WO2023246750A1 - 用于抑制乙型肝炎病毒的双链核糖核酸 - Google Patents

用于抑制乙型肝炎病毒的双链核糖核酸 Download PDF

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WO2023246750A1
WO2023246750A1 PCT/CN2023/101298 CN2023101298W WO2023246750A1 WO 2023246750 A1 WO2023246750 A1 WO 2023246750A1 CN 2023101298 W CN2023101298 W CN 2023101298W WO 2023246750 A1 WO2023246750 A1 WO 2023246750A1
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seq
nucleotides
modified
double
ribonucleic acid
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PCT/CN2023/101298
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English (en)
French (fr)
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徐宏江
葛兴枫
郑佳佳
杨玲
张喜全
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正大天晴药业集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/02—Hepadnaviridae, e.g. hepatitis B virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present application belongs to the field of biomedicine and relates to a double-stranded ribonucleic acid (dsRNA), its pharmaceutically acceptable salt, its ligand conjugate, or a pharmaceutical composition, which can be used to inhibit hepatitis B virus.
  • dsRNA double-stranded ribonucleic acid
  • Viral hepatitis B is a disease caused by the infection of the body by the hepatitis B virus (Hepatitis B Virus, referred to as HBV).
  • Hepatitis B virus is a hepatotropic virus that mainly exists in liver cells and damages liver cells, causing liver cell inflammation, necrosis, fibrosis, etc.
  • Hepatitis B virus is divided into two types: acute and chronic. Acute hepatitis B in adults can mostly resolve spontaneously through their own immune mechanisms.
  • CHB chronic hepatitis B
  • HCC hepatocellular carcinoma
  • anti-HBV drugs currently approved for marketing are mainly immunomodulators (interferon- ⁇ and peginterferon- ⁇ -2a, etc.) and antiviral therapeutic drugs (lamivudine, adefovir dipivoxil, entecavir, Telbivudine, tenofovir, clavudine, etc.).
  • antiviral therapeutic drugs are nucleoside or nucleotide drugs. Their mechanism of action is to inhibit the synthesis of HBV DNA and cannot directly reduce HBsAg levels.
  • nucleoside or nucleotide agents show HBsAg clearance rates similar to those observed in nature (Janssen et al. Lancet (2005), 365, 123-129; Marcellin et al. N. Engl. J. Med. (2004) ), 351, 1206-1217; Buster et al. Hepatology (2007), 46, 388-394.).
  • Small interfering RNA can inhibit or block the translation or transcription of target genes in a sequence-specific manner based on the RNA interference (RNAi) mechanism to inhibit the expression of target genes and exert an inhibitory effect at the mRNA level. , while reducing the levels of HBV DNA and HBsAg, thereby achieving the purpose of treating the disease.
  • RNAi RNA interference
  • the application provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof, the double-stranded ribonucleic acid comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-CUGGCUCAGUUUACUAGUA -3'(SEQ ID NO:1) and the length of the sense strand does not exceed 21 nucleotides, where the antisense strand contains 5'-UACUAGUAAACUGAGCCAGGA-3'(SEQ ID NO:2) and the length of the antisense strand does not exceed 23 nucleotides, the sense or antisense strand is optionally modified.
  • the present application provides a pharmaceutical composition, which includes the double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof and a pharmaceutically acceptable carrier or excipient.
  • the present application provides a kit for treating and/or preventing hepatitis B virus infection, which includes the double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a drug The composition; and optional instructions for use.
  • the present application provides a method for treating and/or preventing hepatitis B virus infection, which includes administering the double-stranded ribonucleic acid of the present application to a treatment and/or prevention subject (hereinafter also referred to as a subject), its pharmaceutical Acceptable salts, ligand conjugates thereof, or pharmaceutical compositions thereof.
  • a treatment and/or prevention subject hereinafter also referred to as a subject
  • the present application provides the use of the double-stranded ribonucleic acid of the present application, pharmaceutically acceptable salts thereof, ligand conjugates thereof, or pharmaceutical compositions in the preparation of medicaments for the treatment and/or prevention of hepatitis B virus infection. use.
  • the present application provides the use of the double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition in treating and/or preventing hepatitis B virus infection.
  • the present application provides the double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition thereof for treating and/or preventing hepatitis B virus infection.
  • the hepatitis B virus infection can be at any stage of the disease, such as acute hepatitis B or chronic hepatitis B, or liver diseases caused by hepatitis B virus infection include hepatitis, liver fibrosis, hepatitis Cirrhosis, liver failure, or liver cancer.
  • the hepatitis B virus infection is chronic hepatitis B.
  • the double-stranded ribonucleic acid, pharmaceutically acceptable salts thereof, ligand conjugates thereof, or pharmaceutical compositions of the present application are used as a single therapeutic agent for the treatment and/or prevention of hepatitis B virus infection. use.
  • the double-stranded ribonucleic acid, pharmaceutically acceptable salts thereof, ligand conjugates thereof, or pharmaceutical compositions of the present application are combined with other therapeutic agents for treating and/or preventing hepatitis B virus infection. shared use.
  • the present application provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof.
  • the double-stranded ribonucleic acid includes a sense strand and an antisense strand, wherein the sense strand includes 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO:1) has at least 15 consecutive nucleotides and the length of the sense strand does not exceed 21 nucleotides, in which the antisense strand contains at least 5'-UACUAGUAAACUGAGCCAGGA-3'(SEQ ID NO:2) 15 consecutive nucleotides and the length of the antisense strand does not exceed 23 nucleotides, and the sense or antisense strand is optionally modified.
  • the present application provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof.
  • the double-stranded ribonucleic acid includes a sense strand and an antisense strand, wherein the sense strand includes 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO:1) and the length of the sense strand does not exceed 21 nucleotides, where the antisense strand contains 5'-UACUAGUAAACUGAGCCAGGA-3' (SEQ ID NO:2) and the length of the antisense strand does not exceed 23 nucleotides
  • the sense strand or antisense strand is optionally modified.
  • the present application also provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof.
  • the double-stranded ribonucleic acid includes a sense strand and an antisense strand, and the sense strand and the antisense strand are identical to the above-mentioned
  • the full length of the nucleotide sequences of the sense strand and the antisense strand respectively have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. sex.
  • the double-stranded ribonucleic acid of the present application can be used to inhibit hepatitis B virus, especially to inhibit hepatitis B virus gene expression.
  • the double-stranded ribonucleic acid of the present application is an RNAi drug.
  • the double-stranded region of the double-stranded ribonucleic acid has a length of 19-21 pairs of nucleotides, for example, the double-stranded region of the double-stranded ribonucleic acid has a length of 19, 20 or 21 pairs of nucleotides. .
  • the sense strand or antisense strand optionally includes an overhang located at the 5' end and/or the 3' end.
  • the overhang contains 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the overhang contains 1 or 2 nucleotides.
  • the sense strand optionally includes an overhang located at the 5' end and/or the 3' end. In some embodiments, the sense strand optionally includes an overhang of 1, 2, 3, 4, or 5 nucleotides located at the 5' end and/or the 3' end. In some embodiments, the sense strand optionally includes a 1 or 2 nucleotide overhang located at the 5' end and/or the 3' end.
  • the antisense strand optionally includes an overhang located at the 5' end and/or the 3' end. In some embodiments, the antisense strand optionally includes a 1, 2, 3, 4, or 5 nucleotide overhang located at the 5' end and/or the 3' end. In some embodiments, the antisense strand optionally includes a 1 or 2 nucleotide overhang located at the 5' end and/or the 3' end.
  • the overhang is selected from unmodified or modified A, G, C, U, or T.
  • the antisense strand optionally includes an overhang located at the 5' end and/or the 3' end, and the overhang is selected from unmodified or modified GA. In some embodiments, the antisense strand optionally includes an overhang at the 3' end, the overhang being selected from unmodified or modified GA.
  • the overhang is selected from unmodified or modified U or T.
  • the overhang when the overhang is 1 nucleotide, the overhang is selected from unmodified or modified U or T. In some embodiments, when the overhang is 1 nucleotide, the overhang is selected from 2'-O-methyl modified U (hereinafter also referred to as u) or deoxythymine nucleotides (Hereinafter also referred to as dT).
  • u 2'-O-methyl modified U
  • dT deoxythymine nucleotides
  • the overhang when the overhang is 2 nucleotides, the overhang is selected from unmodified or modified UU or TT. In some embodiments, when the overhang is 2 nucleotides, the overhang is selected from uu or dTdT.
  • the sense strand optionally includes an overhang located at the 5' end and/or the 3' end, and the overhang is selected from uu or dTdT.
  • the antisense strand optionally includes an overhang located at the 5' end and/or the 3' end, and the overhang is selected from uu or dTdT.
  • the overhang is connected to its adjacent nucleotide through a phosphate group or a phosphorothioate group.
  • one or more nucleotides in the overhang are connected through a phosphate group or a phosphorothioate group.
  • the sense strand or antisense strand is optionally modified.
  • one or more nucleotides of the sense or antisense strand are modified.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides are modified; or nucleotides within the range formed by any of the aforementioned values are modified, such as 1 -21 pcs, 1-20 pcs, 1-19 pcs, 1-18 pcs, 1-17 pcs, 1-16 pcs, 1-15 pcs, 1-14 pcs, 1-13 pcs, 1-12 pcs, 1 -11 or 1-10 nucleotides are modified.
  • more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, more than 19, more than 20 or more than 21 nucleotides are modified ;
  • the justice chain has more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18 or more than 19 cores
  • the glycolic acid is modified.
  • all nucleotides of the sense strand are modified.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 of the antisense strands , 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides are modified; or nucleotides within the range formed by any of the aforementioned values is modified, such as 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15 , 1-14, 1-13, 1-12, 1-11 or 1-10 nucleotides are modified.
  • all nucleotides of the antisense strand are modified.
  • all nucleotides of the sense strand and all nucleotides of the antisense strand are modified.
  • the modification is selected from the group consisting of sugar modification of the nucleotide, modification of the linkage between nucleotides, or terminal modification.
  • the glycosyl modification of the nucleotide is selected from dehydroxylation, fluorination, amination, alkylation, hydroxyalkylation or hydroxyalkenylation.
  • the sugar modification of the nucleotide occurs at the 2' position of the sugar group.
  • the glycosyl modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-alkyl, 2'-O-alkyl, 2'-O- Ether group, 2'-O-alkenyl group.
  • the glycosyl modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O -Methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl or 2'-O-ene propyl.
  • the linkage modification between nucleotides is selected from phosphorothioate (PS), phosphorodithioate (PS2), methylphosphonate (MP), methoxypropylphosphine acid ester (MOP) or aminophosphonate.
  • the inter-nucleotide linkage modification is selected from phosphorothioate (PS).
  • the terminal modification is selected from 5'-terminal modification or 3'-terminal modification. In some embodiments, the terminal modification is selected from 5'-phosphate, 5'-methylphosphonate (5'-MP), 5'-phosphorothioate (5'-PS) or 5'- (E)-Vinylphosphonate (5'-(E)-VP).
  • the modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl group, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-allyl, phosphorothioate group, methylphosphonate group , aminophosphonate group, 5'-phosphate group, 3'-phosphate group, 5'-(E)-vinyl phosphate or 3'-(E)-vinyl phosphate.
  • the modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O- Methyl, 2'-O-allyl, phosphorothioate or 5'-(E)-vinyl phosphate.
  • the modification is selected from 2'-fluoro, 2'-O-methyl, phosphorothioate, or 5'-(E)-vinyl phosphate.
  • counting is from the 5' end.
  • a phosphorothioate group is used to modify the space between the 1st and 2nd nucleotides of the sense strand and/or the 2nd and 3rd nucleotides of the sense strand.
  • between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, and between the 19th and 20th nucleotides of the antisense strand and/or between nucleotides 20 and 21 of the antisense strand are modified with phosphorothioate groups.
  • the 5'-end of the antisense strand is modified with 5'-(E)-vinyl phosphate.
  • nucleotides of the sense strand are modified with 2'-fluoro. In some embodiments, 14, 15, 16 or 17 nucleotides of the sense strand are modified with 2'-O-methyl.
  • nucleotides in nucleotides 1 to 6 of the sense strand are modified with 2'-fluoro.
  • nucleotides in nucleotides 1 to 6 of the sense strand are modified with 2’-O-methyl.
  • 2 or 3 of the nucleotides 7 to 9 of the sense strand are modified with 2'-fluoro.
  • 0 or 1 nucleotide among the 7th to 9th nucleotides of the sense strand is modified with 2'-O-methyl.
  • 0 or 1 nucleotide among the 10th to 19th nucleotides of the sense strand is modified with 2'-fluoro.
  • 8, 9 or 10 nucleotides among nucleotides 10 to 19 of the sense strand are modified with 2’-O-methyl.
  • the 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and the 1st to 6th and/or 10th to 19th nucleotides of the sense strand are There are 0, 1 or 2 nucleotides also modified with 2'-fluoro.
  • nucleotides 7, 8, and 9 of the sense strand are modified with 2'-fluoro, and nucleotides 1 to 6 and 10 to 19 of the sense strand have 0 One, one or two nucleotides are also modified with 2'-fluoro.
  • nucleotides 7, 8, and 9 of the sense strand are modified with 2'-fluoro, and 1 of nucleotides 1 to 6 and 10 to 19 of the sense strand is Each nucleotide is also modified with 2'-fluoro.
  • 2, 3, 4, 5, 6 or 7 nucleotides of the antisense strand are modified with 2'-fluoro.
  • 14, 15, 16, 17, 18 or 19 nucleotides of the antisense strand are modified with 2'-O-methyl.
  • 3 or 4 of the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are modified with 2'-fluoro. In some embodiments, 4 of the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are modified with 2'-fluoro.
  • 3, 4, 5 or 6 of the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense strand are 2'-fluorinated. Grooming. In some embodiments, 4 or 6 of the 2, 6, 8, 9, 14, and 16 nucleotides of the antisense strand are modified with 2'-fluoro.
  • nucleotides 2, 6, 14 and 16 of the antisense strand are modified with 2'-fluoro, and nucleotides 1, 3 to 5, 7 to 13, Any one or both of nucleotides 15 and/or 17 to 21 are also modified with 2'-fluoro.
  • nucleotides 2, 6, 14 and 16 of the antisense strand are modified with 2'-fluoro, and nucleotides 1, 3 to 5, 7 to 13, Any one or both of nucleotides 15 and 17 to 21 are also modified with 2'-fluoro.
  • 14, 15, 16 or 17 of the 1, 3 to 5, 7 to 13, 15, 17 to 21 nucleotides of the antisense strand adopt 2'-O -Methyl modification.
  • the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and the other nucleotides of the sense strand are modified with 2'-O-methyl Modified, and the 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14 and 16 of the antisense strand are modified with 2'-O-methyl. base modification.
  • the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro
  • the other nucleotides of the sense strand are modified with 2'-O-methyl Modification
  • the 2'-fluoro modification is used on the 2nd, 6th, 14th and 16th nucleotides of the antisense strand, and the other nucleotides of the antisense strand are modified with 2'-O-methyl.
  • the 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and other nucleotides of the sense strand are modified with 2'-O-methyl, And the 2'-fluoro modification is used on the 2nd, 6th, 14th and 16th nucleotides of the antisense strand, and the other nucleotides of the antisense strand are modified with 2'-O-methyl.
  • the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and the other nucleotides of the sense strand are modified with 2'-O-methyl Modified, and the 2'-fluoro modified nucleotides at positions 2, 6, 8, 9, 14 and 16 of the antisense strand are modified with 2'-O-methyl. base modification, and between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, and between the 1st and 2nd nucleotides of the antisense strand.
  • a phosphorothioate group is used for modification .
  • the 5'-end of the antisense strand is modified with 5'-(E)-vinyl phosphate.
  • the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro
  • the other nucleotides of the sense strand are modified with 2'-O-methyl Modification
  • the 2'-fluoro modification is used on the 2nd, 6th, 14th and 16th nucleotides of the antisense strand, and the other nucleotides of the antisense strand are modified with 2'-O-methyl
  • Phosphorothioate groups are used for modification between the 3 nucleotides, between the 19th and 20th nucleotides of the antisense strand, and/or between the 20th and 21s
  • the 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and other nucleotides of the sense strand are modified with 2'-O-methyl, Nucleotides at positions 2, 6, 14 and 16 of the antisense strand are modified with 2'-fluoro, other nucleotides of the antisense strand are modified with 2'-O-methyl, and the nucleotides of the sense strand are modified with 2'-fluoro.
  • the 20th and 21st nucleotides of the sense strand are modified with a phosphorothioate group.
  • the 5'-end of the antisense strand is modified with 5'-(E)-vinyl phosphate.
  • the ligand includes a branching group and a linking group.
  • the ligand is connected to the double-stranded ribonucleic acid through a linking group.
  • the double-stranded ribonucleic acid to which the ligand is connected is called a double-stranded ribonucleic acid ligand conjugate.
  • the ligand is connected to one or more targeting groups through a branching group.
  • the branching group includes a targeting group.
  • the ligand contains at least one targeting group. In some embodiments, the ligand contains one, two, three, four or five targeting groups. In some embodiments, the ligand contains two, three, or four targeting groups. In some embodiments, the ligand contains three targeting groups.
  • the targeting group is selected from GalNAc groups.
  • the ligand contains one, two, three, four or five GalNAc groups. In some embodiments, the ligand contains two, three or four GalNAc groups. In some embodiments, the ligand contains three GalNAc groups.
  • the branching group is selected from:
  • the linking group is selected from:
  • the ligand is selected from:
  • the ligand is linked to the sense strand or the antisense strand. In some embodiments, the ligand is linked to the 5' or 3' end of the sense strand or antisense strand. In some embodiments, the ligand is attached to the 5' or 3' end of the sense strand. In some embodiments, the ligand is linked to the 3' end of the sense strand.
  • the ligand is connected to the sense strand or antisense strand of the double-stranded ribonucleic acid through a phosphate group or a phosphorothioate group. In some embodiments, the ligand is connected to the sense strand through a phosphate group or a phosphorothioate group. In some embodiments, the ligand is connected to the 3′ end of the sense strand through a phosphate group or a phosphorothioate group.
  • the double-stranded ribonucleic acid ligand conjugate is as follows:
  • the double-stranded ribonucleic acid ligand conjugate is selected from:
  • the double-stranded ribonucleic acid ligand conjugate is selected from:
  • the unmodified sense strand of the double-stranded ribonucleic acid is selected from:
  • the unmodified antisense strand of the double-stranded ribonucleic acid is selected from:
  • the modified sense strand of the double-stranded ribonucleic acid is selected from:
  • the modified antisense strand of the double-stranded ribonucleic acid is selected from:
  • the double-stranded ribonucleic acid of the present application can be formed by including any one of the above-mentioned sense strands and any one of the above-mentioned antisense strands.
  • the double-stranded ribonucleic acid includes any one of the following sense strands: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16.
  • the double-stranded ribonucleic acid includes any one of the following antisense strands: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO :45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 , SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65.
  • the double-stranded ribonucleic acid (unmodified) is selected from:
  • the double-stranded ribonucleic acid (modified) is selected from:
  • the double-stranded ribonucleic acid ligand conjugate is selected from:
  • the L represents a ligand
  • the ligand is as described above; in some embodiments, the ligand is selected from L01 ligand or L02 ligand.
  • VP- indicates that the nucleotide on the right side of the hyphen is an (E)-vinyl phosphate modified nucleotide, for example, the 5'- of SEQ ID NO:64 and SEQ ID NO:65 above
  • the terminal u is modified with 5'-(E)-vinyl phosphate.
  • the sequence ID number of the sense strand refers to the sequence ID number of the nucleic acid sequence in the sense strand.
  • the dsRNA ligand conjugate number and the L at the 3' end of the sense strand sequence indicate that in the dsRNA ligand conjugate, a ligand is connected to the 3' end of the sense strand sequence.
  • the salts described above are selected from base addition salts, acid addition salts, and combinations thereof.
  • the base addition salt is selected from sodium, potassium, calcium, ammonium, organic amines, magnesium salts and combinations thereof
  • the acid addition salt is selected from inorganic acid salts, organic acid salts and combinations thereof.
  • the inorganic acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydriodic acid, phosphorous acid, and the like.
  • the organic acid is selected from acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzene Sulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and combinations thereof.
  • the double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate of the present application has a high coverage rate in different HBV genotypes and is suitable for the treatment and/or prevention of patients with multiple genotypes.
  • the double-stranded ribonucleic acid of the present application, its pharmaceutically acceptable salt or its ligand conjugate also has good HBV inhibitory activity and stability.
  • the double-stranded ribonucleic acid of the present application, its pharmaceutically acceptable salt or its ligand conjugate exhibits excellent HBV mRNA inhibition efficiency at both the cellular level and the animal level, and can effectively reduce the hepatitis B virus.
  • the expression of surface antigen and e-antigen shows a good therapeutic effect on hepatitis B virus infection, especially chronic hepatitis B.
  • nucleotide When any variable occurs more than once in a compound, nucleotide, single- or double-stranded structure, its definition in each case is independent.
  • modifications described in this application occur independently, that is, unless otherwise indicated, modifications to the sense strand will not affect modifications to the antisense strand, and modifications to a certain nucleotide will not affect modifications to another nucleotide.
  • the modification on the sugar group of a certain nucleotide will not affect the modification of another sugar group on the same nucleotide. The influence includes whether it is modified, but also what type of modification is used.
  • HBV genome sequence or mRNA sequence of the present application can be obtained through published databases, such as Genbank, etc., such as the HBV genome sequence (GenBank accession#EU554538.1).
  • HBV as used herein also refers to any naturally occurring DNA sequence variation of the HBV genome.
  • interfering RNA or “RNAi” or “interfering RNA sequence” refers to such single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (e.g., duplex RNA such as siRNA, aiRNA or pre-miRNA), when the interfering RNA is in the same cell as the target gene or sequence, it can reduce or inhibit the expression of the target gene or sequence (e.g., by mediating with the interfering RNA sequence Degradation of complementary mRNA or inhibition of translation or transcription of mRNA complementary to the interfering RNA sequence).
  • the interfering RNA may have substantial or complete identity with the target gene or sequence, or may include mismatched regions (i.e., mismatched sequences).
  • the double-stranded ribonucleic acid of the present application functions as interfering RNA.
  • the double-stranded ribonucleic acid of the present application may be referred to as siRNA.
  • mismatch region refers to a portion of an interfering RNA (eg, siRNA, aiRNA, miRNA) sequence that does not have 100% complementarity to its target sequence.
  • Interfering RNA eg, siRNA, aiRNA, miRNA
  • the mismatched regions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides.
  • a mismatched region may include a single nucleotide or may include 2, 3, 4, 5, 6, or more nucleotides.
  • identity refers to the similarity between two nucleotide sequences or between two amino acid sequences. Sequence identity preferably relates to the percentage of nucleotides or amino acids in the sequence that have the same position in two or more sequences of the same length. Specifically, the "% identity" of two amino acid sequences or two nucleotide sequences can be determined by aligning the sequences for optimal comparison (e.g., gaps can be introduced in either sequence to best match the other sequence). (Ultimate alignment) and compare the amino acids or nucleotides at corresponding positions. Gaps are generally considered to be non-identical positions, regardless of their actual position in the alignment. An “optimal alignment” is generally the alignment of two sequences that results in the highest percent identity.
  • the sequence identity of the present application is at least 80%, 85%, 90% or 95%, preferably at least 90%.
  • Non-limiting examples include: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art.
  • interfering RNA includes "small interfering RNA (siRNA)", the length of which is, for example, about 15-60, 15-50, 15-40, 15-30, 15-25 , 19-25, 19-23 or 19-21 nucleotides.
  • siRNA small interfering RNA
  • the double-stranded ribonucleic acid of the present application can function as such siRNA and can have corresponding lengths.
  • the nucleotide positions of the sense strand or antisense strand are counted from the 5' end.
  • the 1st position of 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO:1) is C and the 19th position is A ;
  • the 1st position of 5'-UACUAGUAAACUGAGCCAGGA-3' (SEQ ID NO:2) is U, and the 21st position is A.
  • the sense strand adds several nucleotides at the end based on SEQ ID NO:1, and the antisense strand has SEQ ID NO:1.
  • the counting method of the nucleotide position of the sense strand or antisense strand will not change because of this, that is, the first position of the sense strand is still SEQ ID NO: The first position of 1, the first position of the antisense strand is still the first position of SEQ ID NO:2.
  • complementary when used to describe the relationship of a first nucleic acid sequence and a second nucleic acid sequence refers to an oligonucleotide or polynucleotide that contains the first nucleic acid sequence under specific conditions. The ability to hybridize to and form a double-stranded structure with an oligonucleotide or polynucleotide containing a second nucleic acid sequence.
  • “complementary” sequences may also include double-stranded structures formed by base pairings formed by non-Watson-Crick base pairing and/or non-natural or modified nucleotides, or may be entirely composed of non-Watson-Crick base pairs. - Double-stranded structures formed by Crick base pairing and/or base pairing of non-natural or modified nucleotides, as long as the above requirements with respect to their ability to hybridize are met.
  • a "perfectly complementary" sequence includes an oligonucleotide or polynucleotide containing a first nucleic acid sequence and an oligonucleotide or polynucleotide containing a second nucleic acid sequence that are between the first nucleic acid sequence and the second nucleic acid sequence. Base pairing over the entire length of the two nucleic acid sequences.
  • substantially complementary means complete complementarity or at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) between two nucleic acid sequences. %, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the overlapping nucleotides are complementary.
  • double-stranded ribonucleic acid double-stranded RNA
  • double-stranded RNA double-stranded RNA
  • dsRNA encompasses two antiparallel and complementary nucleic acid strands with a “sense” or “antisense” orientation relative to a target RNA (eg, HBV gene).
  • dsRNA can degrade target RNA (eg, mRNA) through RNA interference (RNAi) mechanism.
  • RNAi RNA interference
  • the double-stranded ribonucleic acid of the present application contains a sense strand and an antisense strand.
  • sense strand or “sense strand” refers to the single strand of the dsRNA duplex that is substantially complementary to a region of the antisense strand.
  • antisense strand or “guide strand” refer to the single strand of a dsRNA duplex that is substantially complementary to a region of the target sequence. If the sense strand is not completely complementary to the antisense strand, mismatches may occur within the molecule or in the terminal region. Typically, the most tolerated mismatches are within the terminal region. If the antisense strand is not completely complementary to the target sequence, mismatches may occur within the molecule or in the terminal region. Typically, the most tolerated mismatches are within the terminal region.
  • the double strands of dsRNA can have the same or different numbers of nucleotides.
  • the length of the double-stranded region formed by complementation can be any length that allows degradation of the target RNA, and possible lengths range from about 9 to 36 nucleotide pairs, such as 15 to 30 pairs, 16 to 28 pairs, 19 to 21 pairs Right and so on.
  • a dsRNA may include one or more nucleotide overhangs, which refer to at least one unpaired nucleotide/nucleoside analog.
  • nucleotide overhangs refer to at least one unpaired nucleotide/nucleoside analog.
  • the nucleotides at the "overhang” can include 0-5 nucleotides, where "0” means no "overhang” and "5" means there are 5 additional nucleotides on the single strand of the dsRNA duplex. (that is, a nucleotide that does not form a pair with another single strand).
  • an “overhang” can be located at the 5' and/or 3' end of either strand of the dsRNA.
  • an “overhang” includes 0-5 nucleotides.
  • an “overhang” includes 0-2 nucleotides.
  • the "overhang” at the 3' and/or 5' end of the sense strand of the dsRNA has 0-2 nucleotides.
  • the "overhang” at the 3' and/or 5' end of the antisense strand of the dsRNA has 0-2 nucleotides.
  • the nucleotides forming the "overhang” may be A, G, C, U or T or modified structures thereof.
  • the nucleotide forming the "overhang” may be U, T or dT or modified structures thereof.
  • “overhangs” include, but are not limited to, “TT”, “dTdT”, “UU” or their corresponding modified structures, such as UU modified with 2' methoxy, that is, uu.
  • the "overhang” at the 3' and/or 5' end of the antisense strand of the dsRNA is substantially complementary to the target RNA.
  • the "overhang” at the 3' and/or 5' end of the antisense strand of the dsRNA is completely complementary to the target RNA.
  • the "overhang" at the 3' end of the antisense strand of the dsRNA is completely complementary to the target RNA. In some embodiments, the "overhang” at the 3' end of the antisense strand of the dsRNA is selected from unmodified or modified GA.
  • the term “blunt” or “blunt end” means that there are no unpaired nucleotides at the end of the dsRNA, i.e. there are no nucleotide overhangs.
  • a dsRNA that has "blunt ends” at both ends is a dsRNA that is double-stranded throughout its length, that is, there are no nucleotide overhangs at either end of the molecule.
  • the dsRNA or any single strand thereof is optionally modified, and both unmodified and modified ribonucleic acids are within the protection scope of this application.
  • the modification will not cause the dsRNA to significantly weaken or lose its function of inhibiting HBV gene expression.
  • the modification of the dsRNA or any single strand thereof can be located at the 5' end and/or the 3' end, nucleotides, or the connecting bonds between nucleotides. Methods known in the art can be used for synthesis or modification.
  • the modification of the nucleotide includes, but is not limited to, occurring on the sugar moiety of the nucleotide, including one or more substituted or removed sugar moiety groups, such as removing the hydroxyl group on the carbonyl group, Or fluorination, amination, alkylation, hydroxyalkylation or hydroxyalkenylation may occur. Modifications on the sugar moiety can occur at various positions on the sugar ring.
  • modifications on the sugar moiety of the nucleotide include, but are not limited to, 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2 '-Methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl or 2'-O-allyl, the structure can be as follows:
  • Base represents the base.
  • the modification of the bond between nucleotides includes substitution or replacement of atoms or functional groups of the phosphate group, such as phosphorothioate (PS), phosphorodithioate (PS2), methylphosphine acid ester (MP), methoxypropylphosphonate (MOP) or aminophosphonate.
  • PS phosphorothioate
  • PS2 phosphorodithioate
  • MP methylphosphine acid ester
  • MOP methoxypropylphosphonate
  • the capital letters G, C, A, U or T each generally represent a substance containing guanine, cytosine, adenine, uracil, Thymine serves as the base of the nucleotide.
  • the lowercase letters g, c, a, and u respectively represent that the nucleotide represented by the corresponding uppercase letter is modified by 2'-methoxy, that is, g, c, a, and u respectively represent 2'-O-methyl G, 2 '-O-methyl C, 2'-O-methyl A, 2'-O-methyl U.
  • the uppercase letter plus the lowercase letter f to the right represents that the nucleotide represented by the corresponding uppercase letter is modified by 2'-fluorine, that is, Gf, Cf, Af, and Uf represent 2'-fluoroG and 2'-fluorine respectively.
  • the lowercase letter s indicates that the two nucleotide residues adjacent to the left and right of s are connected by a phosphorothioate group. For example, "csu” indicates that the c and u residues are connected by a phosphorothioate group.
  • VP- indicates that the nucleotide to the right of the hyphen is an (E)-vinyl phosphate modified nucleotide, for example, "VP-u” indicates (E)-vinyl phosphate modified 2'-O- Methyl U.
  • the 5' end and/or 3' end modification refers to the modification that occurs at the 5' end and/or 3' end of dsRNA or any single strand thereof, such as phosphorylation, conjugation or reverse linkage, etc.
  • the 5' end includes but is not limited to 5'-phosphate, 5'-methylphosphonate (5'-MP), 5'-phosphorothioate (5'-PS) or 5'-( E)-Vinylphosphonate (5'-(E)-VP), the structure can be as follows:
  • Base represents a base
  • X is selected from the hydroxyl group or the 2’ modification on the sugar group.
  • the ligand is a group connected to dsRNA.
  • the ligand includes a branched group and a linker, and the dsRNA, linking group and branched group are sequentially connection (for example, shown in Equation 104).
  • the branched group contains at least one (such as one, two, three, four or five) pharmaceutically acceptable targeting group (targeting group), which targets dsRNA to specific tissues or enhances cellular uptake.
  • the targeting group is for example but not limited to GalNAc (N-acetylgalactosamine, N-Acetylgalactosamine, for example, shown in Formula 105) group. Multiple targeting groups are connected in series or parallel through branching groups.
  • the GalNAc group can be monovalent, divalent, trivalent, or tetravalent.
  • the monovalent, bivalent, trivalent and tetravalent terms mentioned here respectively refer to the dsRNA molecules in the dsRNA ligand conjugate after the dsRNA molecule and the ligand containing GalNAc as the targeting group form a dsRNA ligand conjugate.
  • the molar ratios to GalNAc molecules are 1:1, 1:2, 1:3 and 1:4.
  • the GalNAc molecule when the dsRNA of the present application is conjugated to a GalNAc-containing ligand, the GalNAc molecule is trivalent or tetravalent.
  • the GalNAc molecule when the dsRNA of the present application is conjugated to a GalNAc-containing ligand, the GalNAc molecule is trivalent.
  • the ligand can be connected to the phosphate group, the 2’-position hydroxyl group, the 3’-position hydroxyl group or the base of the nucleotide.
  • the ligand can be connected to any nucleotide of the dsRNA, including but not limited to the 5' or 3' terminal nucleotides of the sense strand or the antisense strand or the non-terminal intermediate nucleotides.
  • the types or preparation methods of the ligands may refer to methods known in the art, including but not limited to ligands and preparation methods described in WO2009082607, WO2014025805, WO2015006740, and WO2021249484, which are incorporated by reference. The entire disclosure is incorporated into this application.
  • Exemplary ligands include, but are not limited to, L01 or L02 described above.
  • conjugation means that two or more chemical moieties each having a specific function are connected to each other in a non-covalent connection or a covalent connection; accordingly, “conjugation””Compound” refers to a compound formed by non-covalent connection or covalent connection between various chemical parts. In this application, it is preferred to use conjugates that are linked to each other in a covalently linked manner.
  • the ligand is connected to the 5' or 3' end of the sense strand or the antisense strand.
  • the ligand is attached to the 5' or 3' end of the sense strand. More preferably, the ligand is attached to the 3' end of the sense strand.
  • the dsRNA ligand conjugate formed by connecting the dsRNA and the ligand is represented by the following formula 102 or formula 103:
  • the compounds of the present application may exist in specific geometric or stereoisomeric forms, and all of these geometric or stereoisomeric forms All fall within the scope of this application.
  • This application contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic and other mixtures thereof, such as enantiomers or diastereomers. body-enriched mixtures, all of which are within the scope of this application.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this application.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • diastereomer refers to stereoisomers whose molecules have two or more chiral centers and are in a non-mirror image relationship between the molecules.
  • use wedge-shaped solid line keys and wedge-shaped dotted keys Represents the absolute configuration of a three-dimensional center
  • using straight solid line keys and straight dotted keys Represent the relative configuration of the three-dimensional center with a wavy line
  • wedge-shaped solid line key or wedge-shaped dotted key or use tilde Represents a straight solid line key and/or straight dotted keys
  • the terms “enriched in an isomer,” “enantiomerically enriched,” “enriched in an enantiomer,” or “enantiomerically enriched” refer to one of the isomers or enantiomers.
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • isomeric excess or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If it is desired to obtain an enantiomer of a compound of the present application, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereomeric salt is formed with a suitable optically active acid or base, and then the salt is formed by conventional methods known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g., generation of amino groups from amines). formate).
  • the compounds of the present application may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • compounds can be labeled with radioactive isotopes, such as tritium ( 3H ), iodine-125 ( 125I ), or C-14 ( 14C ).
  • deuterated drugs can be replaced by heavy hydrogen to form deuterated drugs.
  • the bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon.
  • deuterated drugs can reduce side effects and increase drug stability. , enhance efficacy, extend drug biological half-life and other advantages. All transformations of the isotopic composition of the compounds of this application, whether radioactive or not, are included within the scope of this application.
  • treatment means administering a compound or formulation described herein (eg, a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition herein) to improve or Eliminating a disease or one or more symptoms associated with said disease, and includes: (i) inhibiting the disease or disease state, i.e., arresting its progression; (ii) alleviating the disease or disease state, i.e., ie, resolving the disease or disease state.
  • a compound or formulation described herein eg, a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition herein
  • prevention means administering a compound or formulation described herein (eg, a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition of the present application) to prevent disease. or one or more symptoms associated with said disease, and includes: preventing the occurrence of a disease or disease state in a subject, particularly where such subject is susceptible to the disease state but has not been diagnosed as having it When you have this disease state.
  • a compound or formulation described herein eg, a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition of the present application
  • subject refers to an animal that has been the subject of treatment, observation, or experimentation.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • terapéuticaally effective amount means (i) treating or preventing a specified disease, condition, or disorder, (ii) alleviating, ameliorating, or eliminating one or more symptoms of a specified disease, condition, or disorder, or (iii) preventing or delaying The amount of a compound of the present application (e.g., a double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof) for the onset of one or more symptoms of a particular disease, condition, or disorder described herein .
  • a compound of the present application e.g., a double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof
  • the amount of a compound of the present application that constitutes a "therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the subject to be treated, but can be routinely determined by one skilled in the art. based on its own knowledge and the contents of this application.
  • Therapeutic dosages of the compounds of the present application may be determined based, for example, on the specific use of the treatment, the manner in which the compound is administered, the health and condition of the patient, and the judgment of the prescribing physician.
  • the proportions or concentrations of the compounds of the present application in pharmaceutical compositions may not be fixed and depend on a variety of factors, including dosage, chemical properties (eg, hydrophobicity), and route of administration.
  • a compound of the present application may be provided for parenteral administration in a physiologically buffered saline solution containing about 0.1 to 10% w/v of the compound.
  • Some typical dosage ranges are from about 1 ⁇ g/kg to about 1 g/kg body weight per day.
  • the dosage range is from about 0.01 mg/kg to about 100 mg/kg body weight per day.
  • the dosage will likely depend on such variables as the type and extent of the disease or condition, the general health of the particular patient, the relative biological potency of the compound selected, the formulation of the excipients, and their route of administration. Effective doses can be obtained by extrapolation of dose-response curves derived from in vitro or animal model test systems.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and other animal tissues without Excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • Examples of pharmaceutically acceptable salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. .
  • pharmaceutical composition refers to one or more compounds of the present application (such as the double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof) and a pharmaceutically acceptable carrier, A mixture of excipients or excipients.
  • a pharmaceutically acceptable carrier such as the double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof
  • pharmaceutically acceptable carrier A mixture of excipients or excipients.
  • pharmaceutical compositions are to facilitate The compounds of the present application are administered to an organism.
  • pharmaceutical composition and “preparation” have the same meaning and are used interchangeably.
  • Carriers, excipients or excipients as used herein include any and all solvents, diluents or other liquid excipients, dispersing or suspending agents, surfactants, isotonicity enhancing agents, suitable for the particular dosage form desired. Thickeners or emulsifiers, preservatives, solid binders, lubricants, etc.
  • Various carriers, excipients or excipients for formulating pharmaceutically acceptable compositions and methods for their preparation may employ known techniques. Except for any conventional carrier medium that is incompatible with the compounds of the present application (e.g., produces any undesirable biological effect or otherwise interacts in a deleterious manner with any other component of the pharmaceutically acceptable composition), its use is contemplated in within the scope of this application.
  • the carrier, auxiliary material or excipient used herein is a carrier, auxiliary material or excipient commonly used in the field of dsRNA administration.
  • compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable carriers, auxiliary materials or excipients.
  • suitable pharmaceutically acceptable carriers such as tablets, Pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.
  • Typical routes of administration of the compounds of the present application or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration. medicine.
  • the pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolving methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.
  • kit of the present application it includes the double-stranded ribonucleic acid of the present application, its pharmaceutically acceptable salt, its ligand conjugate, or a pharmaceutical composition; and optionally using the double-stranded ribonucleic acid of the present application. , pharmaceutically acceptable salts thereof, ligand conjugates thereof, or pharmaceutical compositions, instructions for treating and/or preventing hepatitis B virus infection.
  • the solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this application are volume ratios.
  • modified nucleotide groups can be introduced into the dsRNA described herein by using nucleoside monomers with corresponding modifications. Those skilled in the art can learn methods for preparing corresponding modified nucleoside monomers and methods for introducing modified nucleotide groups into dsRNA from the prior art. All modified nucleoside monomers can be obtained from commercial sources or prepared by known methods.
  • the required ribonucleic acid can be obtained by conventional ribonucleic acid preparation methods in the art (such as solid phase synthesis and liquid phase synthesis).
  • the required ribonucleic acid can be synthesized by phosphoramidite solid phase synthesis technology.
  • the preparation method of the double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate of the present application includes the following steps: according to the nucleotide type or sequence of the sense strand or antisense strand of the double-stranded ribonucleic acid, to The nucleotide monomers are sequentially linked in the 3' to 5' direction to synthesize the sense and antisense strands.
  • connection of each nucleotide monomer involves four steps of deprotection, coupling, capping, oxidation or sulfation.
  • Those skilled in the art can adopt conventional reaction conditions, types and dosages of reagents, or make adjustments according to experimental conditions to achieve the deprotection, coupling, capping, oxidation or sulfidation reaction.
  • the ligand for the synthesis of ribonucleic acid containing ligands, can be connected to the ribonucleic acid through a coupling reaction during the synthesis of the oligoribonucleotide or after the synthesis is completed, or the ligand can be connected first. to the solid phase carrier, and then connect the nucleoside monomer and the ligand-solid phase carrier sequentially in the 3' to 5' direction.
  • purification and desalting are well known to those skilled in the art.
  • purification of ribonucleic acid can be accomplished by preparative ion chromatography methods.
  • desalting of ribonucleic acid can be accomplished by reversed-phase chromatography purification method or ultrafiltration centrifugation method.
  • the sense strand and the antisense strand can be mixed in a 1:1 molar amount, heated to 70-95°C, and then lowered to room temperature to form a double-stranded structure.
  • the ribonucleic acid concentration can be detected by, for example, ion exchange chromatography, or the molecular weight can be determined by liquid mass spectrophotometry, or the concentration can be measured by a micro-spectrophotometer to control the synthesis quality.
  • detection methods are well known to those skilled in the art. of.
  • FIG 1 shows experimental serum HBV DNA levels in AAV-HBV mice.
  • Figure 2 shows experimental serum HBsAg levels in AAV-HBV mice.
  • FIG. 3 shows experimental serum HBeAg levels in AAV-HBV mice.
  • DMT removal First wash the resin twice with acetonitrile, use 3% trichloroacetic acid/DCM to remove DMT on the resin, and wash with acetonitrile 4 to 5 times.
  • Blocking Use Capping A first, then Capping B to block unreacted hydroxyl groups. Wash with acetonitrile 4 to 5 times.
  • Oxidation Add oxidation reagent, oxidation time is 2 minutes, and then wash with acetonitrile 4 to 5 times.
  • Formation of phosphorothioate bonds replace the oxidizing reagent with a thio reagent, set the thio time to 10 minutes, and complete the formation of phosphorothioate bonds.
  • DMT removal Wash the resin twice with acetonitrile first, use 3% trichloroacetic acid/DCM to remove DMT on the resin, and wash it 4 to 5 times with acetonitrile.
  • Blocking Use Capping A first, then Capping B to block unreacted hydroxyl groups. Wash with acetonitrile 4 to 5 times.
  • Oxidation Add oxidation reagent, oxidation time is 2 minutes, and then wash with acetonitrile 4 to 5 times.
  • Formation of phosphorothioate bonds replace the oxidizing reagent with a thio reagent, set the thio time to 10 minutes, and complete the formation of phosphorothioate bonds.
  • Mobile phase A 20mm ammonium formate, pH 6.2
  • Mobile phase B 20mm ammonium formate + 95% ACN
  • siRNA dsRNA
  • mobile phase A is 0.1mol/L triethylamine, pH8.0
  • mobile phase B is 0.1mol/L triethylamine, pH8.0+50% ACN; detection wavelength 215nm, 260nm.
  • the main peak was collected and evaporated to remove most of the solvent to obtain the target product.
  • Concentration determination After concentration, the concentration of the sample is measured using a micro-volume spectrophotometer.
  • Double-stranded ribonucleic acid looks like this:
  • the pharmaceutically acceptable salt of the above-mentioned double-stranded ribonucleic acid or its ligand conjugate (the ligand is selected from L01 ligand or L02 ligand) can be prepared according to the method described in this application or methods known in the art.
  • Test Example 1 In vitro screening of double-stranded RNA
  • Cos7 cells (purchased from Nanjing) were cultured in DMEM complete medium (Hyclone, cat. No. SH30243.01) containing 10% fetal calf serum (Gibco, cat. no. 10099-141) at 37°C in an incubator containing 5% CO2 . Kebai Biotechnology Co., Ltd.). When the cells grow to near confluence, trypsin is used to release the cells from the culture flask. Cos7 cells were seeded in a 96-well plate at 2 ⁇ 10 4 cells/well and cultured overnight. When the cell density reached 70-80%, they were transfected with pmirGLO-containing 2.2kb HBV genome sequence (GenBank accession#EU554538.1).
  • HBV plasmid into Cos7 cells Transfections were performed with 0.3 ⁇ L Lipofectamine TM 3000 transfection reagent (Invitrogen, Cat. No. L3000015) at a concentration of 50 ng plasmid/well as described by the manufacturer's instructions. After 6-8 hours of plasmid transfection, the medium was removed. Then siRNA was transfected into Cos7 cells that had been transfected with pmirGLO-HBV plasmid. The siRNA transfection method is to add 0.5 ⁇ L siRNA to 5 ⁇ L OPTI-MEM medium, and add 0.3 ⁇ L Lipofectamine TM RNAiMAX transfection reagent (Invitrogen, Cat. No.
  • Cos7 cells (purchased from Nanjing Branch) were cultured in DMEM complete medium (Hyclone, catalog number SH30243.01) containing 10% fetal calf serum (Gibco, catalog number 10099-141) at 37°C in an incubator containing 5% CO2 . Bai Biotechnology Co., Ltd.). When the cells grow to near confluence, trypsin is used to release the cells from the culture flask. Cos7 cells were seeded in a 96-well plate at 2 ⁇ 10 4 cells/well and cultured overnight. When the cell density reached 70-80%, they were transfected with pmirGLO-containing 2.2kb HBV genome sequence (GenBank accession#EU554538.1).
  • HBV plasmid into Cos7 cells Transfections were performed with 0.3 ⁇ L Lipofectamine TM 3000 transfection reagent (Invitrogen, Cat. No. L3000015) at a concentration of 50 ng plasmid/well as described by the manufacturer's instructions. After 6-8 hours of plasmid transfection, the medium was removed. Then siRNA was transfected into Cos7 cells that had been transfected with pmirGLO-HBV plasmid.
  • the transfection method of siRNA is to use RNase-free water to serially dilute siRNA 5 times to a concentration range of 2-0.0000256 ⁇ M; add 0.5 ⁇ L siRNA to 5 ⁇ L OPTI-MEM medium, and take 0.3 ⁇ L Lipofectamine TM RNAiMAX for transfection Reagent (Invitrogen, Cat. No. 13778150) was added to 5 ⁇ L of OPTI-MEM culture medium; mix the two by pipetting and let stand at room temperature for 5 minutes; add the complex to the 96-well plate, and then add 90 ⁇ L of DMEM to each well to complete culture medium and continue culturing for 48 hours; set 8 concentrations for each siRNA. Set 3 duplicate wells for each concentration. Select a dsRNA that cannot pair with the target sequence as a control.
  • the transfection method of siRNA is to use RNase-free water to serially dilute siRNA 5 times to a concentration range of 2-0.0000256 ⁇ M; add 0.5 ⁇ L siRNA to a solution containing 0.3 ⁇ L Lipofectamine TM RNAiMAX transfection reagent (Invitrogen, Cat. No. 13778150) Add 10 ⁇ L of OPTI-MEM culture medium, mix by pipetting; let stand at room temperature for 5 minutes; discard the MEM culture medium in the 96-well plate, and add 90 ⁇ L of MEM complete culture medium to each well.
  • the mixed solution was added into the 96-well plate at a volume of 10 ⁇ L/well, and the 96-well plate was placed in a 37°C incubator containing 5% CO2 for 6 days. The medium was changed every 3 days, and the culture supernatant was collected on the last day. Detection; set 8 concentrations for each siRNA. Set 3 duplicate wells for each concentration. Select a dsRNA that cannot pair with the target sequence as a control.
  • HBsAg hepatitis B virus surface antigen assay kit
  • Beijing Kemei Diagnostic Technology Co., Ltd., Cat. No. 1010803042 was used to detect the HBsAg content in the cell supernatant.
  • the operation was carried out according to the instructions, and the Spark multifunctional microplate reader (Tecan) was used to read the chemiluminescence value; a standard curve was drawn with the standard substance, and then the concentration of HBsAg in the sample (ng/mL) was calculated, and the concentration-inhibition rate () was processed using EXCEL software. %) data, the EC50 of siRNA's inhibitory effect on HBsAg was calculated through a four-parameter nonlinear regression model.
  • Inhibition rate (%) (1-siRNA group concentration (ng/mL)/control group concentration (ng/mL)) ⁇ 100
  • HBeAg hepatitis B virus e antigen
  • Beijing Kemei Diagnostic Technology Co., Ltd., Cat. No. 1010801042 was used to detect the HBeAg content in the cell supernatant.
  • the operation was carried out according to the instructions, and the Spark multifunctional microplate reader (Tecan) was used to read the chemiluminescence value; a standard curve was drawn with the standard substance, and then the concentration of HBeAg in the sample (NCU/mL) was calculated, and the concentration-inhibition rate (NCU/mL) was processed using EXCEL software. %) data, the EC50 of the inhibitory effect of siRNA on HBeAg was calculated through a four-parameter nonlinear regression model.
  • Inhibition rate (%) (1-siRNA group concentration (NCU/mL)/control group concentration (NCU/mL)) ⁇ 100.
  • dsRNA of the present application shows excellent activity at the cellular level and has a low EC50 value in both HBsAg inhibitory effect and HBeAg inhibitory effect.
  • this result also shows that the dsRNA of the present application has a significant inhibitory effect on HBsAg, indicating the possibility of functional cure of viral hepatitis B by the dsRNA of the present application.
  • the AAV-HBV mouse model was prepared according to the literature method (Dong Xiaoyan et al., Chin J Biotech 2010, May 25;26(5):679-686).
  • the rAAV8-1.3HBV, type D (ayw) used was purchased from Beijing Five Jiahe Gene Technology Co., Ltd., 1.78 ⁇ 10 12 viral genome (vg)/mL, batch number A2020081701.
  • rAAV8-1.3HBV was pre-prepared with sterile PBS into a solution with a concentration of 5 ⁇ 10 11 vg/mL before injection.
  • mice Male C57BL/6 mice aged 6-7 weeks (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were taken and each mouse was injected with 200 ⁇ L rAAV8-1.3HBV through the tail vein, that is, each mouse was injected with 1 ⁇ 10 11 vg rAAV8-1.3HBV.
  • blood was collected from all mice through the orbit to collect serum, and the levels of HBV DNA, HBsAg and HBeAg in the serum were detected.
  • mice were randomly divided into groups (3 mice in each group) according to the serum HBV DNA, HBsAg and HBeAg levels. There were 7 groups in total, namely: PBS control group, M01L01 group, M01EVPL01 group, M21L01 group, M21EVPL01 group, M22L01 group and M22EVPL01 group.
  • the first day of administration was set as day 0 of the experiment.
  • Mice in the administration group were given a single subcutaneous injection of the corresponding dsRNA ligand conjugate solution according to the animal's body weight, with a dosage of 9 mg/kg and a dosage volume of 5 mL/kg body weight; mice in the PBS control group were given a single subcutaneous injection of PBS.
  • the drug volume is 5mL/kg body weight.
  • blood was collected from the orbit, serum was separated, and the HBV DNA, HBsAg, and HBeAg contents in the serum were detected.
  • 100 ⁇ L of blood was collected from the orbital vein. After the whole blood was left to stand at room temperature for 30 minutes, the serum was collected by centrifugation at 3000 rpm and 4°C for 10 minutes.
  • the Hepatitis B Virus Nucleic Acid Assay Kit Shengxiang Biotechnology, Cat. No.
  • the HBV DNA inhibition rate is calculated according to the following equation:
  • HBV DNA inhibition rate (1-HBV DNA content after administration/HBV DNA content before administration) ⁇ 100%.
  • the HBV DNA content is expressed by the number of equivalents (IU) of HBV DNA per milliliter (mL) of serum.
  • the HBsAg inhibition rate is calculated according to the following equation:
  • HBsAg inhibition rate (1-HBsAg content after administration/HBsAg content before administration) ⁇ 100%.
  • the HBsAg content is expressed by the number of equivalents (IU) of HBsAg per milliliter (mL) of serum.
  • the HBeAg inhibition rate is calculated according to the following equation:
  • HBeAg inhibition rate (1 - HBeAg content after administration / HBeAg content before administration) ⁇ 100%.
  • the HBeAg content is expressed by the number of equivalents (U) of HBeAg per milliliter (mL) of serum.
  • dsRNA ligand conjugates are shown in the table below:
  • VP- indicates that the nucleotide on the right side of the hyphen is an (E)-vinyl phosphate modified nucleotide, for example, the 5'- of SEQ ID NO:64 and SEQ ID NO:65 above
  • the terminal u is modified with 5'-(E)-vinyl phosphate.
  • the sequence ID number of the sense strand refers to the sequence ID number of the nucleic acid sequence in the sense strand.
  • the dsRNA ligand conjugate number and L01 at the 3' end of the sense strand sequence indicate that in the dsRNA ligand conjugate, the L01 ligand is connected to the 3' end of the sense strand sequence.
  • the PBS control group showed no inhibitory effect on serum HBV DNA, HBsAg and HBeAg during the entire 84-day monitoring process.
  • the dsRNA ligand conjugate of the present application also shows excellent activity at the animal level, and has a high inhibition rate in serum HBV DNA inhibition, serum HBsAg inhibition and serum HBeAg inhibition, showing Clinical application potential.
  • this result also shows that the dsRNA ligand conjugate of the present application has a significant inhibitory effect on HBsAg, indicating the possibility of functional cure of viral hepatitis B by the dsRNA of the present application.

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Abstract

提供一种用于抑制乙型肝炎病毒的双链核糖核酸、其药学上可接受的盐或其配体缀合物，还提供了包含所述双链核糖核酸、其药学上可接受的盐或其配体缀合物的药物组合物，以及所述双链核糖核酸、其药学上可接受的盐或其配体缀合物的治疗用途。

Description

用于抑制乙型肝炎病毒的双链核糖核酸
相关申请的交叉引用
本申请要求于2022年06月21日向中国国家知识产权局提交的第202210708546.7号中国专利申请的优先权和权益，在此通过引用将所述申请公开的内容整体并入本文。
技术领域
本申请属于生物医药领域，涉及一种双链核糖核酸(dsRNA)、其药学上可接受的盐、其配体缀合物、或药物组合物，其可用于抑制乙型肝炎病毒。
背景技术
乙型病毒性肝炎，简称乙肝，是一种由乙型肝炎病毒(Hepatitis B Virus，简称HBV)感染机体后所引起的疾病。乙型肝炎病毒是一种嗜肝病毒，主要存在于肝细胞内并损害肝细胞，引起肝细胞炎症、坏死、纤维化等。乙型病毒性肝炎分急性和慢性两种。急性乙型肝炎在成年人中大多数可通过其自身的免疫机制而自愈。但是慢性乙型肝炎(CHB)已成为全球健康保健所面临的极大挑战，同时也是引起慢性肝病、肝硬化(cirrhosis)和肝癌(HCC)的主要原因(Edward J.G.,et al.,The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection.Journal of Hepatology(2015)；63:320-328)。
目前被批准上市的抗HBV药物主要是免疫调节剂(干扰素-α和聚乙二醇干扰素-α-2a等)和抗病毒治疗药物(拉米夫定、阿德福韦酯、恩替卡韦、替比夫定、替诺福韦、克拉夫定等)。其中，抗病毒治疗药物属于核苷或核苷酸类药物，其作用机制是抑制HBV DNA的合成，并不能直接减少HBsAg水平。与延长治疗一样，核苷或核苷酸类药物显示HBsAg清除速度类似于自然观察结果(Janssen et al.Lancet(2005),365,123-129；Marcellin et al.N.Engl.J.Med.(2004),351,1206-1217；Buster et al.Hepatology(2007),46,388-394.)。
小干扰RNA(small interfering RNA，siRNA)可以基于RNA干扰(RNA interference，RNAi)机制，以序列特异性的方式抑制或阻断目标基因的翻译或转录来抑制目标基因表达，从mRNA水平发挥抑制作用，同时降低HBV DNA和HBsAg的水平，从而达到治疗疾病的目的。
发明概述
一方面，本申请提供一种双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述双链核糖核酸包含正义链和反义链，其中正义链包含5’-CUGGCUCAGUUUACUAGUA-3’(SEQ ID NO:1)且正义链的长度不超过21个核苷酸，其中反义链包含5’-UACUAGUAAACUGAGCCAGGA-3’(SEQ ID NO:2)且反义链的长度不超过23个核苷酸，所述正义链或反义链任选地是经修饰的。
另一方面，本申请提供一种药物组合物，其包括本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物和药学上可接受的载体或赋形剂。
另一方面，本申请提供一种治疗和/或预防乙型肝炎病毒感染的试剂盒，其包括本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物；以及任选的使用说明书。
另一方面，本申请提供一种治疗和/或预防乙型肝炎病毒感染的方法，其包括给予治疗和/或预防对象(下文也称为受试者)本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物。
另一方面，本申请提供本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物在制备治疗和/或预防乙型肝炎病毒感染的药物中的用途。
另一方面，本申请提供本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物在治疗和/或预防乙型肝炎病毒感染中的用途。
另一方面，本申请提供用于治疗和/或预防乙型肝炎病毒感染的本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物。
在部分实施方式中，所述乙型肝炎病毒感染可以处于疾病的任意阶段，例如急性乙型肝炎或慢性乙型肝炎，或由乙型肝炎病毒感染引起的肝脏疾病包括肝炎、肝纤维化、肝硬化、肝衰竭或肝癌。在部分实施方式中，乙型肝炎病毒感染是慢性乙型肝炎。
在部分实施方式中，本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物作为用于治疗和/或预防乙型肝炎病毒感染的单一治疗剂使用。
在部分实施方式中，本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物与用于治疗和/或预防乙型肝炎病毒感染的其他治疗剂共同使用。
发明详述
以下将对本申请的示例性实施方式进行描述，但是本领域技术人员将理解的是，本申请的保护范围并不限于此，而是可基于本申请的精神和构思进行各种修饰、修改或改变，这些修饰、修改或改变后的内容仍然落在本申请的范围内。
本申请提供一种双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述双链核糖核酸包含正义链和反义链，其中正义链包含5’-CUGGCUCAGUUUACUAGUA-3’(SEQ ID NO:1)中至少15个连续的核苷酸且正义链的长度不超过21个核苷酸，其中反义链包含5’-UACUAGUAAACUGAGCCAGGA-3’(SEQ ID NO:2)中至少15个连续的核苷酸且反义链的长度不超过23个核苷酸，所述正义链或反义链任选地是经修饰的。
本申请提供一种双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述双链核糖核酸包含正义链和反义链，其中正义链包含5’-CUGGCUCAGUUUACUAGUA-3’(SEQ ID NO:1)且正义链的长度不超过21个核苷酸，其中反义链包含5’-UACUAGUAAACUGAGCCAGGA-3’(SEQ ID NO:2)且反义链的长度不超过23个核苷酸，所述正义链或反义链任选地是经修饰的。
本申请还提供一种双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述双链核糖核酸包含正义链和反义链，所述正义链和反义链与上述正义链和反义链的核苷酸序列的全长分别具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在部分实施方式中，本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物可用于抑制乙型肝炎病毒，特别是抑制乙型肝炎病毒基因表达。
在部分实施方式中，本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物是一种RNAi药物。
双链区长度
在部分实施方式中，所述双链核糖核酸的双链区具有19-21对核苷酸的长度，例如所述双链核糖核酸的双链区具有19、20或21对核苷酸的长度。
突出端
在部分实施方式中，所述正义链或反义链任选地包括位于5’端和/或3’端的突出端。
在部分实施方式中，所述突出端含有1、2、3、4或5个核苷酸。在部分实施方式中，所述突出端含有1或2个核苷酸。
在部分实施方式中，所述正义链任选地包括位于5’端和/或3’端的突出端。在一些实施方式中，所述正义链任选地包括位于5’端和/或3’端的1、2、3、4或5个核苷酸的突出端。在一些实施方式中，所述正义链任选地包括位于5’端和/或3’端的1或2个核苷酸的突出端。
在部分实施方式中，所述反义链任选地包括位于5’端和/或3’端的突出端。在一些实施方式中，所述反义链任选地包括位于5’端和/或3’端的1、2、3、4或5个核苷酸的突出端。在一些实施方式中，所述反义链任选地包括位于5’端和/或3’端的1或2个核苷酸的突出端。
在部分实施方式中，所述突出端选自未修饰或经修饰的A、G、C、U或T。
在部分实施方式中，所述反义链任选地包括位于5’端和/或3’端的突出端，所述突出端选自未修饰或经修饰的GA。在部分实施方式中，所述反义链任选地包括位于3’端的突出端，所述突出端选自未修饰或经修饰的GA。
在部分实施方式中，所述突出端选自未修饰或经修饰的U或T。
在部分实施方式中，当所述突出端为1个核苷酸时，所述突出端选自未修饰或经修饰的U或T。在部分实施方式中，当所述突出端为1个核苷酸时，所述突出端选自经2’-O-甲基修饰的U(下文也称为u)或脱氧胸腺嘧啶核苷酸(下文也称为dT)。
在部分实施方式中，当所述突出端为2个核苷酸时，所述突出端选自未修饰或经修饰的UU或TT。在部分实施方式中，当所述突出端为2个核苷酸时，所述突出端选自uu或dTdT。
在部分实施方式中，所述正义链任选地包括位于5’端和/或3’端的突出端，所述突出端选自uu或dTdT。
在部分实施方式中，所述反义链任选地包括位于5’端和/或3’端的突出端，所述突出端选自uu或dTdT。
在部分实施方式中，所述突出端与其相邻的核苷酸通过磷酸酯基或硫代磷酸酯基连接。
在部分实施方式中，所述突出端中的一个或多个核苷酸之间通过磷酸酯基或硫代磷酸酯基连接。
修饰
在部分实施方式中，所述正义链或反义链任选地是经修饰的。
在部分实施方式中，所述正义链或反义链的一个或多个核苷酸是经修饰的。
在部分实施方式中，所述正义链的1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个或21个核苷酸是经修饰的；或者前述任意值形成的范围内的核苷酸是经修饰的，例如1-21个、1-20个、1-19个、1-18个、1-17个、1-16个、1-15个、1-14个、1-13个、1-12个、1-11个或1-10个核苷酸是经修饰的。
在部分实施方式中，所述正义链的1个以上、2个以上、3个以上、4个以上、5个以上、6个以上、7个以上、8个以上、9个以上、10个以上、11个以上、12个以上、13个以上、14个以上、15个以上、16个以上、17个以上、18个以上、19个以上、20个以上或21个以上核苷酸是经修饰的；典型地，所述正义链的10个以上、11个以上、12个以上、13个以上、14个以上、15个以上、16个以上、17个以上、18个以上或19个以上核苷酸是经修饰的。
在部分实施方式中，所述正义链的所有核苷酸是经修饰的。
在部分实施方式中，所述反义链的1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个或23个核苷酸是经修饰的；或者前述任意值形成的范围内的核苷酸是经修饰的，例如1-23个、1-22个、1-21个、1-20个、1-19个、1-18个、1-17个、1-16个、1-15个、1-14个、1-13个、1-12个、1-11个或1-10个核苷酸是经修饰的。
在部分实施方式中，所述反义链的1个以上、2个以上、3个以上、4个以上、5个以上、6个以上、7个以上、8个以上、9个以上、10个以上、11个以上、12个以上、13个以上、14个以上、15个以上、16个以上、17个以上、18个以上、19个以上、20个以上、21个以上、22个以上或23个以上核苷酸是经修饰的；典型地，所述反义链的10个以上、11个以上、12个以上、13个以上、14个以上、15个以上、16个以上、17个以上、18个以上、19个以上、20个以上或21个以上核苷酸是经修饰的。
在部分实施方式中，所述反义链的所有核苷酸是经修饰的。
在部分实施方式中，所述正义链的所有核苷酸和所述反义链的所有核苷酸是经修饰的。
在部分实施方式中，所述修饰选自核苷酸的糖基修饰、核苷酸间的连接键修饰或末端修饰。
在部分实施方式中，所述核苷酸的糖基修饰选自脱羟基化、氟代、氨基化、烷基化、羟基烷基化或羟基烯基化。
在部分实施方式中，所述核苷酸的糖基修饰发生在糖基的2’位。
在部分实施方式中，所述糖基修饰选自2’-脱羟基化、2’-氟代、2’-氨基、2’-烷基、2’-O-烷基、2’-O-醚基、2’-O-烯基。在部分实施方式中，所述糖基修饰选自2’-脱羟基化、2’-氟代、2’-氨基、2’-甲基、2’-乙基、2’-甲基-O-甲基、2’-乙基-O-甲基、2’-O-甲基、2’-O-乙基、2’-O-乙基-O-甲基或2’-O-烯丙基。
在部分实施方式中，所述核苷酸间的连接键修饰选自硫代磷酸酯(PS)、二硫代磷酸酯(PS2)、甲基膦酸酯(MP)、甲氧基丙基膦酸酯(MOP)或氨基膦酸酯。在部分实施方式中，所述核苷酸间的连接键修饰选自硫代磷酸酯(PS)。
在部分实施方式中，所述末端修饰选自5’-末端修饰或3’-末端修饰。在部分实施方式中，所述末端修饰选自5’-磷酸酯、5’-甲基膦酸酯(5’-MP)、5’-硫代磷酸酯(5’-PS)或5’-(E)-乙烯基膦酸酯(5’-(E)-VP)。
在部分实施方式中，所述修饰选自2’-脱羟基化、2’-氟代、2’-氨基、2’-甲基、2’-乙基、2’-甲基-O-甲基、2’-乙基-O-甲基、2’-O-甲基、2’-O-乙基、2’-O-烯丙基、硫代磷酸酯基、甲基膦酸酯基、氨基膦酸酯基、5’-磷酸酯基、3’-磷酸酯基、5’-(E)-乙烯基磷酸酯或3’-(E)-乙烯基磷酸酯。
在部分实施方式中，所述修饰选自2’-脱羟基化、2’-氟代、2’-O-甲基、2’-O-乙基、2’-O-乙基-O-甲基、2’-O-烯丙基、硫代磷酸酯基或5’-(E)-乙烯基磷酸酯。
在部分实施方式中，所述修饰选自2’-氟代、2’-O-甲基、硫代磷酸酯基或5’-(E)-乙烯基磷酸酯。
如下文中将详述的，当提及正义链和反义链的第n位核苷酸时，均从5’端开始计数。
在部分实施方式中，所述正义链的第1、2位核苷酸之间和/或正义链的第2、3位核苷酸之间采用硫代磷酸酯基修饰。
在部分实施方式中，所述反义链的第1、2位核苷酸之间、反义链的第2、3位核苷酸之间、反义链的第19、20位核苷酸之间和/或反义链的第20、21位核苷酸之间采用硫代磷酸酯基修饰。
在部分实施方式中，所述反义链的5’-末端采用5’-(E)-乙烯基磷酸酯修饰。
在部分实施方式中，所述正义链有2、3、4或5个核苷酸采用2’-氟代修饰。在部分实施方式中，所述正义链有14、15、16或17个核苷酸采用2’-O-甲基修饰。
在部分实施方式中，所述正义链的第1至6位核苷酸中有0个、1个或2个核苷酸采用2’-氟代修饰。
在部分实施方式中，所述正义链的第1至6位核苷酸中有4个、5个或6个核苷酸采用2’-O-甲基修饰。
在部分实施方式中，所述正义链的第7至9位核苷酸中有2个或3个核苷酸采用2’-氟代修饰。
在部分实施方式中，所述正义链的第7至9位核苷酸中有0个或1个核苷酸采用2’-O-甲基修饰。
在部分实施方式中，所述正义链的第10至19位核苷酸中有0个或1个核苷酸采用2’-氟代修饰。
在部分实施方式中，所述正义链的第10至19位核苷酸中有8个、9个或10个核苷酸采用2’-O-甲基修饰。
在部分实施方式中，所述正义链的第7、8和9位核苷酸采用2’-氟代修饰，并且所述正义链的第1至6和/或10至19位核苷酸中有0个、1个或2个核苷酸也采用2’-氟代修饰。在部分实施方式中，所述正义链的第7、8和9位核苷酸采用2’-氟代修饰，并且所述正义链的第1至6和10至19位核苷酸中有0个、1个或2个核苷酸也采用2’-氟代修饰。在一些实施方式中，所述正义链的第7、8和9位核苷酸采用2’-氟代修饰，并且所述正义链的第1至6和10至19位核苷酸中有1个核苷酸也采用2’-氟代修饰。
在部分实施方式中，所述反义链有2、3、4、5、6或7个核苷酸采用2’-氟代修饰。
在部分实施方式中，所述反义链有14、15、16、17、18或19个核苷酸采用2’-O-甲基修饰。
在部分实施方式中，所述反义链的第2、6、14、16位核苷酸中有3个或4个核苷酸采用2’-氟代修饰。在一些实施方式中，所述反义链的第2、6、14、16位核苷酸中有4个核苷酸采用2’-氟代修饰。
在部分实施方式中，所述反义链的第2、6、8、9、14、16位核苷酸中有3个、4个、5个或6个核苷酸采用2’-氟代修饰。在一些实施方式中，所述反义链的第2、6、8、9、14、16位核苷酸中有4个或6个核苷酸采用2’-氟代修饰。
在一些实施方案中，所述反义链的第2、6、14和16位核苷酸采用2’-氟代修饰，并且所述反义链的第1、3至5、7至13、15和/或17至21位核苷酸中任意一个或两个也采用2’-氟代修饰。在一些实施方案中，所述反义链的第2、6、14和16位核苷酸采用2’-氟代修饰，并且所述反义链的第1、3至5、7至13、15和17至21位核苷酸中任意一个或两个也采用2’-氟代修饰。
在部分实施方式中，所述反义链的第1、3至5、7至13、15、17至21位核苷酸中有14、15、16或17个核苷酸采用2’-O-甲基修饰。
在一个具体的实施方式中，所述正义链的第5、7、8和9位核苷酸采用2’-氟代修饰，所述正义链的其他核苷酸采用2’-O-甲基修饰，并且所述反义链的第2、6、8、9、14和16位核苷酸采用2’-氟代修饰，所述反义链的其他核苷酸采用2’-O-甲基修饰。
在一个具体的实施方式中，所述正义链的第5、7、8和9位核苷酸采用2’-氟代修饰，所述正义链的其他核苷酸采用2’-O-甲基修饰，并且所述反义链的第2、6、14和16位核苷酸采用2’-氟代修饰，所述反义链的其他核苷酸采用2’-O-甲基修饰。
在一个具体的实施方式中，所述正义链的第7、8和9位核苷酸采用2’-氟代修饰，所述正义链的其他核苷酸采用2’-O-甲基修饰，并且所述反义链的第2、6、14和16位核苷酸采用2’-氟代修饰，所述反义链的其他核苷酸采用2’-O-甲基修饰。
在一个具体的实施方式中，所述正义链的第5、7、8和9位核苷酸采用2’-氟代修饰，所述正义链的其他核苷酸采用2’-O-甲基修饰，并且所述反义链的第2、6、8、9、14和16位核苷酸采用2’-氟代修饰，所述反义链的其他核苷酸采用2’-O-甲基修饰，并且正义链的第1、2位核苷酸之间、正义链的第2、3位核苷酸之间、反义链的第1、2位核苷酸之间、反义链的第2、3位核苷酸之间、反义链的第19、20位核苷酸之间和/或反义链的第20、21位核苷酸之间采用硫代磷酸酯基修饰。任选地，所述反义链的5’-末端采用5’-(E)-乙烯基磷酸酯修饰。
在一个具体的实施方式中，所述正义链的第5、7、8和9位核苷酸采用2’-氟代修饰，所述正义链的其他核苷酸采用2’-O-甲基修饰，并且所述反义链的第2、6、14和16位核苷酸采用2’-氟代修饰，所述反义链的其他核苷酸采用2’-O-甲基修饰，并且正义链的第1、2位核苷酸之间、正义链的第2、3位核苷酸之间、反义链的第1、2位核苷酸之间、反义链的第2、3位核苷酸之间、反义链的第19、20位核苷酸之间和/或反义链的第20、21位核苷酸之间采用硫代磷酸酯基修饰。任选地，所述反义链的5’-末端采用5’-(E)-乙烯基磷酸酯修饰。
在一个具体的实施方式中，所述正义链的第7、8和9位核苷酸采用2’-氟代修饰，所述正义链的其他核苷酸采用2’-O-甲基修饰，所述反义链的第2、6、14和16位核苷酸采用2’-氟代修饰，所述反义链的其他核苷酸采用2’-O-甲基修饰，并且正义链的第1、2位核苷酸之间、正义链的第2、3位核苷酸之间、反义链的第1、2位核苷酸之间、反义链的第2、3位核苷酸之间、反义链的第19、20位核苷酸之间和/或反 义链的第20、21位核苷酸之间采用硫代磷酸酯基修饰。任选地，所述反义链的5’-末端采用5’-(E)-乙烯基磷酸酯修饰。
配体
在部分实施方式中，所述配体包括分支基团和连接基团。
在部分实施方式中，所述配体通过连接基团与双链核糖核酸连接。本文中，将连接有配体的双链核糖核酸称为双链核糖核酸配体缀合物。
在部分实施方式中，所述配体通过分支基团连接一个或多个靶向基团。在部分实施方式中，所述分支基团包含靶向基团。
在部分实施方式中，所述配体含有至少一个靶向基团。在部分实施方式中，所述配体含有一个、二个、三个、四个或五个靶向基团。在部分实施方式中，所述配体含有二个、三个或四个靶向基团。在部分实施方式中，所述配体含有三个靶向基团。
在部分实施方式中，所述靶向基团选自GalNAc基团。
在部分实施方式中，所述配体含有一个、二个、三个、四个或五个GalNAc基团。在部分实施方式中，所述配体含有二个、三个或四个GalNAc基团。在部分实施方式中，所述配体含有三个GalNAc基团。
在部分实施方式中，所述分支基团选自：
在部分实施方式中，所述连接基团选自：
在部分实施方式中，所述配体选自：
在部分实施方式中，所述配体与正义链或反义链连接。在部分实施方式中，所述配体与正义链或反义链的5’或3’末端连接。在部分实施方式中，所述配体与正义链的5’或3’末端连接。在部分实施方式中，所述配体与正义链的3’末端连接。
在部分实施方式中，所述配体通过磷酸酯基或硫代磷酸酯基与双链核糖核酸的正义链或反义链连接。在部分实施方式中，所述配体通过磷酸酯基或硫代磷酸酯基与正义链连接。在部分实施方式中，所述配体通过磷酸酯基或硫代磷酸酯基与正义链的3’末端连接。
在部分实施方式中，所述双链核糖核酸配体缀合物如下所示：
在部分实施方式中，所述双链核糖核酸配体缀合物选自：
在部分实施方式中，所述双链核糖核酸配体缀合物选自：
在部分实施方式中，所述双链核糖核酸的未修饰的正义链选自：
在部分实施方式中，所述双链核糖核酸的未修饰的反义链选自：
在部分实施方式中，所述双链核糖核酸的经修饰的正义链选自：

在部分实施方式中，所述双链核糖核酸的经修饰的反义链选自：

本申请的双链核糖核酸可以通过包含上述正义链中的任一者以及上述反义链中的任一者而形成。
例如，所述双链核糖核酸包含如下正义链中的任一者：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41或SEQ ID NO:42；并且
所述双链核糖核酸包含如下反义链中的任一者：SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:24、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65。
在部分实施方式中，所述双链核糖核酸(未经修饰)选自：
在部分实施方式中，所述双链核糖核酸(经修饰)选自：


在部分实施方式中，所述双链核糖核酸配体缀合物选自：


其中，所述L代表配体，所述配体如前所述；在部分实施方式中，所述配体选自L01配体或L02配体。其中，VP-表示该连字符右侧的一个核苷酸为(E)-乙烯基磷酸酯修饰的核苷酸，例如，上文中的SEQ ID NO:64和SEQ ID NO:65的5’-末端的u采用5’-(E)-乙烯基磷酸酯修饰。其中，正义链的序列ID编号是指正义链中核酸序列的序列ID编号。dsRNA配体缀合物序号及正义链序列3’端处的L表示在该dsRNA配体缀合物中，在正义链序列3’端处连接有配体。
药学上可接受的盐
在部分实施方式中，如上所述的盐选自碱加成盐、酸加成盐及其组合。
在部分实施方式中，碱加成盐选自钠、钾、钙、铵、有机胺、镁盐及其组合，酸加成盐选自无机酸盐、有机酸盐及其组合。
在部分实施方式中，无机酸选自盐酸、氢溴酸、硝酸、碳酸、碳酸氢根、磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸及其组合，有机酸选自乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸、甲磺酸及其组合。
有益效果
本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物在HBV不同基因型中有较高的覆盖率，适用于多种基因型患者的治疗和/或预防。本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物还具有良好的HBV抑制活性和稳定性。特别地，本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物在细胞水平和动物水平均表现出优异的HBV mRNA抑制效率，同时能够有效地降低乙型肝炎病毒表面抗原和e抗原表达，显示出对乙型肝炎病毒感染、特别是慢性乙型肝炎良好的治疗作用。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通技术人员所理解的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
本申请中，除非另有说明，否则术语“包含、包括和含有”或等同物为开放式表述，意味着除所列出的要素、组分或步骤外，还可涵盖其他未指明的要素、组分或步骤。
当任何变量在化合物、核苷酸、单链或双链结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，本申请所述的修饰是独立的发生，即除非另有所指，正义链的修饰不会影响反义链的修饰，某一核苷酸的修饰不会影响另一核苷酸的修饰，某一核苷酸的糖基上的修饰不会影响同一核苷酸的糖基的另一处的修饰。所述影响包括是否经修饰，也包括采用何种修饰类型。
本申请的HBV基因组序列或mRNA序列的实例可以通过已公开的数据库取得，例如Genbank等，例如HBV基因组序列(GenBank accession#EU554538.1)。本申请所采用的术语“HBV”也指HBV基因组的任何天然产生的DNA序列变异。
如本领域所知，术语“干扰RNA”或“RNAi”或“干扰RNA序列”指这样的单链RNA(例如，成熟的miRNA)或双链RNA(例如，双链体RNA诸如siRNA、aiRNA或前-miRNA)，当干扰RNA与靶基因或序列处于相同细胞中时，其能够降低或抑制该靶基因或序列的表达(例如，通过介导与干扰RNA序 列互补的mRNA的降解或抑制与干扰RNA序列互补的mRNA的翻译或转录)。干扰RNA可以与靶基因或序列具有基本或完全的同一性，或可以包括错配区(即，错配序列)。
本申请的双链核糖核酸作为干扰RNA发挥作用。下文中，有时也将本申请的双链核糖核酸称为siRNA。
如本领域所知，术语“错配区”或“错配序列”指干扰RNA(例如，siRNA、aiRNA、miRNA)序列的一部分，该部分与其靶序列不具有100％的互补性。干扰RNA(例如，siRNA、aiRNA、miRNA)可以具有至少1、2、3、4、5、6或更多个错配区。错配区可以是连续的或可以被1、2、3、4、5、6、7、8、9、10、11、12或更多个核苷酸分开。错配区可以包括单个核苷酸或可以包括2、3、4、5、6或更多个核苷酸。
术语“同一性”是指两个核苷酸序列之间或两个氨基酸序列之间的相似性。序列的同一性优选地涉及序列中的、在具有相同长度的两个或更多个序列中具有相同的位置的核苷酸或氨基酸的百分比。具体地，两个氨基酸序列或两个核苷酸序列的“％同一性”可以通过如下确定：以最优比较为目的比对序列(例如，可以在任一序列中引入空位以与另一序列最优比对)和比较相应位置的氨基酸或核苷酸。空位通常被视为不相同的位置，而无论其在比对中的实际位置。“最优比对”一般是导致最高同一性百分比的两个序列的比对。同一性百分比通过所比较的序列中相同核苷酸的数量来确定(即，％同一性＝相同位置数/总位置数×100)。本申请的序列同一性至少为80％、85％、90％或95％，优选为至少90％，非限制性实例包括：80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。可以利用本领域技术人员已知的数学算法完成两个序列之间同一性百分比的确定。
如本领域所知，干扰RNA包括“小干扰RNA(small interfering RNA,siRNA)”，其长度例如为约15-60个、15-50个、15-40个、15-30个、15-25个、19-25个、19-23个或19-21个核苷酸。本申请的双链核糖核酸可作为此类siRNA发挥作用，并可具有相应的长度。
如本申请所用，所述正义链或反义链的核苷酸的位次均从5’端开始计数。对于正义链为19个核苷酸，反义链为21个核苷酸的dsRNA，例如，5’-CUGGCUCAGUUUACUAGUA-3’(SEQ ID NO:1)的第1位是C，第19位是A；5’-UACUAGUAAACUGAGCCAGGA-3’(SEQ ID NO:2)的第1位是U，第21位是A。对于正义链为19个以上核苷酸，反义链为21个以上核苷酸的dsRNA，例如，正义链在SEQ ID NO:1基础上于末端增加若干个核苷酸，反义链在SEQ ID NO:2基础上于末端增加若干个核苷酸，其正义链或反义链的核苷酸位次的计数方法不会因此而改变，即正义链的第1位仍是SEQ ID NO:1的第1位，反义链的第1位仍是SEQ ID NO:2的第1位。
如本领域所知，并且除非另外说明，使用术语“互补”描述第一核酸序列和第二核酸序列的关系时，是指在特定条件下含有第一核酸序列的寡核苷酸或多核苷酸与含有第二核酸序列的寡核苷酸或多核苷酸杂交并形成双链结构的能力。如本申请所述，“互补”序列也可以包括由非Watson-Crick碱基配对和/或非天然或修饰的核苷酸形成的碱基配对形成的双链结构，或可以为完全由非Watson-Crick碱基配对和/或非天然或修饰的核苷酸形成的碱基配对形成的双链结构，只要满足相对于它们杂交的能力的上述要求。
如本领域所知，“完全互补”的序列包括含有第一核酸序列的寡核苷酸或多核苷酸与含有第二核酸序列的寡核苷酸或多核苷酸的在第一核酸序列和第二核酸序列的全长上的碱基配对。
如本领域所知，“基本互补”表示两条核酸序列之间完全互补或至少85％(例如85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)的重叠核苷酸是互补的。
本申请的术语“互补”、“完全互补”和“基本互补”可以根据dsRNA的正义链和反义链之间，或dsRNA的反义链和目标序列之间的碱基配对使用，如从它们使用的内容中可以理解。
如本领域所知，所述“双链核糖核酸”、“双链RNA”或“dsRNA”可相互替换使用。术语“dsRNA”包含两条反向平行且互补的核酸链，相对于靶标RNA(例如HBV基因)具有“正义”或“反义”的取向。在本申请的实施例中，dsRNA可通过RNA干扰(RNAi)机制降解靶标RNA(例如mRNA)。
本申请的双链核糖核酸含有正义链和反义链。术语“正义链”或“有义链”是指dsRNA双链中，与反义链的一个区域基本互补的单链。术语“反义链”、“导向链”或“引导链”是指dsRNA双链中，与目标序列的一个区域基本互补的单链。若当正义链未与反义链完全互补时，可能在分子内部或末端区发生错配。通常，最能忍受的错配是在末端区内。若当反义链未与目标序列完全互补时，可能在分子内部或末端区发生错配。通常，最能忍受的错配是在末端区内。
如本领域所知，dsRNA的双链可以具有相同或不同的核苷酸的数量。通过互补形成的双链区的长度可为容许降解靶标RNA的任何长度，且可能的长度在约9至36对核苷酸的范围内，例如15至30对、16至28对、19至21对等等。
如本领域所知，在双链区以外，dsRNA可以包括一个或多个核苷酸突出端，所述突出端是指至少一个未配对的核苷酸/核苷类似物。例如，当dsRNA的一条链的3’端超出另一链5’端(或反之亦然)，即存在 突出端。在所述“突出端”的核苷酸可以包括0-5个核苷酸，其中“0”表示没有“突出端”，而“5”表示在dsRNA双链的单链上有5个额外的(即未与另一单链形成配对的)核苷酸。这些任选的“突出端”可以位于dsRNA两条链中的任意单链的5’和/或3’末端。在部分实施方式中，“突出端”包括0-5个核苷酸。在部分实施方式中，“突出端”包括0-2个核苷酸。在部分实施方式中，dsRNA的正义链3’和/或5’端的“突出端”具有0-2个核苷酸。在部分实施方式中，dsRNA的反义链3’和/或5’端的“突出端”具有0-2个核苷酸。形成“突出端”的核苷酸可以是A、G、C、U或T或其修饰结构。形成“突出端”的核苷酸可以是U、T或dT或其修饰结构。在部分实施方式中，“突出端”包括但不限于“TT”、“dTdT”、“UU”或其相应的经修饰结构，例如经2’甲氧基修饰的UU，即uu。在部分实施方式中，dsRNA的反义链3’和/或5’端的“突出端”与靶标RNA基本互补。在部分实施方式中，dsRNA的反义链3’和/或5’端的“突出端”与靶标RNA完全互补。在部分实施方式中，dsRNA的反义链3’端的“突出端”与靶标RNA完全互补。在部分实施方式中，dsRNA的反义链3’端的“突出端”选自未修饰或经修饰的GA。术语“钝”或“钝端”是指在dsRNA的末端没有未配对的核苷酸，即没有核苷酸突出端。两端都是“钝端”的dsRNA是全长都是双链区的dsRNA，即在分子的任意一端都没有核苷酸突出端。
本申请中，所述dsRNA或其中任一单链任选地是被修饰的，无论是未修饰或经修饰的核糖核酸都在本申请的保护范围内。所述修饰不会导致所述dsRNA抑制HBV基因表达的功能明显削弱或丧失。所述dsRNA或其中任一单链的修饰可位于5’端和/或3’端末端、核苷酸或核苷酸间的连接键。可采用本领域中公知的方法进行合成或修饰。
本申请中，所述核苷酸的修饰包括但不限于发生在核苷酸的糖基上，包括一个或多个经取代或脱除的糖基部分基团，如脱除羰基上的羟基，或发生氟代、氨基化、烷基化、羟基烷基化或羟基烯基化。糖基上的修饰可发生在糖环上的各个位置。示例性地，所述核苷酸的糖基上的修饰包括但不限于2’-脱羟基化、2’-氟代、2’-氨基、2’-甲基、2’-乙基、2’-甲基-O-甲基、2’-乙基-O-甲基、2’-O-甲基、2’-O-乙基、2’-O-乙基-O-甲基或2’-O-烯丙基，结构可如下所示：
其中Base表示碱基。
本申请中，所述核苷酸间的连接键的修饰包括对磷酸酯基的原子或官能团的取代或替换，例如硫代磷酸酯(PS)、二硫代磷酸酯(PS2)、甲基膦酸酯(MP)、甲氧基丙基膦酸酯(MOP)或氨基膦酸酯。
本申请中，大写字母G、C、A、U或T分别各自通常代表含有鸟嘌呤、胞嘧啶、腺嘌呤、尿嘧啶、 胸腺嘧啶作为碱基的核苷酸。小写字母g、c、a、u分别代表其相应大写字母所代表的核苷酸被2’-甲氧基修饰，即g、c、a、u分别代表2’-O-甲基G、2’-O-甲基C、2’-O-甲基A、2’-O-甲基U。大写字母加上其右侧的小写字母f代表其相应的大写字母所代表的核苷酸被2’-氟修饰，即Gf、Cf、Af、Uf分别代表2’-氟G、2’-氟C、2’-氟A、2’-氟U。小写字母s表示与s左右相邻的两个核苷酸残基之间为硫代磷酸酯基连接，例如“csu”表示c和u残基之间通过硫代磷酸酯基连接。VP-表示该连字符右侧的一个核苷酸为(E)-乙烯基磷酸酯修饰的核苷酸，例如“VP-u”表示(E)-乙烯基磷酸酯修饰的2’-O-甲基U。
本申请中，所述5’端和/或3’端修饰指发生在dsRNA或其中任一单链5’端和/或3’端的修饰，例如磷酸化、接合或反向键联等。以5’端为例，包括但不限于5’-磷酸酯、5’-甲基膦酸酯(5’-MP)、5’-硫代磷酸酯(5’-PS)或5’-(E)-乙烯基膦酸酯(5’-(E)-VP)，结构可如下所示：
其中Base表示碱基，X选自羟基或糖基上的2’修饰。
本申请中，所述配体是一种与dsRNA连接的基团，该配体包括分支基团(branched group)和连接基团(linker)，并且所述dsRNA、连接基团和分支基团依次连接(例如式104所示)。所述分支基团含有药学上可接受的至少一个(例如一个、二个、三个、四个或五个)靶向基团(targeting group)，其将dsRNA靶向特定组织或增强细胞吸收作用。所述靶向基团例如但不限于GalNAc(N-乙酰半乳糖胺，N-Acetylgalactosamine，例如式105所示)基团。多个所述靶向基团之间通过分支基团以串联或并联的方式连接。其中，GalNAc基团可以是一价、二价、三价、四价。这里所述的一价、二价、三价、四价分别指dsRNA分子与含有作为靶向基团的GalNAc的配体形成dsRNA配体缀合物后，该dsRNA配体缀合物中dsRNA分子与GalNAc分子的摩尔比为1:1、1:2、1:3和1:4。在部分实施方式中，当本申请的dsRNA与含有GalNAc的配体缀合时，GalNAc分子是三价或四价。在部分实施方式中，当本申请的dsRNA与含有GalNAc的配体缀合时，GalNAc分子是三价。
本申请中，所述配体可以连接在核苷酸的磷酸酯基、2’-位羟基、3’-位羟基或者碱基上。所述配体可以连接在dsRNA的任一核苷酸上，包括但不限于正义链或反义链的5’或3’末端核苷酸或非末端的中间位置核苷酸。当配体连接在dsRNA链的末端时，所述配体可以连接在核苷酸的磷酸酯基上；当配体连接在dsRNA的中间位置核苷酸时，所述配体可以连接在核苷酸的糖环或者碱基上。
本申请中，所述配体的种类或制备方法可参考本领域已知的方法，包括但不限于记载于WO2009082607、WO2014025805、WO2015006740、WO2021249484中的配体及其制备方法，以引用的方式将其全部公开内容并入本申请中。示例性的配体包括但不限于上文所述的L01或L02。
本申请中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以非共价连接的方式或共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过非共价连接或共价连接而形成的化合物。本申请中，优选使用以共价连接的方式彼此连接的缀合物。
本申请中，所述配体与正义链或反义链的5’或3’末端连接。优选地，所述配体与正义链的5’或3’末端连接。更优选地，所述配体与正义链的3’末端连接。示例性地，所述dsRNA与配体连接形成的dsRNA配体缀合物如下式102或式103所示：
本申请的化合物(例如本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物)可以存在特定的几何或立体异构体形式，所有这些几何或立体异构体形式都属于本申请的范围之内。本申请设想所有的这类化合物，包括(R)-和(S)-对映体、非对映异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本申请的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本申请的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，用楔形实线键和楔形虚线键表示一个立体中心的绝对构型，用直形实线键和直形虚线键表示立体中心的相对构型，用波浪线表示楔形实线键或楔形虚线键或用波浪线表示直形实线键和/或直形虚线键
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本申请某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。本申请的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)、碘-125(125I)或C-14(14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本申请的化合物的所有同位素组成的变换，无论是否具备放射性，都包括在本申请的范围之内。
术语“治疗”意为将本申请所述化合物或制剂(例如本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物)进行给药以改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：(i)抑制疾病或疾病状态，即遏制其发展；(ii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
术语“预防”意为将本申请所述化合物或制剂(例如本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物)进行给药以预防疾病或与所述疾病相关的一个或多个症状，且包括：预防疾病或疾病状态在受试者中出现，特别是当这类受试者易患有该疾病状态，但尚未被诊断为已患有该疾病状态时。
术语“受试者”、“患者”或“对象”在本文中可互换使用，是指已成为治疗、观察或实验对象的动物。在部分实施方式中，所述受试者为哺乳动物，优选为灵长类动物，更优选为人。
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请化合物(例如本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物)的用量。构成“治疗有效量”的本申请化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的受试者的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本申请内容而确定。
本申请的化合物的治疗剂量可根据例如以下而定：治疗的具体用途、给予化合物的方式、患者的健康和状态，以及签处方医师的判断。本申请的化合物在药物组合物中的比例或浓度可不固定，取决于多种因素，它们包括剂量、化学特性(例如疏水性)和给药途径。例如可通过含约0.1～10％w/v该化合物的生理缓冲盐水溶液提供本申请化合物，用于肠胃外给药。某些典型剂量范围为约1μg/kg～约1g/kg体重/日。在某些实施方式中，剂量范围为约0.01mg/kg～约100mg/kg体重/日。剂量很可能取决于此类变量，如疾病或病症的种类和发展程度、具体患者的一般健康状态、所选择的化合物的相对生物学效力、赋形剂制剂及其给药途径。可通过由体外或动物模型试验系统导出的剂量-反应曲线外推，得到有效剂量。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和其他动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
作为药学上可接受的盐，例如，可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。
术语“药物组合物”是指一种或多种本申请的化合物(例如本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物)与药学上可接受的载体、辅料或赋形剂组成的混合物。药物组合物的目的是有利于对 有机体给予本申请的化合物。在本文中，术语“药物组合物”和“制剂”具有相同的含义，并可互换使用。本文所用的载体、辅料或赋形剂包括适合于期望的特定剂型的任意的和所有的溶剂，稀释剂或其它液体赋形剂，分散剂或助悬剂，表面活性剂，等渗剂，增稠剂或乳化剂，防腐剂，固体粘合剂，润滑剂等。用于配制药学上可接受的组合物的各种载体、辅料或赋形剂和用于其制备的方法可采用已知技术。除了与本申请的化合物不相容(例如产生任何不良的生物学效应或者其它以有害方式与药学上可接受的组合物的任何其它组分相互作用)的任何常规载体介质，其使用都涵盖在本申请的范围内。在一些具体的实施方式中，本文所用的载体、辅料或赋形剂是dsRNA给药领域常规使用的载体、辅料或赋形剂。
本申请的药物组合物可通过将本申请的化合物与适宜的药学上可接受的载体、辅料或赋形剂组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等。
给予本申请化合物或其药物组合物的典型途径包括但不限于口服、直肠、局部、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。
本申请的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
在本申请的试剂盒中，其包括本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物；以及任选的使用本申请的双链核糖核酸、其药学上可接受的盐、其配体缀合物、或药物组合物治疗和/或预防乙型肝炎病毒感染的说明书。
本申请所使用的溶剂可经市售获得。
如无特殊说明，本申请柱层析、制备薄层硅胶色谱所用溶剂配比均为体积比。
本领域知晓，可以通过使用具有相应修饰的核苷单体将修饰的核苷酸基团引入本申请所述的dsRNA。本领域技术人员可以从现有技术中获知相应修饰的核苷单体的制备方法和将修饰的核苷酸基团引入dsRNA的方法。所有修饰的核苷单体可以从商业途径获取或采用已知的方法制备。
本领域知晓，可以通过本领域常规的核糖核酸制备方法(例如固相合成和液相合成)得到所需的核糖核酸，例如可以通过亚磷酰胺固相合成技术合成所需的核糖核酸。本申请的双链核糖核酸、其药学上可接受的盐或其配体缀合物的制备方法包含以下步骤：按照双链核糖核酸的正义链或反义链的核苷酸种类或顺序，以3’到5’的方向将核苷酸单体依次连接以合成正义链和反义链。每个核苷酸单体的连接包括脱保护、偶连、盖帽、氧化或硫化四步反应。本领域技术人员可以采用常规的反应条件、试剂的种类和用量，或根据试验情况进行调整以实现所述脱保护、偶连、盖帽、氧化或硫化反应。
在一些实施方式中，对于含有配体的核糖核酸的合成，可以在寡核糖核苷酸的合成过程中或合成完成后通过偶连反应将配体与核糖核酸连接，也可以先将配体连接到固相载体，然后按照3’到5’的方向将核苷单体与配体-固相载体依次连接。
纯化和脱盐的方法是本领域技术人员所公知的。例如，可以通过制备型离子色谱方法完成核糖核酸的纯化。又例如，可以通过反相色谱纯化方法或超滤离心方法完成核糖核酸的脱盐。
退火的方法是本领域技术人员所公知的。例如可以将正义链和反义链以1:1摩尔量混合，加热至70-95℃，随后降至室温以形成双链结构。
在合成过程中，可以通过例如离子交换色谱检测核糖核酸浓度，或通过液质联用色谱测定分子量，或通过微量分光光度计测定浓度以控制合成质量，此类检测方法是本领域技术人员所公知的。
除非另外特别说明，否则单数术语涵盖复数术语，并且复数术语涵盖单数术语。除非另外特别说明，否则词语“一个”或“一种”意指“至少一个”或“至少一种”。除非另外说明，否则“或”的使用意指“和/或”。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它已确定的出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请人可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本文中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。
附图说明
图1示出了AAV-HBV小鼠实验血清HBV DNA水平。
图2示出了AAV-HBV小鼠实验血清HBsAg水平。
图3示出了AAV-HBV小鼠实验血清HBeAg水平-。
具体实施方式
下面通过实施例对本申请进行详细描述，但并不意味着对本申请构成任何不利限制。本申请的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术人员所熟知的等同替换方式，优选的实施方式包括但不限于本申请的实施例。对本领域的技术人员而言，在不脱离本申请精神和范围的情况下针对本申请具体实施方式进行各种变化和改进将是显而易见的。
试剂与材料
合成所用的市售保护单体
合成所用的试剂
制备实施例1将GalNAc配体(含有GalNAc基团作为靶向基团的配体)连接到固相载体上
称取氨基-CPG 1.0g(氨基载量：30-40μmol/g)，加入5mL脱帽剂，反应1min，排干试剂，上述操作循环重复4次。按载量为35μmol计算，加入2eq GalNAc配体/乙腈溶液，再加入2.5eq DIC、2.5eq DMAP组成的缩合试剂。25-30℃反应24h。结束后，滤除溶剂，无水乙腈清洗，再按上述用量加入GalNAc配体和缩合试剂，再反应24h。反应结束后，无水乙腈清洗固相载体，加入4mL Capping A和Capping B的1:1混合试剂，反应2min，排干后，再加入4mL混合试剂，反应2min，排干，无水乙腈洗涤，30℃真空干燥1h，备用。此实施例采用L01配体。
制备实施例2双链核糖核酸的制备
2.1.1 GalNAc配体-ssRNA(正义链)的合成
在K&A核酸合成仪上，将上述GalNAc配体-固相载体放入合成柱中，按照标准的亚磷酰胺技术合成ssRNA。具体步骤包括：
1)DMT脱除：先用乙腈洗涤树脂两次，用3％三氯乙酸/DCM脱除树脂上的DMT，乙腈洗涤4～5次。
2)缩合：合成柱中加入单体，加入缩合试剂，室温缩合10分钟。用乙腈洗涤4～5次。
3)封闭：先用Capping A，再用Capping B封闭未反应的羟基。用乙腈洗涤4～5次。
4)氧化：加入氧化试剂，氧化时间2分钟，后用乙腈洗涤4～5次。
重复1)～4)的步骤，至所有序列合成完成，最后用脱帽剂脱除DMT，乙腈洗涤4～5次。
硫代磷酸酯键的形成：用硫代试剂替换氧化试剂，硫代时间设置为10分钟，完成硫代磷酸酯键的形成。
2.1.2 ssRNA(无配体正义链)的合成
在K&A核酸合成仪上，将通用性CPG固相载体放入合成柱中，按照标准的亚磷酰胺技术合成ssRNA。具体步骤包括：
1)DMT脱除：树脂先用乙腈洗涤两次，用3％三氯乙酸/DCM脱除树脂上的DMT，乙腈洗涤4～5次。
2)缩合：合成柱中加入单体，加入缩合试剂，室温缩合10分钟。用乙腈洗涤4～5次。
3)封闭：先用Capping A，再用Capping B封闭未反应的羟基。用乙腈洗涤4～5次。
4)氧化：加入氧化试剂，氧化时间2分钟，后用乙腈洗涤4～5次。
重复1)～4)的步骤，至所有序列合成完成，最后用脱帽剂脱除DMT，乙腈洗涤4～5次。
硫代磷酸酯键的形成：用硫代试剂替换氧化试剂，硫代时间设置为10分钟，完成硫代磷酸酯键的形成。
2.2 asRNA(无配体反义链)的合成
在K&A核酸合成仪上，将上述CPG固相载体放入合成柱中，参考2.1.1或2.1.2所述方法进行合成。
2.3 ssRNA与固相载体的分离
裂解试剂为氨水：乙醇＝3:1，每100mg固相载体加入5ml裂解试剂，65-70℃搅拌反应3h。反应结束，放至室温，用冰正丁醇沉淀，置于-20℃冰箱30min。离心得到沉淀，再加入正丁醇洗涤，重复2次。最后沉淀用丙酮洗涤，离心得到沉淀，真空干燥，得到粗品，进行质谱检测。
LC-MS(负离子模式)条件如下：
色谱柱：HILIC色谱柱
流动相A：20mm甲酸铵，pH6.2
流动相B：20mm甲酸铵+95％ACN
柱温：45℃
流速：1mL/min。
2.4退火形成siRNA(dsRNA)
加水溶解单链RNA粗品，膜过滤除去载体。用微量分光光度计测定正义链和反义链的浓度，加入正义链和反义链(使二者摩尔含量比＝1:1)，混合，变性温度：94℃，维持4min；退火温度：自然降温至室温。
2.5 siRNA(dsRNA)的纯化
通过施用DNAPac RP 10*150mm 4μm色谱柱纯化，流动相A是0.1mol/L三乙胺，pH8.0，流动相B是0.1mol/L三乙胺，pH8.0+50％ACN；检测波长215nm，260nm。收集主峰，旋蒸以除去大部分溶剂得到目标产物。
2.6脱盐以及含量标定
使用3K的超滤离心管，12000×g，离心时间12min，进行超滤浓缩并脱盐；旋蒸液浓缩完毕后，再 加入水进行置换，重复5次以上。
浓度测定：浓缩后的样品，使用微量分光光度计进行浓度测定。
双链核糖核酸如下所示：

上述双链核糖核酸的药学上可接受的盐或其配体缀合物(所述配体选自L01配体或L02配体)可以根据本申请所述方法或本领域已知方法制备得到。
试验例1活体外筛选双链核糖核酸
细胞培养与转染
用含有10％的胎牛血清(Gibco，货号为10099-141)的DMEM完全培养基(Hyclone，货号SH30243.01)于37℃，含有5％CO2的培养箱中培养Cos7细胞(购自南京科佰生物科技有限公司)。待细胞生长至接近汇合时，使用胰蛋白酶处理，从培养瓶中释出细胞。将Cos7细胞以2×104细胞/孔接种于96孔板内，培养过夜，待细胞密度达到70-80％时，转染包含2.2kb HBV基因组序列(GenBank accession#EU554538.1)的pmirGLO-HBV质粒至Cos7细胞。如制造商说明书所述，在50ng质粒/孔的浓度下用0.3μL LipofectamineTM 3000转染试剂(Invitrogen，货号L3000015)进行转染。在质粒转染6-8小时之后，去除培养基。再将siRNA转染至已经过pmirGLO-HBV质粒转染的Cos7细胞中。siRNA的转染方法是取0.5μL siRNA加入到5μL的OPTI-MEM培养基中，取0.3μL LipofectamineTM RNAiMAX转染试剂(Invitrogen，货号13778150)加入到5μL的OPTI-MEM培养基中；将两者吹打混匀，室温静置5分钟；将复合物加入到96孔板内，每孔 再加入90μL的DMEM完全培养基，继续培养48小时；每个siRNA设置2个浓度。每个浓度设置3个复孔。以10nM及1nM最终dsRNA浓度进行单剂量实验。选择不能与靶标序列配对的dsRNA作为对照。
检测
去除培养孔中培养基，每孔加入150μL的Luciferase试剂(Promega)与DMEM完全培养基的混合溶液(二者体积比＝1:1)，充分混匀，室温孵育10分钟后，使用Spark多功能酶标仪(Tecan)读取化学发光值(Fir)；再向每孔加入75μL的试剂(Promega)，充分混匀，室温孵育10min后，使用Spark多功能酶标仪读取化学发光值(Ren)。
计算每孔发光比值Ratio＝Fir/Ren，各测试组或对照组的发光比值Ratio(测试)或Ratio(对照)为培养孔Ratio的平均值；以对照组的发光比值为基准，对各测试组的发光比值进行归一化，获得Ratio(测试)/Ratio(对照)的比值R，以此表达firefly luciferase报告基因的表达水平。siRNA的抑制率(％)＝(1-R)×100％。结果如下所示：
该结果表明，本申请的dsRNA在细胞水平显示出了优异活性，并且抑制活性呈剂量依赖性。
试验例2双荧光素酶实验
细胞培养与转染
用含有10％的胎牛血清(Gibco，货号10099-141)的DMEM完全培养基(Hyclone，货号SH30243.01)于37℃，含有5％CO2的培养箱中培养Cos7细胞(购自南京科佰生物科技有限公司)。待细胞生长至接近汇合时，使用胰蛋白酶处理，从培养瓶中释出细胞。将Cos7细胞以2×104细胞/孔接种于96孔板内，培养过夜，待细胞密度达到70-80％时，转染包含2.2kb HBV基因组序列(GenBank accession#EU554538.1)的pmirGLO-HBV质粒至Cos7细胞。如制造商说明书所述，在50ng质粒/孔的浓度下用0.3μL LipofectamineTM 3000转染试剂(Invitrogen，货号L3000015)进行转染。在质粒转染6-8小时之后，去除培养基。再将siRNA转染至已经过pmirGLO-HBV质粒转染的Cos7细胞中。siRNA的转染方法是用无RNA酶水对siRNA进行5倍连续稀释至浓度范围为2-0.0000256μM；取0.5μL siRNA加入到5μL的OPTI-MEM培养基中，取0.3μL LipofectamineTM RNAiMAX转染试剂(Invitrogen，货号为13778150)加入到5μL的OPTI-MEM培养基中；将两者吹打混匀，室温静置5分钟；将复合物加入到96孔板内，每孔再加入90μL的DMEM完全培养基，继续培养48小时；每个siRNA设置8个浓度。每个浓度设置3个复孔。选择不能与靶标序列配对的dsRNA作为对照。
检测
去除培养孔中培养基，每孔加入150μL的Luciferase试剂(Promega)与DMEM完全培养基的混合溶液(二者体积比＝1:1)，充分混匀，室温孵育10分钟后，使用Spark多功能酶标仪(Tecan)读取化学发光值(Fir)；再向每孔加入75μL的试剂(Promega)，充分混匀，室温孵育10min后，使用Spark多功能酶标仪读取化学发光值(Ren)。
计算每孔发光比值Ratio＝Fir/Ren，各测试组或对照组的发光比值Ratio(测试)或Ratio(对照)为培养孔Ratio的平均值；以对照组的发光比值为基准，对各测试组的发光比值进行归一化，获得Ratio(测试)/Ratio(对照)的比值R，以此表达firefly luciferase报告基因的表达水平。siRNA的抑制率(％)＝(1-R)×100％。
用EXCEL软件处理浓度-抑制率(％)数据，通过四参数非线性回归模型计算siRNA的EC50，结果如下所示：

该结果表明，本申请的dsRNA在细胞水平显示出了优异活性，具有较低的EC50值。
试验例3 HepG2.2.15细胞中的抑制活性
细胞培养与转染
用含有10％胎牛血清(Gibco，货号10099-141)、380μg/mL G418(Gibco，货号10131-027)的MEM完全培养基(Gibco，货号11095-080)，在37℃，5％CO2的培养箱中培养HepG2.2.15细胞。待细胞生长至接近汇合时，使用胰蛋白酶处理，从培养瓶中释出细胞，将HepG2.2.15细胞以2×104细胞/孔接种于96孔板内，培养过夜，待细胞密度达到70-80％时，转染siRNA。siRNA的转染方法是用无RNA酶水对siRNA进行5倍连续稀释至浓度范围为2-0.0000256μM；取0.5μL siRNA加入到含有0.3μL LipofectamineTM RNAiMAX转染试剂(Invitrogen，货号为13778150)的10μL的OPTI-MEM培养基中，吹打混匀；室温静置5分钟；弃去96孔板内的MEM培养基，每孔加入90μL的MEM完全培养基。混合液按照10μL/孔的体积加入到96孔板内，将96孔板置于37℃，含有5％CO2培养箱继续培养6天，每3天换液一次，最后一天收集培养上清进行检测；每个siRNA设置8个浓度。每个浓度设置3个复孔。选择不能与靶标序列配对的dsRNA作为对照。
ELISA法检测siRNA对HBsAg的抑制作用
收集细胞上清，使用乙型肝炎病毒表面抗原(HBsAg)测定试剂盒(北京科美诊断技术股份有限公司，货号1010803042)检测细胞上清中HBsAg的含量。操作按照说明书进行，使用Spark多功能酶标仪(Tecan)读取化学发光值；以标准品绘制标准曲线，然后计算样品中HBsAg的浓度(ng/mL)，用EXCEL软件处理浓度-抑制率(％)数据，通过四参数非线性回归模型计算siRNA对HBsAg抑制作用的EC50。
抑制率(％)＝(1-siRNA组浓度(ng/mL)/对照组浓度(ng/mL))×100
ELISA法检测siRNA对HBeAg的抑制作用
收集细胞上清，使用乙型肝炎病毒e抗原(HBeAg)测定试剂盒(北京科美诊断技术股份有限公司，货号1010801042)检测细胞上清中HBeAg的含量。操作按照说明书进行，使用Spark多功能酶标仪(Tecan)读取化学发光值；以标准品绘制标准曲线，然后计算样品中HBeAg的浓度(NCU/mL)，用EXCEL软件处理浓度-抑制率(％)数据，通过四参数非线性回归模型计算siRNA对HBeAg抑制作用的EC50。
抑制率(％)＝(1-siRNA组浓度(NCU/mL)/对照组浓度(NCU/mL))×100。
结果如下所示：



该结果表明，本申请的dsRNA在细胞水平显示出了优异活性，在HBsAg抑制作用和HBeAg抑制作用中均具有较低的EC50值。
另一方面，该结果还表明，本申请的dsRNA对HBsAg有明显的抑制作用，表明了本申请的dsRNA对病毒性乙型肝炎功能性治愈的可能性。
试验例4 AAV-HBV小鼠实验
按照文献方法(董小岩等，Chin J Biotech 2010,May 25；26(5):679-686)制备AAV-HBV小鼠模型，所使用的rAAV8-1.3HBV,D型(ayw)购于北京五加和基因科技有限公司，1.78×1012viral genome(v.g)/mL，批号A2020081701。rAAV8-1.3HBV在注射前预先用无菌PBS配制成浓度为5×1011v.g/mL的溶液。取6-7周龄雄性C57BL/6小鼠(购自上海斯莱克实验动物有限责任公司)，每只小鼠经尾静脉注射200μL rAAV8-1.3HBV，即每只小鼠注射1×1011v.g rAAV8-1.3HBV。病毒注射后第28天，所有小鼠通过眼眶采血用于收集血清，并检测血清中的HBV DNA、HBsAg和HBeAg含量。
动物造模成功后，按血清HBV DNA、HBsAg和HBeAg含量将小鼠随机分组(每组3只)，一共7组，分别是：PBS对照组、M01L01组、M01EVPL01组、M21L01组、M21EVPL01组、M22L01组和M22EVPL01组。首次给药当天设为实验第0天。给药组小鼠根据动物体重单次皮下注射相应dsRNA配体缀合物溶液，给药剂量为9mg/kg，给药体积为5mL/kg体重；PBS对照组小鼠单次皮下注射PBS，给药体积为5mL/kg体重。在给药前2天和给药后第7、14、21、28、35、42、56、70、84天，分别眼眶采血，分离血清，检测血清中HBV DNA、HBsAg、HBeAg含量。眼眶静脉采血100μL，全血置于室温静置30分钟后，3000rpm，4℃离心10分钟收集血清。参照乙型肝炎病毒核酸测定试剂盒(圣湘生物，货号101604042)说明书裂解、释放血清中DNA，进行定量PCR，检测血清中HBV DNA水平；利用乙肝病毒表面抗原定量检测试剂盒(Roche，08814899190)和乙肝e抗原检测试剂盒(Roche，货号11820583122)，在Cobas e免疫分析仪(Roche，货号Cobas8000)上检测血清中HBsAg和HBeAg水平。
HBV DNA抑制率按如下等式计算：
HBV DNA抑制率＝(1-给药后HBV DNA含量/给药前HBV DNA含量)×100％。
其中，HBV DNA含量用每毫升(mL)血清含多少当量(IU)HBV DNA表示。
HBsAg抑制率按如下等式计算：
HBsAg抑制率＝(1-给药后HBsAg含量/给药前HBsAg含量)×100％。其中，HBsAg含量用每毫升(mL)血清含多少当量(IU)HBsAg表示。
HBeAg抑制率按如下等式计算：
HBeAg抑制率＝(1-给药后HBeAg含量/给药前HBeAg含量)×100％。其中，HBeAg含量用每毫升(mL)血清含多少当量(U)HBeAg表示。
dsRNA配体缀合物如下表所示：
其中，VP-表示该连字符右侧的一个核苷酸为(E)-乙烯基磷酸酯修饰的核苷酸，例如，上文中的SEQ ID NO:64和SEQ ID NO:65的5’-末端的u采用5’-(E)-乙烯基磷酸酯修饰。其中，正义链的序列ID编号是指正义链中核酸序列的序列ID编号。dsRNA配体缀合物序号及正义链序列3’端处的L01表示在该dsRNA配体缀合物中，在正义链序列3’端处连接有L01配体。
结果如下表和图1-图3所示：
小鼠血清HBV DNA抑制率
小鼠血清HBsAg抑制率
小鼠血清HBeAg抑制率
另外，PBS对照组在整个84天的监测过程中均未显示出血清HBV DNA、HBsAg和HBeAg抑制作用。
该结果表明，本申请的dsRNA配体缀合物在动物水平也显示出了优异活性，在血清HBV DNA抑制作用、血清HBsAg抑制作用和血清HBeAg抑制作用中均具有较高的抑制率，显示出临床应用潜能。
另一方面，该结果还表明，本申请的dsRNA配体缀合物对HBsAg有明显的抑制作用，表明了本申请的dsRNA对病毒性乙型肝炎功能性治愈的可能性。

Claims (22)

  1. 一种双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述双链核糖核酸包含正义链和反义链，其中正义链包含5’-CUGGCUCAGUUUACUAGUA-3’(SEQ ID NO:1)且正义链的长度不超过21个核苷酸，其中反义链包含5’-UACUAGUAAACUGAGCCAGGA-3’(SEQ ID NO:2)且反义链的长度不超过23个核苷酸，所述正义链或反义链任选地是经修饰的。
  2. 根据权利要求1所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述正义链或反义链任选地包括位于5’端和/或3’端的突出端。
  3. 根据权利要求2所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述突出端含有1、2、3、4或5个核苷酸；或者，所述突出端含有1或2个核苷酸。
  4. 根据权利要求2或3所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述突出端选自未修饰或经修饰的A、G、C、U或T；或者选自未修饰或经修饰的U或T；或者当所述突出端为1个核苷酸时，所述突出端选自u或dT；或者当所述突出端为2个核苷酸时，所述突出端选自uu或dTdT；或者当所述突出端为2个核苷酸时，所述突出端选自未修饰或经修饰的GA。
  5. 根据权利要求2至4任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述突出端与其相邻的核苷酸通过磷酸酯基或硫代磷酸酯基连接；或者，所述突出端中的一个或多个核苷酸之间通过磷酸酯基或硫代磷酸酯基连接。
  6. 根据权利要求1至5任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述正义链的1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个或21个核苷酸是经修饰的，或者前述任意值形成的范围内的核苷酸是经修饰的，例如1-21个、1-20个、1-19个、1-18个、1-17个、1-16个、1-15个、1-14个、1-13个、1-12个、1-11个或1-10个核苷酸是经修饰的；或者所述正义链的1个以上、2个以上、3个以上、4个以上、5个以上、6个以上、7个以上、8个以上、9个以上、10个以上、11个以上、12个以上、13个以上、14个以上、15个以上、16个以上、17个以上、18个以上、19个以上或20个以上核苷酸是经修饰的；或者，所述正义链的所有核苷酸是经修饰的；和/或
    所述反义链的1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个或23个核苷酸是经修饰的，或者前述任意值形成的范围内的核苷酸是经修饰的，例如1-23个、1-22个、1-21个、1-20个、1-19个、1-18个、1-17个、1-16个、1-15个、1-14个、1-13个、1-12个、1-11个或1-10个核苷酸是经修饰的；或者所述反义链的1个以上、2个以上、3个以上、4个以上、5个以上、6个以上、7个以上、8个以上、9个以上、10个以上、11个以上、12个以上、13个以上、14个以上、15个以上、16个以上、17个以上、18个以上、19个以上、20个以上、21个以上或22个以上核苷酸是经修饰的；或者，所述反义链的所有核苷酸是经修饰的。
  7. 根据权利要求1至6任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述修饰选自核苷酸的糖基修饰、核苷酸间的连接键修饰或末端修饰。
  8. 根据权利要求1至7任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述核苷酸的糖基修饰选自2’-脱羟基化、2’-氟代、2’-氨基、2’-烷基、2’-O-烷基、2’-O-醚基、2’-O-烯基；或者，所述核苷酸的糖基修饰选自2’-脱羟基化、2’-氟代、2’-氨基、2’-甲基、2’-乙基、2’-甲基-O-甲基、2’-乙基-O-甲基、2’-O-甲基、2’-O-乙基、2’-O-乙基-O-甲基或2’-O-烯丙基；
    所述核苷酸间的连接键修饰选自硫代磷酸酯(PS)、二硫代磷酸酯(PS2)、甲基膦酸酯(MP)、甲氧基丙基膦酸酯(MOP)或氨基膦酸酯；或者，所述核苷酸间的连接键修饰选自硫代磷酸酯(PS)；和/或所述末端修饰选自5’-末端修饰或3’-末端修饰；或者，所述末端修饰选自5’-磷酸酯、5’-甲基膦酸酯(5’-MP)、5’-硫代磷酸酯(5’-PS)或5’-(E)-乙烯基膦酸酯(5’-(E)-VP)。
  9. 根据权利要求1至8任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述正义链的第1、2位核苷酸之间和/或正义链的第2、3位核苷酸之间采用硫代磷酸酯基修饰，所述反义链的第1、2位核苷酸之间、反义链的第2、3位核苷酸之间、反义链的第19、20位核苷酸之间和/或反义链的第20、21位核苷酸之间采用硫代磷酸酯基修饰。
  10. 根据权利要求1至9任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述正义链有2、3、4或5个核苷酸采用2’-氟代修饰，所述正义链有14、15、16或17个核苷酸采用2’-O-甲基修饰；和/或；
    所述反义链有2、3、4、5、6或7个核苷酸采用2’-氟代修饰，所述反义链有14、15、16、17、18或19个核苷酸采用2’-O-甲基修饰。
  11. 根据权利要求1至10任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述正义链的第1至6位核苷酸中有0个、1个或2个核苷酸采用2’-氟代修饰，所述正义链的第1至6位核苷酸 中有4个、5个或6个核苷酸采用2’-O-甲基修饰，所述正义链的第7至9位核苷酸中有2个或3个核苷酸采用2’-氟代修饰，所述正义链的第7至9位核苷酸中有0个或1个核苷酸采用2’-O-甲基修饰，所述正义链的第10至19位核苷酸中有0个或1个核苷酸采用2’-氟代修饰，所述正义链的第10至19位核苷酸中有8个、9个或10个核苷酸采用2’-O-甲基修饰；或者所述正义链的第7、8和9位核苷酸采用2’-氟代修饰，并且所述正义链的第1至6和/或10至19位核苷酸中有0个、1个或2个核苷酸也采用2’-氟代修饰。
  12. 根据权利要求1至11任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述反义链的第2、6、14、16位核苷酸中有3个或4个核苷酸采用2’-氟代修饰；或者反义链的第2、6、8、9、14、16位核苷酸中有3个、4个、5个或6个核苷酸采用2’-氟代修饰；或者反义链的第2、6、14和16位核苷酸采用2’-氟代修饰，并且所述反义链的第1、3至5、7至13、15和17至21位核苷酸中任意一个或两个也采用2’-氟代修饰；或者反义链的第1、3至5、7至13、15、17至21位核苷酸中有14、15、16或17个核苷酸采用2’-O-甲基修饰。
  13. 根据权利要求1至12任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述配体含有至少一个靶向基团；或者所述配体含有一个、二个、三个、四个或五个靶向基团。
  14. 根据权利要求13所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述靶向基团为GalNAc基团。
  15. 根据权利要求1至14任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述配体包括如下分支基团：
  16. 根据权利要求1至15任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述配体选自：
  17. 根据权利要求1至16任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述配体与正义链或反义链的5’或3’末端连接；或者，所述配体与正义链的5’或3’末端连接；或者，所述配体与正义链的3’末端连接。
  18. 根据权利要求1至17任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述双链核糖核酸包含如下正义链中的任一者：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41或SEQ ID NO:42；并且
    所述双链核糖核酸包含如下反义链中的任一者：SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:24、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID  NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65。
  19. 根据权利要求1至18任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，所述双链核糖核酸包含如下正义链和反义链的序列对：SEQ ID NO:1/SEQ ID NO:2、SEQ ID NO:3/SEQ ID NO:4、SEQ ID NO:5/SEQ ID NO:4、SEQ ID NO:6/SEQ ID NO:4、SEQ ID NO:7/SEQ ID NO:4、SEQ ID NO:8/SEQ ID NO:4、SEQ ID NO:9/SEQ ID NO:4、SEQ ID NO:10/SEQ ID NO:4、SEQ ID NO:11/SEQ ID NO:4、SEQ ID NO:12/SEQ ID NO:4、SEQ ID NO:13/SEQ ID NO:4、SEQ ID NO:14/SEQ ID NO:4、SEQ ID NO:15/SEQ ID NO:4、SEQ ID NO:16/SEQ ID NO:4、SEQ ID NO:17/SEQ ID NO:4、SEQ ID NO:18/SEQ ID NO:4、SEQ ID NO:19/SEQ ID NO:4、SEQ ID NO:20/SEQ ID NO:4、SEQ ID NO:21/SEQ ID NO:4、SEQ ID NO:22/SEQ ID NO:4、SEQ ID NO:23/SEQ ID NO:4、SEQ ID NO:3/SEQ ID NO:24、SEQ ID NO:9/SEQ ID NO:24、SEQ ID NO:25/SEQ ID NO:24、SEQ ID NO:26/SEQ ID NO:24、SEQ ID NO:27/SEQ ID NO:24、SEQ ID NO:28/SEQ ID NO:24、SEQ ID NO:29/SEQ ID NO:24、SEQ ID NO:30/SEQ ID NO:24、SEQ ID NO:31/SEQ ID NO:24、SEQ ID NO:32/SEQ ID NO:24、SEQ ID NO:33/SEQ ID NO:24、SEQ ID NO:34/SEQ ID NO:24、SEQ ID NO:35/SEQ ID NO:24、SEQ ID NO:36/SEQ ID NO:24、SEQ ID NO:37/SEQ ID NO:24、SEQ ID NO:38/SEQ ID NO:24、SEQ ID NO:39/SEQ ID NO:24、SEQ ID NO:40/SEQ ID NO:24、SEQ ID NO:41/SEQ ID NO:24、SEQ ID NO:42/SEQ ID NO:24、SEQ ID NO:9/SEQ ID NO:43、SEQ ID NO:9/SEQ ID NO:44、SEQ ID NO:9/SEQ ID NO:45、SEQ ID NO:9/SEQ ID NO:46、SEQ ID NO:9/SEQ ID NO:47、SEQ ID NO:9/SEQ ID NO:48、SEQ ID NO:9/SEQ ID NO:49、SEQ ID NO:9/SEQ ID NO:50、SEQ ID NO:9/SEQ ID NO:51、SEQ ID NO:9/SEQ ID NO:52、SEQ ID NO:9/SEQ ID NO:53、SEQ ID NO:9/SEQ ID NO:54、SEQ ID NO:9/SEQ ID NO:55、SEQ ID NO:9/SEQ ID NO:56、SEQ ID NO:9/SEQ ID NO:57、SEQ ID NO:9/SEQ ID NO:58、SEQ ID NO:9/SEQ ID NO:59、SEQ ID NO:9/SEQ ID NO:60、SEQ ID NO:9/SEQ ID NO:61、SEQ ID NO:9/SEQ ID NO:62、SEQ ID NO:9/SEQ ID NO:63、SEQ ID NO:27/SEQ ID NO:61、SEQ ID NO:28/SEQ ID NO:61、SEQ ID NO:34/SEQ ID NO:61、SEQ ID NO:36/SEQ ID NO:61、SEQ ID NO:42/SEQ ID NO:61、SEQ ID NO:27/SEQ ID NO:62、SEQ ID NO:28/SEQ ID NO:62、SEQ ID NO:34/SEQ ID NO:62、SEQ ID NO:36/SEQ ID NO:62、SEQ ID NO:42/SEQ ID NO:62、SEQ ID NO:3/SEQ ID NO:64、SEQ ID NO:3/SEQ ID NO:65或SEQ ID NO:9/SEQ ID NO:65。
  20. 一种药物组合物，其包含根据权利要求1-19任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物，任选地包括药学上可接受的载体或赋形剂。
  21. 权利要求1-19任一项所述的双链核糖核酸、其药学上可接受的盐或其配体缀合物或权利要求20所述的药物组合物在制备用于治疗和/或预防乙型肝炎病毒感染的药物中的用途。
  22. 根据权利要求21所述的用途，所述双链核糖核酸、其药学上可接受的盐或其配体缀合物、或药物组合物与其他治疗剂共同使用。
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