBV/Cは急性肝炎罹患後の慢性化率は低い。
「C遺伝子」とは、コアタンパク質をコードする遺伝子で、前述のようにHBVゲノムにコードされた4種類の遺伝子のうちの1つである。
本発明のHBV複製阻害剤は、ペプチド断片又は発現ベクターからなる。それぞれの構成を以下で具体的に説明する。
本発明のHBV複製阻害剤を構成する「ペプチド断片」は、HBVのスパイク領域、又はそのスパイク領域のN末端及び/又はC末端に前記HBcとは異なる任意のアミノ酸配列が付加されたペプチド断片からなる。スパイク領域は、前述のように、HBcのアッセンブリドメインのN末端側に含まれる構成要素である。HBc/A~HBc/F、及びHBc/Hのスパイク領域は、HBcの1位~111位からなる111アミノ酸残基に、またHBc/Gのスパイク領域はHBcの1位~123位からなる123アミノ酸残基に相当する。各ジェノタイプ間におけるスパイク領域のアミノ酸は高度に保存されており、HBc/A~HBc/F、及びHBc/H間であれば、アミノ酸類似性で95%以上、アミノ酸同一性で84%以上を有する。
本発明のHBV複製阻害剤を構成する「発現ベクター」は、前記ペプチド断片をコードする核酸、及びプロモーターを含み、細胞内で前記スパイク領域を発現可能な発現ベクターである。発現ベクターは、前記構成要素である核酸及びプロモーターに加えて、必要に応じて、標識遺伝子(選抜マーカー)、エンハンサー、ターミネーター、複製起点、及びポリAシグナル等の構成要素を含んでいてもよい。以下、各構成要素について説明をする。
「HBcのスパイク領域を構成するペプチド断片をコードする核酸」は、前記各ジェノタイプのいずれかのスパイク領域をコードする核酸を含むものであればよい。そのような核酸の塩基配列として、限定はしない。例えば、ゲノム上にコードされた各ジェノタイプの遺伝子の塩基配列が挙げられる。具体的には、アクセッションNo.AY707087.1のHBc/Aのスパイク領域をコードする核酸の塩基配列、アクセッションNo.GU357842.1のHBc/Bのスパイク領域をコードする核酸の塩基配列、アクセッションNo.AB033556.1のHBc/Cのスパイク領域をコードする核酸の塩基配列、アクセッションNo.GU357846.1のHBc/Dのスパイク領域をコードする核酸の塩基配列、アクセッションNo.X75664.1のHBc/Eのスパイク領域をコードする核酸の塩基配列、アクセッションNo.JN792913.1のHBc/Fのスパイク領域をコードする核酸の塩基配列、アクセッションNo.AB625342.1のHBc/Gのスパイク領域をコードする核酸の塩基配列、及びアクセッションNo.AP007261.1のHBc/Hのスパイク領域をコードする核酸の塩基配列である。また、上記ゲノム上の遺伝子のコドンをヒト細胞での発現に最適化した塩基配列であってもよい。そのような具体例として、配列番号8で示すHBc/Aのスパイク領域をコードする核酸の塩基配列、配列番号9で示すHBc/B又はHBc/Cのスパイク領域をコードする核酸の塩基配列、配列番号10で示すHBc/Dのスパイク領域をコードする核酸の塩基配列、配列番号11で示すHBc/Eのスパイク領域をコードする核酸の塩基配列、配列番号12で示すHBc/Fのスパイク領域をコードする核酸の塩基配列、配列番号13で示すHBc/Hのスパイク領域をコードする核酸の塩基配列、又は配列番号14で示すHBc/Gのスパイク領域をコードする核酸の塩基配列が挙げられる。なお、配列番号8~14には、スパイク領域のみを発現させるために、それぞれの3'末端に終始コドン(TGA)が付加されている。その他、上記いずれかの塩基配列において、1又は複数個の塩基が付加、欠失、又は置換された塩基配列や、上記いずれかの塩基配列と80%以上、82%以上、85%以上、88%以上、90%以上、93%以上、95%以上、98%以上、99%以上の塩基同一性を有する塩基配列、又は上記いずれかの塩基配列に相補的な塩基配列と高ストリンジェントな条件下でハイブリダイズする塩基配列が挙げられる。
本明細書においてプロモーターは、前記ペプチド断片をコードする核酸を細胞内で発現誘導できるプロモーターである。本発明のHBV複製阻害剤を適用する、すなわち発現ベクターを導入する標的細胞は、原則として哺乳動物細胞、特にヒト又はチンパンジー由来の細胞であることから、それらの細胞内で下流の遺伝子を発現できるプロモーターであればよい。例えば、CMVプロモーター(CMV-IEプロモーター)、SV40初期プロモーター、RSVプロモーター、EF1αプロモーター、Ubプロモーター等が挙げられる。
本明細書において「標識遺伝子」は、選抜マーカー又はレポータータンパク質とも呼ばれる標識タンパク質をコードする遺伝子である。「標識タンパク質」とは、その活性に基づいて標識遺伝子の発現の有無を判別することのできるペプチドをいう。活性の検出は、標識タンパク質の活性そのものを直接的に検出するものであってもよいし、色素のような標識タンパク質の活性によって発生する代謝物を介して間接的に検出するものであってもよい。検出は、生物学的検出(抗体、アプタマー等のペプチドや核酸の結合による検出を含む)、化学的検出(酵素反応的検出を含む)、物理的検出(行動分析的検出を含む)、又は検出者の感覚的検出(視覚、触覚、嗅覚、聴覚、味覚による検出を含む)のいずれであってもよい。
本明細書において「エンハンサー」は、ベクター内の遺伝子又はその断片の発現効率を増強できるものであれば特に限定はされない。
本明細書において「ターミネーター」は、前記プロモーターの活性により発現した遺伝子等の転写を終結できる配列である。ターミネーターの種類は、特に限定はしない。好ましくはプロモーターと同一生物種由来のターミネーターである。一遺伝子発現制御系においてゲノム上で前記プロモーターと対になっているターミネーターは特に好ましい。
本発明における発現ベクターは、HBcのスパイク領域を哺乳動物細胞内で一過的に発現することができればよい。したがって、哺乳動物細胞用の複製起点は不要である。しかし、シャトルベクターとして、例えば、大腸菌等の細菌内で発現させる場合には、その複製起点が必須となる。複製起点は、公知の配列を利用することができる。例えば、大腸菌用の複製起点であればf1 origin等を利用すればよい。
HBVヌクレオカプシド形成阻害剤は、HBcを標的分子とする。HBcは、前述のように、HBVゲノム複製に不可欠なタンパク質であると同時に、HBV粒子の骨幹をなすヌクレオカプシドの主成分でもある。つまり、HBVヌクレオカプシド形成の阻害効果は、同時にHBVの複製阻害効果をもたらす。本明細書の実施例においても、その効果は実証されている。したがって、本発明のHBV複製阻害剤は、HBVヌクレオカプシド形成阻害剤としても機能し得る。HBVヌクレオカプシド形成阻害剤の構成は、HBV複製阻害剤に記載の構成と同一で良い。
2-1.概要
本発明の第2の態様は、B型肝炎治療用医薬組成物である。本発明のB型肝炎治療用医薬組成物は、前記第1態様のHBV複製阻害剤を必須の有効成分とし、HBV感染後のHBVの複製を阻害することでHBVの増殖を抑制し、B型肝炎を治療することができる。
2-2-1.構成成分
本発明のB型肝炎治療用医薬組成物の構成成分について説明をする。本発明のB型肝炎治療用医薬組成物は、必須の構成成分として一以上の有効成分、及び溶媒及び/又は担体、さらに薬剤送達系(DDS;Drug Delivery System)粒子を含む。以下、各構成成分について具体的に説明をする。
本発明のB型肝炎治療用医薬組成物は、必須の有効成分として第1態様に記載のHBV複製阻害剤を包含する。また、必要に応じて、1又は複数の抗B型肝炎ウイルス剤(抗HBV剤)を包含していてもよい。
必須の有効成分であるHBV複製阻害剤の構成については、第1態様で詳述していることから、ここでの具体的な説明は省略する。本発明のB型肝炎治療用医薬組成物は、1種又は複数種のHBV複製阻害剤を含むことができる。
本発明のB型肝炎治療用医薬組成物は、必要に応じて薬学的に許容可能な溶媒を含むことができる。「薬学的に許容可能な溶媒」とは、製剤技術分野において通常使用する溶媒をいう。例えば、水若しくは水溶液、又は有機溶剤が挙げられる。水溶液には、例えば、生理食塩水、ブドウ糖又はその他の補助剤を含む等張液、リン酸塩緩衝液、酢酸ナトリウム緩衝液が挙げられる。補助剤には、例えば、D-ソルビトール、D-マンノース、D-マンニトール、塩化ナトリウム、その他にも低濃度の非イオン性界面活性剤、ポリオキシエチレンソルビタン脂肪酸エステル類等が挙げられる。有機溶剤には、エタノールが挙げられる。
本発明のB型肝炎治療用医薬組成物は、必要に応じて薬学的に許容可能な担体を含むことができる。「薬学的に許容可能な担体」とは、製剤技術分野において通常使用する添加剤をいう。例えば、賦形剤、結合剤、崩壊剤、充填剤、乳化剤、流動添加調節剤、滑沢剤、ヒト血清アルブミン等が挙げられる。
本発明のB型肝炎治療用医薬組成物は、必要に応じてDDS粒子を含むことができる。DDS粒子は、その内部等に有効成分や他の担体等を包含して、標的部位にまで内容物、特に有効成分を分解させることなく送達し、また生体内での薬物分布を時間的に、量的に制御し得る粒子をいう。本発明のB型肝炎治療用医薬組成物の有効成分はペプチド又は核酸であることから、投与後に生体内でプロテアーゼやヌクレアーゼによる分解から保護するためにも、DDS粒子の使用は好適である。DDS粒子の種類は問わない。例えば、リポソーム、高分子ミセル、ウイルス粒子等が挙げられる。
本発明のB型肝炎治療用医薬組成物の剤形は、特に限定しない。被験体の体内で有効成分を失活させることなく目的の部位にまで送達される形態であればよい。
具体的な剤形は、後述する適用方法によって異なる。適用方法は、非経口投与と経口投与に大別することができるので、それぞれの投与法に適した剤形にすればよい。
本発明のB型肝炎治療用医薬組成物の適用方法は、経口投与でも、非経口投与でもよい。一般に経口投与法は全身投与となるが、非経口投与法は、さらに全身投与と局所投与に細分できる。局所投与には、例えば、筋肉内投与、皮下投与、組織投与、及び器官投与が該当し、非経口投与法の全身投与には、循環器内投与、例えば、静脈内投与(静注)、動脈内投与及びリンパ管内投与が挙げられる。本発明のB型肝炎治療用医薬組成物を局所投与する場合には、注射等で肝臓に直接投与すればよい。また、全身投与する場合には、静注等の循環器内に投与すればよい。投与量は、有効成分が奏効する上で有効な量であればよい。有効量は、前述のように被験体情報に応じて適宜選択される。
3-1.概要
本発明の第3の態様は、HBV複製阻害方法である。本発明のHBV複製阻害方法は、HBV感染細胞若しくはその恐れのある細胞に、(I)B型肝炎ウイルスのコアタンパク質におけるスパイク領域を構成するペプチド断片、(II)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片、又は(III)前記(I)又は(II)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクターを導入することで細胞内におけるHBVの複製を阻害し、それによってHBVの複製を阻害し、増殖を抑制することができる。また、この方法をB型肝炎罹患患者若しくはその罹患の恐れがある者に投与すれば、B型肝炎の治療方法となり得る。
本態様におけるHBV複製阻害方法は、必須の工程として導入工程を含む。
「導入工程」は、(I)HBcのスパイク領域を構成するペプチド断片、(II)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片、又は(III)前記(I)又は(II)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクター、すなわち、第1態様に記載のHBV複製阻害剤を宿主内に導入する工程である。
(目的)
HBcを様々な形で欠失させた変異型HBcを作製し、HBV複製に対する阻害効果を検証する。
1.欠失変異型HBcの作製
アミノ酸配列番号16で示されるHBcのジェノタイプC(HBc/C)を検証対象として、図4に示す以下の11種類の欠失変異型HBc(ΔHBc)を作製した。
(1)ΔHBc#1は、RNA/DNA結合ドメイン(RDBD)欠失型で、配列番号16の1~144位からなるアッセンブリドメインのみで構成されている。実施例では「AD」と表記する。
(2)ΔHBc#2は、配列番号16の1~111位からなる。ΔHBc#1から第5αヘリックス(α5)が欠失した構造で、α1~α4bを含む。実施例では「α1-4b」と表記する。このα1-4bは、ADからハンド領域(HR)が欠失したHBcのスパイク領域に相当する。
(3)ΔHBc#3は、配列番号16の1~91位からなる。ΔHBc#1からα4b~α5が欠失した構造で、α1~α4aを含む。実施例では「α1-4a」と表記する。
(4)ΔHBc#4は、配列番号16の1~78位からなる。ΔHBc#1からα4a~α5が欠失した構造で、α1~α3を含む。実施例では「α1-3」と表記する。
(5)ΔHBc#5は、配列番号16の1~49位からなる。ΔHBc#1からα3~α5が欠失した構造で、α1~α2を含む。実施例では「α1-2b」と表記する。
(6)ΔHBc#6は、配列番号16の1~26位からなる。ΔHBc#1からα2~α5が欠失した構造で、α1のみを含む。実施例では「α1」と表記する。
(7)ΔHBc#7は、配列番号16の1位(開始メチオニン)及び18~111位からなる。ΔHBc#1からα1及びα5が欠失した構造で、α2~α4bヘリックスを含む。実施例では「α2-4b」と表記する。
(8)ΔHBc#8は、配列番号16の1位(開始メチオニン)及び44~111位からなる。ΔHBc#1からα1、α2、及びα5が欠失した構造で、α3~α4bを含む。実施例では「α3-4b」と表記する。
(9)ΔHBc#9は、配列番号16の1位(開始メチオニン)及び74~111位からなる。ΔHBc#1からα1~α3、及びα5が欠失した構造で、α4a及びα4bを含む。実施例では「α4ab」と表記する。
(10)ΔHBc#10は、配列番号16の1位(開始メチオニン)及び111~144位からなる。ΔHBc#1からα1~α4bが欠失した構造で、α5のみを含む。実施例では「HR」と表記する。このHRは、ADからスパイク領域が欠失したHBcのハンド領域(HR)に相当する。
(11)ΔHBc#11は、配列番号16の1位(開始メチオニン)及び111~183位からなる。HBcからα1~α4bが欠失した構造で、HRに相当するα5と、RNA/DNA結合ドメイン(RDBD)とを含む。実施例では「HR-RDBD」と表記する。
前記各ΔHBc発現ベクター(pCI-ΔHBc)によるHBV複製への影響は、本発明者らが開発し、WO2018/030534に記載のHBV複製活性評価システムを用いて検証した。
図7A~Cに結果を示す。これらの図からα1-4bで示すスパイク領域を導入したHeLa細胞においてのみ、HBVの複製が著しく抑制されることが明らかとなった。一方、スパイク領域のα1-4bからさらにC末端側のアミノ酸配列を欠失させたΔHBc(α1-4a、α1-3、α1-2b、及びα1)(図7A)や、α1-4bのN末端側のアミノ酸配列を欠失させたΔHBc(α2-4b、α3-4b、及びα4ab)(図7B)では、HBV複製阻害活性が消失した。これらの結果から、HBVの複製阻害活性には、スパイク領域に含まれる全てのαヘリックス(α1~α4b)が必要であることが明らかとなった。
(目的)
スパイク領域がHBV複製を量依存的に阻害するか否かを検証する。
基本的な操作は実施例1に準じた。HeLa細胞にHBV複製活性評価システムと共に導入するpCI-ΔHBcとしてpCI-ΔHBc(α1-4b)のみを用いた。各発現ベクターの導入比率は、pCI-HBc:pCI-HBV-Pol:pCI-HBxの比率が9:3:1で、かつpBB-intron:pCI-HBc/pCI-HBV-Pol/pCI-HBxの比率が1:1となるように設定した上で、pCI-ΔHBc(α1-4b):pBB-intron/pCI-HBc/pCI-HBV-Pol/pCI-HBxの比率が1:26、3:26、9:26、及び26:26に調整して導入したHBV複製活性評価システムのみを陽性対照として導入した。各試料で、導入するベクター量が同一となるように空ベクター(pCI)で調製した。
図8に結果を示す。この図で示すように、導入するpCI-ΔHBc(α1-4b)の量依存的にHBVの複製が抑制され、野生型HBcに対して約3倍量(26/9量)のΔHBc(α1-4b)を導入するとHBVの複製活性がほとんど失われてしまうことが明らかとなった。この結果からHBcのスパイク領域は、その発現量に依存してHBV複製を阻害することが示された。
(目的)
HBcには8種類のジェノタイプ(HBc/A~HBc/H)が存在する。実施例1で得られたHBc/Cのスパイク領域によるHBV複製阻害活性の効果が他のジェノタイプのスパイク領域でも得られることを検証する。
基本的な操作は、実施例1に準じた。HBc/C以外のジェノタイプとして、HBc/A、HBc/D、HBc/E、及びHBc/Fを用いた。なお、HBc/Bは、HBc/Cとアミノ酸配列が完全一致することから、HBc/B/Cとして対照用に用いた。
図9に結果を示す。この図で示すように、いずれのジェノタイプのスパイク領域も著しいHBV複製阻害活性が認められた。この結果は、HBcのスパイク領域はジェノタイプに関係なく、HBV阻害活性を有することが明らかとなった。
(目的)
HBcの構造解析からスパイク領域(α1-4b)内の23位(HBc/Gでは35位)のフェニルアラニン(F)、又は42位(HBc/Gでは54位)のロイシン(L)をそれぞれアラニン(A)に置換することで、HBcのヌクレオカプシド形成が阻害され得ることが報告されている(Alexander C.G., et al., 2013, PNAS, 110(30): E2782-E2791)。そこで、これらの点変異がヌクレオカプシド形成、及びHBV複製に関して阻害効果を有するか否かについて検証する。
(1)F24A又はL42Aを導入したHBc発現ベクターの作製
実施例1で用いたジェノタイプCの全長HBc発現ベクター(pCI-HBc)を鋳型として、PrimeSTAR Mutagenesis Basal Kit(TaKaRa)を用いた部位特異的変異誘発法により、23番目のF残基又は42番目のL残基をそれぞれA残基に置換した(それぞれF23A及びL42Aと表記する)。点変異導入をした全長HBcのF23A及びL42A変異体は、それぞれHBc-F23A及びHBc-L42Aとする。
(2)抗HBcモノクローナル抗体
ウェスタンブロッティング等でのHBc検出用の抗HBcモノクローナル抗体を作製した。は、HBc/Cのハンド領域において130~144位に相当する配列番号29(PAYRPPNAPILSTLP)で示されるペプチドを合成し、その合成ペプチドでBALB/cマウス(8週齢、雌)を免疫した。その後、常法により免疫したマウスの脾臓細胞を用いたハイブリドーマ法によりマウス抗ヒトHBcモノクローナル抗体(#511)を作製した。
(3)ウェスタンブロッティング
HeLa細胞に野生型の全長HBc(HBc-WT)、及び変異型全長HBc(HBc-F23A及びHBc-L42A)の各発現ベクターを導入し、24時間培養した後にWB lysis buffer (1%Triton、25mM Tris pH7.4、150mM NaCl)でタンパク質を抽出した。タンパク抽出液はCuSO4 (100μM)を添加して室温で20分間インキュベートした後、1mM EDTAで中和し、β-ME不含SDS-PAGE sample bufferを添加して、10~20%ポリアクリルアミドゲル(スーパーセップTMエース、富士フィルム和光純薬)を用いてSDS-PAGEを施した。電気泳動後、タンパク質をセミドライ式ブロッターによってPVDF膜に転写し、マウス抗ヒトHBcモノクローナル抗体(#511)を用いてウェスタンブロッティングを実施した。
(4)パーティクルブロッティング
HeLa細胞に野生型HBc(HBc-WT)、変異型全長HBc(HBc-F23A及びHBc-L42A)の各発現ベクターを遺伝子導入し、24時間培養後にPB lysis buffer(1% NP40、25mM Tris pH7.4、150mM NaCl、1mM EDTA、50mM NaF)で溶解した。サンプルは1.2%アガロース(TAE)に泳動し、PVDF膜に転写し、マウス抗ヒトHBcモノクローナル抗体(#511)を用いて検出した。
(5)HBV複製活性評価
点変異を導入したHBcのHBV複製活性評価は、実施例1に記載の方法に準じた。ただし、本実施例では、実施例1に記載のHBV複製評価システムに含まれるHBc発現ベクター(pCI-HBc)を、HBc-F23A又はHBc-L42Aに置き換えて実施した。
図10に結果を示す。Aは、非還元条件下でのウェスタンブロッティングの結果である。HBc-WTを導入した細胞では、HBcの単量体バンド(M)がほとんど存在せず、二量体(D)以上の多量体バンド(Mlt)が強く認められた。HBc-F23Aを導入した細胞も、HBc-WTを導入した細胞と同様に、単量体(M)がほとんど存在せず、二量体(D)以上の多量体バンド(Mlt)が強く認められた。この結果は、F23Aの変異は、HBcの多量体形成能を阻害しないことを示唆している。一方、HBc-L42Aを導入した細胞では、単量体バンド(M)が強く認められると共に二量体バンド(D)がHBc-WTやHBc-F23Aと比較して減弱しており、多量体形成能が阻害されているかと思われたが、HBc-WTやHBc-F23Aを導入した細胞と同程度の多量体バンド(Mlt)が認められた。Bは、パーティクルブロッティングの結果である。この結果から、HBc-WT、HBc-F23A、及びHBc-L42Aは、いずれもカプシド形成能を維持していることが示された。Cは、HBV複製活性評価システムで、HBc-WTに代えてHBc-F23A又はHBc-L42Aを細胞導入してHBV複製の阻害効果を検討した結果である。HBc-F23Aは、Aの結果から多量体形成能において、HBc-WTと著しい差異が認められなかったにもかかわらず、HBV複製活性はほぼ完全に失われていることが明らかとなった。HBc-L42Aは、複製活性を残しているものの、HBc-WTのそれと比較して、有意に減弱することが明らかとなった。以上の結果から、全長野生型のHBcにF23A又はL42Aの点変異を導入すると、HBV複製阻害活性に影響を及ぼすことが判明した。
(目的)
ΔHBc(α1-4b)にF23A又はL42A変異を導入した場合、ΔHBc(α1-4b)のHBV複製阻害活性にどのような影響を及ぼすかを検証する。
(1)F24A又はL42Aを導入したΔHBc(α1-4b)発現ベクターの作製
実施例1で用いたジェノタイプCのΔHBc(α1-4b)発現ベクター(pCI-ΔHBc(α1-4b))を鋳型として、PrimeSTAR Mutagenesis Basal Kit(TaKaRa)を用いた部位特異的変異誘発法により、23番目のF残基又は42番目のL残基をそれぞれA残基に置換した(それぞれF23A及びL42Aと表記する)。F23A又はL42Aを導入したΔHBc(α1-4b)を、それぞれΔHBc(α1-4b)-F23A及びΔHBc(α1-4b)-L42Aとする。
(2)HBV複製活性評価
点変異を導入したHBcのHBV複製活性評価は、実施例1に記載の方法に準じた。本実施例では、実施例1に記載のHBV複製評価システムと共に、野生型のpCI-ΔHBc(α1-4b)、点変異型のpCI-ΔHBc(α1-4b)-F23A又はpCI-ΔHBc(α1-4b)-L42AをHeLa細胞に導入した。
またpCI-ΔHBc(α1-4b)、及びpCI-ΔHBc(α1-4b)-F23Aについては、実施例2と同様の操作で、導入するプラスミドを空ベクター(pCI)で段階希釈し、それぞれのHBV複製阻害の量依存的効果を検証した。
図11に結果を示す。A:HBV複製活性評価の結果を示す。この図から、pCI-ΔHBc(α1-4b)-F23A、及びpCI-ΔHBc(α1-4b)-L42Aのいずれを導入した場合にもHBV複製活性の阻害効果は維持されたが、pCI-ΔHBc(α1-4b)-L42Aを導入した細胞では、野生型pCI-ΔHBc(α1-4b)に比してHBV複製阻害活性が減弱していた。一方、pCI-ΔHBc(α1-4b)-F23Aを導入した細胞では、野生型pCI-ΔHBc(α1-4b)を導入した細胞よりもHBV複製が強く阻害された。B: HBV複製阻害の量依存的効果の結果を示す。この結果らΔHBc(α1-4b)-F23AもΔHBc(α1-4b)と同様に、阻害発現量に依存してHBV複製を阻害することが示された。また、Aの結果と同様に、ΔHBc(α1-4b)-F23AがΔHBc(α1-4b)よりも強いHBV複製阻害活性を有することが確認された。
(目的)
スパイク領域ΔHBc(α1-4b)によるHBV複製阻害の作用機序について検証する。
(1)タグ付加ΔHBc発現ベクターの作製
野生型及びF23A変異型ΔHBc(α1-4b)発現ベクター(それぞれ、pCI-ΔHBc(α1-4b)及びpCI-ΔHBc(α1-4b)-F23A)を用いて、それぞれのΔHBc(α1-4b)のC末端に、配列番号30で示す12アミノ酸残基からなるPAタグ(GVAMPGAEDDVV)をコードする核酸断片を挿入した。得られたタグ付加ΔHBc発現ベクターをpCI-ΔHBc(α1-4b)-PA及びpCI-ΔHBc(α1-4b)-F23A-PAとした。
(2)HBV複製アッセイ
実施例1に記載のHBV複製阻害活性システムを用いてpCI-ΔHBc(α1-4b)-PA及びpCI-ΔHBc(α1-4b)-F23A-PAのHBV複製阻害活性を評価した。HBV複製阻害活性システムとΔHBc発現ベクターの細胞導入比率は26:26とした。その結果、ΔHBc(α1-4b)及びΔHBc(α1-4b)-F23AのC末端にPAタグが付加した場合であっても、HBV複製阻害活性が維持されることが示された(図示せず)。
(3)ウェスタンブロッティング
基本操作は、実施例4に記載の方法に準じた。HeLa細胞にpCI-ΔHBc(α1-4b)-PAを単独で、又はpCI-ΔHBc(α1-4b)-PAとpCI-ΔHBc(α1-4b)-F23A-PAを1:1の比率で導入した。ΔHBcの検出には、実施例4に示した抗HBcモノクローナル抗体#511と抗PAモノクローナル抗体NZ-1(富士フィルム和光純薬)を用いた。抗HBcモノクローナル抗体#511は、HBcのハンド領域を認識するため、ここでは全長HBcのみを認識し、ハンド領域が欠失したΔHBc(α1-4b)-PA及びΔHBc(α1-4b)-F23A-PAは認識しない。一方、抗PAモノクローナル抗体NZ-1は、PAタグを認識するため、ΔHBc(α1-4b)-PA及びΔHBc(α1-4b)-F23A-PAのみを認識し、PAタグのない全長HBcは認識しない。
(4)パーティクルブロッティング
基本操作は、実施例4に記載の方法に準じた。pCI-ΔHBc(α1-4b)-PAとpCI-ΔHBc(α1-4b)-F23A-PAの導入比率は1:3とした。24時間培養後に300μLのTNE buffer(10mM Tris pH8.0、100mM NaCl、1mM EDTA)でHeLa細胞を溶解した。サンプルに100μLの4 x PNE buffer(26% PEG 8000、1.4M NaCl、40mM EDTA)を添加し、氷上で2時間インキュベートした後、4℃にて15,000rpmで15分間遠心分離後、実施例4に記載の方法でパーティクルブロッティングを行った。
図12に結果を示す。A:PAタグを付加したΔHBc(α1-4b)又はΔHBc(α1-4b)-F23Aの発現が、HBcの二量体形成及び多量体形成に与える影響を、抗HBcモノクローナル抗体#511を用いたウェスタンブロッティングによって解析した結果である。この結果から、全長HBcのみを発現させた細胞(レーン1~3)では、全長HBcは二量体(D)以上の多量体(Mlt)として存在しており、単量体(M)は、ほとんど検出されなかった。一方、全長HBcとΔHBc(α1-4b)-PA(レーン4~6)及びΔHBc(α1-4b)-F23A-PA(レーン7~9)を共発現させた細胞では、全長HBcの二量体(D)のみならず、四量体(T)以上の多量体(Mlt)が減少する一方で、単量体(M)は多数検出された。この結果から、ΔHBc(α1-4b)及びΔHBc(α1-4b)-F23Aは、全長HBcの正常な多量体形成を阻害することが示唆された。B:Aと同様に、HBcの二量体形成及び多量体形成に与えるΔHBc(α1-4b)又はΔHBc(α1-4b)-F23A発現の影響を、抗PAモノクローナル抗体NZ-1を用いたウェスタンブロッティングによって解析した結果である。Aとは異なり、NZ-1では、PAタグのない全長HBcは認識されていない(レーン1~3)。一方、ΔHBc(α1-4b)-PA(レーン4~6)、及びΔHBc(α1-4b)-F23A-PA(レーン7~9)では、二量体(D)の他、四量体(T)以上も僅かに検出されたが、単量体(M)が圧倒的な量であった。C:Hela細胞でΔHBc(α1-4b)-PAを単独発現、又は全長HBcと共発現させた後、NZ-1抗体を用いたウェスタンブロッティングによる解析結果である。ΔHBc(α1-4b)は、単量体(M)又はホモ二量体(Homo-D)で存在しており、四量体(T)以上の多量体は形成しないことが示された。一方、全長HBcとΔHBc(α1-4b)を共発現させたHeLa細胞では、Bの結果と同様に、四量体(T)及び多量体(Mlt)も検出される他、二量体が2本のバンドで現れた。このうち一方は、ΔHBc(α1-4b)のホモ二量体のバンドと同位置にあり、他方のバンドはそれよりも上部に位置していた。つまり、全長HBcとΔHBc(α1-4b)を共発現させた場合、ΔHBc(α1-4b)は全長HBcとの間でヘテロ二量体(Hetero-D)を形成していることが示唆された。ΔHBc(α1-4b)は単独では二量体より大きな複合体を形成しないにもかかわらず、野生型HBcと共発現させると、野生型HBcとの間で少なくとも4量体以上の複合体を形成していることが明らかとなった。D:パーティクルブロットの結果からも、α1-4bの発現はカプシド形成を阻害することが明らかとなった。以上の結果から、細胞で発現したΔHBc(α1-4b)、すなわちHBcのスパイク領域は、機能的な全長HBcとヘテロ二量体を形成し、引き続く多量体形成、カプシド形成を阻害することによりHBVの複製を阻害することが明らかとなった。
(目的)
T33N変異を有するHBc(HBc-T33N)は、既存の低分子化合物で構成されるヌクレオカプシド形成阻害剤に対して強い抵抗性を示すことが報告されている(Zhou Z. et al., 2017, Sci Rep, 2017 Feb 13;7:42374.)。そこで、HBV複製活性評価システムにおいてHBc-WTに代えてHBc-T33Nを用いることにより、ヌクレオカプシド形成阻害剤耐性ウイルスの複製を再現し、低分子化合物で構成されるヌクレオカプシド形成阻害剤GLS4に対する抵抗性を示すか否かを検証する。
(1)T33N変異を導入したHBc発現ベクターの作製
実施例1で用いたジェノタイプCの全長HBc発現ベクター(pCI-HBc)を鋳型として、PrimeSTAR Mutagenesis Basal Kit(TaKaRa)を用いた部位特異的変異誘発法により、33番目のT残基をN残基に置換した(T33Nと表記する)。点変異導入をした全長HBcのT33N変異体は、HBc-T33Nとする。
(2)HBV複製阻害効率の評価
HBc-WT又はHBc-T33NによるHBV複製活性の測定は、実施例1に記載のHBV複製評価システムに含まれるHBcとしてHBc-WT又はHBc-T33Nを用いて、0μM、0.08μM、0.16μM、0.31μM、0.63μM、1.25μM、2.5μM、又は5μMの濃度のGLS4存在下で、実施例1に記載の方法に準じて行った。得られたHBV複製活性の測定値(逆転写されたHBV DNA量)に基づいて、HBV複製阻害効率(%)を計算した。特定の濃度のGLS4存在下におけるHBV複製阻害効率(%)は、GLS4濃度が0μM(GLS4無添加)の場合の逆転写されたHBV DNA量を「0%阻害」、逆転写されたHBV DNA量が0の場合(逆転写が一切起こらなかった場合)を「100%阻害」として算出した値である。
HBV複製活性評価システムで、HBc-WT、又はHBc-WTに代えてHBc-T33Nを細胞導入して、各種濃度のGLS4の存在下でHBV複製の阻害効果を検討した結果を図13に示す。GLS4は、HBc-WTによるHBV複製を濃度依存的に阻害した一方、HBc-T33NによるHBV複製に対しては阻害効果を示さず、HBc-T33NはGLS4に対して抵抗性を有することが示された。この結果から、HBV複製活性評価システムにおいてHBc-WTに代えてHBc-T33Nを用いることによって、ヌクレオカプシド形成阻害剤耐性ウイルスの複製が再現されることが示された。
(目的)
スパイク領域ΔHBc(α1-4b)が、ヌクレオカプシド形成阻害剤耐性変異型HBc(HBc-T33N)によるHBV複製を阻害するか否かを検証する。
スパイク領域ΔHBc(α1-4b)のHBV複製阻害効果の評価は、実施例2に記載の方法に準じた。ただし、本実施例では、実施例2に記載のHBV複製評価システムに含まれるHBcとしてHBc-WT又はHBc-T33Nを用いた。
図14に結果を示す。野生型ΔHBc(α1-4b)は、HBc-WTによるHBV複製を量依存的に阻害した(図14左側)のと同様に、HBc-T33NによるHBV複製を量依存的に阻害した(図14右側)。この結果から、スパイク領域ΔHBc(α1-4b)は、実施例7においてGLS4耐性を示したHBc-T33Nに対しても、HBV複製阻害効果を有することが示された。
本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。
Claims (9)
- 以下の(1)~(3)のいずれかからなるB型肝炎ウイルス複製阻害剤。
(1)B型肝炎ウイルスのコアタンパク質におけるスパイク領域を構成するペプチド断片
(2)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片
(3)前記(1)又は(2)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクター - 前記スパイク領域を構成するペプチド断片が以下の(a)~(c)のいずれかのアミノ酸配列からなる、請求項1に記載のB型肝炎ウイルス複製阻害剤。
(a)配列番号1~7で示すいずれかのアミノ酸配列
(b)配列番号1~7で示すいずれかのアミノ酸配列において、1又は複数個のアミノ酸が付加、欠失、又は置換されたアミノ酸配列
(c)配列番号1~7で示すいずれかのアミノ酸配列と82%以上のアミノ酸同一性を有するアミノ酸配列 - 前記核酸が以下の(i)~(iv)のいずれかの塩基配列からなる、請求項1に記載のB型肝炎ウイルス複製阻害剤。
(i)配列番号8~14で示すいずれかの塩基配列
(ii)配列番号8~14で示すいずれかの塩基配列において、1又は複数個の塩基が付加、欠失、又は置換された塩基配列
(iii)配列番号8~14で示すいずれかの塩基配列と80%以上の塩基同一性を有する塩基配列
(iv)配列番号8~14で示すいずれかの塩基配列に相補的な塩基配列と高ストリンジェントな条件下でハイブリダイズする塩基配列 - 配列番号1~6で示すいずれかのアミノ酸配列において、
23位のフェニルアラニン(F)残基がアラニン(A)残基に、及び/又は
42位のロイシン(L)残基がアラニン(A)残基に、又は
配列番号7で示すアミノ酸配列において、
35位のフェニルアラニン(F)残基がアラニン(A)残基に、及び/又は
54位のロイシン(L)残基がアラニン(A)残基に
置換された、請求項2に記載のB型肝炎ウイルス複製阻害剤。 - 以下の(1)~(3)のいずれかからなるB型肝炎ウイルスのヌクレオカプシド形成阻害剤。
(1)B型肝炎ウイルスのコアタンパク質におけるスパイク領域を構成するペプチド断片
(2)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片
(3)前記(1)又は(2)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクター - 有効成分としての請求項1~4のいずれか一項に記載のB型肝炎ウイルス複製阻害剤、及び担体及び/又は溶媒を含むB型肝炎治療用医薬組成物。
- 抗B型肝炎ウイルス剤をさらに含む、請求項6に記載のB型肝炎治療用医薬組成物。
- 抗B型肝炎ウイルス剤が核酸アナログ及び/又はHBV-Pol活性阻害剤である、請求項7に記載のB型肝炎治療用医薬組成物。
- (1)B型肝炎ウイルスのコアタンパク質におけるスパイク領域を構成するペプチド断片、
(2)前記スパイク領域のN末端及び/又はC末端に前記コアタンパク質とは異なる任意のアミノ酸配列が付加されたペプチド断片、
又は
(3)前記(1)又は(2)に記載のペプチド断片をコードする核酸を含み、細胞内で前記ペプチド断片を発現可能な発現ベクター
を宿主内に導入する工程を含むB型肝炎ウイルスの複製阻害方法。
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