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WO2024125636A1 - Toll样受体调节剂与dsRNA的联合治疗 - Google Patents

Toll样受体调节剂与dsRNA的联合治疗 Download PDF

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Publication number
WO2024125636A1
WO2024125636A1 PCT/CN2023/139175 CN2023139175W WO2024125636A1 WO 2024125636 A1 WO2024125636 A1 WO 2024125636A1 CN 2023139175 W CN2023139175 W CN 2023139175W WO 2024125636 A1 WO2024125636 A1 WO 2024125636A1
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Prior art keywords
dsrna
nucleotide
hepatitis
group
formula
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PCT/CN2023/139175
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English (en)
French (fr)
Inventor
李云飞
张瑱
林晓燕
黄晓玲
邓永岩
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上海拓界生物医药科技有限公司
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Publication of WO2024125636A1 publication Critical patent/WO2024125636A1/zh

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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 disclosure relates to the use of a Toll-like receptor modulator in combination with dsRNA in the preparation of a drug for preventing and/or treating hepatitis B virus infection or diseases associated with hepatitis B virus, and belongs to the field of medicine.
  • TLR-8 The Toll-like receptor (TLR) family plays an important role in pathogen recognition and innate immune activation.
  • Toll-like receptor 8 (TLR-8) is mainly expressed by bone marrow immune cells, and activation of this receptor stimulates a wide range of immune responses.
  • TLR-8 agonists activate myeloid dendritic cells, monocytes, monocyte-derived dendritic cells, and Kupffer cells, leading to the production of proinflammatory cytokines and chemokines, such as interleukin 18 (IL-18), interleukin 12 (IL-12), tumor necrosis factor- ⁇ (TNF-a), and interferon- ⁇ (IFN-g).
  • IL-18 interleukin 18
  • IL-12 interleukin 12
  • TNF-a tumor necrosis factor- ⁇
  • IFN-g interferon- ⁇
  • Such agonists also promote increased expression of co-stimulatory molecules such as CD8+ cells, major histocompatibility complex molecules (MAIT, NK cells), and chemokine receptors.
  • TLR8 modulating compounds include those described in WO2020007275A. Collectively, activation of these innate and adaptive immune responses induces immune responses and provides therapeutic benefits in a variety of conditions involving autoimmunity, inflammation, allergy, asthma, transplant rejection, graft-versus-host disease (GvHD), infection, cancer, and immunodeficiency.
  • TLR8 on professional antigen-presenting cells (pAPCs) and other intrahepatic immune cells is associated with the induction of IL-12 and proinflammatory cytokines, which are expected to enhance HBV-specific T cell responses, activate intrahepatic NK cells, and drive reconstitution of antiviral immunity.
  • pAPCs professional antigen-presenting cells
  • cytokines proinflammatory cytokines
  • RNA interference is an effective way to silence gene expression.
  • appropriate siRNA can be designed according to the mRNA encoding these proteins, specifically targeting the target mRNA and degrading the target mRNA, thereby achieving the purpose of inhibiting the production of related proteins.
  • It is an effective drug delivery method to use targeting ligands to conjugate siRNA and use the receptor molecular structure of the targeting ligand on the cell membrane surface to enter the cell through endocytosis.
  • asialoglycoprotein receptor is a receptor specifically expressed by hepatocytes, with high abundance on the surface of hepatocytes and rapid intracellular and extracellular conversion.
  • GalNAc aminogalactose molecular clusters
  • NRTIs nucleoside/nucleotide reverse transcriptase inhibitors
  • NRTIs nucleoside/nucleotide reverse transcriptase inhibitors
  • NRTIs do not directly eliminate cccDNA, so viral proteins continue to be transcribed and translated. Therefore, the expression of viral epitopes on hepatocytes, the secretion of subviral particles, and immune dysfunction remain largely unaffected by NRTI therapy. The result is that long-term or even lifelong treatment is required. Therefore, there is still a demand for new HBV therapies that are effective, well-tolerated, and do not require lifelong medication in the clinic.
  • WO2022241134A discloses a method for treating and/or preventing hepatitis B virus infection in a subject using a TLR8 modulator combined with dsRNA and a PD-1/PD-L1 inhibitor.
  • WO2020232024A discloses a composition and method for treating HBV infection, specifically involving dsRNA and PEG-INF.
  • the present disclosure relates to use of a double-stranded ribonucleic acid (dsRNA) combined with a Toll-like receptor 8 (TLR8) regulator in the preparation of a drug for preventing and/or treating hepatitis B virus infection or a disease associated with hepatitis B virus.
  • dsRNA double-stranded ribonucleic acid
  • TLR8 Toll-like receptor 8
  • the present disclosure also provides a method for preventing and/or treating hepatitis B virus (HBV) infection or a disease associated with hepatitis B virus in a subject, comprising administering dsRNA and a TLR8 modulator to the subject.
  • HBV hepatitis B virus
  • the present disclosure also provides a TLR8 regulator, which is combined with dsRNA for preventing and/or treating hepatitis B virus infection or diseases associated with hepatitis B virus.
  • the present disclosure also provides a dsRNA, which is used in combination with a TLR8 modulator or a pharmaceutically acceptable salt thereof for preventing and/or treating hepatitis B virus infection or a disease associated with hepatitis B virus.
  • the present disclosure also provides a method for inhibiting the expression of a target gene or its mRNA, which comprises administering an effective amount or an effective dose of the dsRNA described in the present disclosure and a TLR8 modulator to a subject.
  • the disease associated with hepatitis B virus is selected from chronic hepatitis, acute hepatitis B, chronic hepatitis B, hepatitis D virus infection, hepatitis D, liver fibrosis, advanced liver disease or hepatocellular carcinoma.
  • the disease associated with hepatitis B virus is chronic hepatitis, and the subject is HBeAg positive or HBeAg negative.
  • the Toll-like receptor 8 (TLR8) modulator is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • the dsRNA comprises:
  • siRNA and one or more ligands conjugated thereto siRNA and one or more ligands conjugated thereto;
  • the siRNA comprises a sense strand and an antisense strand
  • the antisense strand comprises a chemical modification represented by formula (II), a tautomer thereof or a pharmaceutically acceptable salt thereof at the 7th nucleotide position from the 5' end thereof:
  • B is the same as the base when the 7th nucleotide from the 5' end of the antisense strand is not modified;
  • the ligand is shown in the following structure or a pharmaceutically acceptable salt thereof:
  • the sense strand and antisense strand are selected from any one of the following groups:
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 2;
  • the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 3;
  • the 3' end of the sense strand is conjugated to the ligand
  • the B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • SEQ ID NO: 1 is GUGUGCACUUCGCUUCACC
  • SEQ ID NO:2 is AGUGAAGCGAAGUGCACACGG
  • SEQ ID NO:3 is IGUGAAGCGAAGUGCACACGG.
  • the ligand in the dsRNA is linked to the end of the siRNA via a phosphate group or a phosphorothioate group.
  • the ligand in the dsRNA is linked to the end of the siRNA via a phosphodiester group or a phosphorothioate diester group.
  • the ligand in the dsRNA is linked to the end of the siRNA via a phosphodiester group.
  • the ligand in the dsRNA is indirectly linked to the end of the siRNA via a phosphate group or a phosphorothioate group.
  • the ligand in the dsRNA is directly linked to the end of the siRNA via a phosphate group or a phosphorothioate group.
  • the ligand in the dsRNA is linked to a phosphate group or a phosphorothioate group.
  • the group is directly linked to the 3' end of the sense strand of the siRNA.
  • the phosphate group in the dsRNA is a phosphoester group or a phosphodiester group. In some embodiments, the phosphate group is a phosphodiester group.
  • the phosphorothioate group in the dsRNA is a phosphorothioate monoester group or a phosphorothioate diester group.
  • the phosphorothioate group in the dsRNA is a phosphorothioate diester group.
  • the dsRNA may be the following structure or a pharmaceutically acceptable salt thereof,
  • Z is siRNA
  • the 3' end of the sense strand of the siRNA is directly linked to the ligand via a phosphodiester group
  • the siRNA is as defined in the present disclosure.
  • At positions other than the chemical modification shown in formula (I) in the dsRNA at positions other than the chemical modification shown in formula (I) in the dsRNA, at least one additional nucleotide in the sense strand and/or antisense strand is a modified nucleotide.
  • the modified nucleotides in the dsRNA are selected from: 2'-methoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 3'-deoxy-thymine nucleotides, isonucleotides, LNA, ENA, cET, UNA, GNA.
  • the modified nucleotides in the dsRNA are independently selected from: 2'-methoxy modified nucleotides or 2'-fluoro modified nucleotides.
  • the sense strand of the dsRNA contains three consecutive nucleotides having the same modification.
  • the three nucleotides having the same modification are 2'-fluoro modified nucleotides.
  • the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.
  • the antisense strand of the siRNA is at least partially reverse complementary to the target sequence. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch between the antisense strand and the target sequence. In some embodiments, the antisense strand is completely reverse complementary to the target sequence.
  • the sense strand and the antisense strand of the siRNA are at least partially reverse complementary to form a double-stranded region. In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch between the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand are completely reverse complementary.
  • the sense strand and the antisense strand of the siRNA each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (e.g., 19, 20, 21, 22, 23 nucleotides).
  • the sense strand and antisense strand of the siRNA are of the same or different lengths, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length.
  • the length ratio of the sense strand and the antisense strand in the dsRNA provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/19, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense strand to the antisense strand of the siRNA is 19/21, 21/23, or 23/25. In some embodiments, the length ratio of the sense strand to the antisense strand is 19/21.
  • the siRNA comprises one or two blunt ends.
  • each strand of the siRNA independently comprises 1 to 2 unpaired nucleotides forming an overhang.
  • the siRNA comprises an overhang at the 3' end of the antisense strand.
  • three consecutive nucleotides at positions 7-9 at the 5' end of the sense strand of the siRNA are 2'-fluoro-modified nucleotides.
  • the sense strand of the siRNA contains a nucleotide sequence (5'-3') as shown in the following formula: N a N a N a N a N a XN a N b N b N b N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a N a
  • each X is independently Na or Nb ; Na is a 2'-methoxy-modified nucleotide, and Nb is a 2'-fluoro-modified nucleotide.
  • the sense strand of the siRNA contains a nucleotide sequence as shown in the following formula: 5'-N a N a N a N a N a N b N b N b N a N a N a N a N a N a N a N a -3'; or, 5′-N a N a N a N a N b N a N b N b N b N a N a N a N a N a N a N a a a a a a a a a -3′;
  • Na is a 2'-methoxy-modified nucleotide
  • Nb is a 2'-fluoro-modified nucleotide
  • the siRNA antisense strand contains a nucleotide sequence as shown in the following formula: 5'-N a 'N b 'N a 'N b 'N a 'N b 'W'N a 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N a 'N a '-3';
  • Na ' is a 2'-methoxy modified nucleotide
  • Nb ' is a 2'-fluoro modified nucleotide
  • W' represents a 2'-methoxy modified nucleotide or a nucleotide modified with the chemical modification shown in formula (I), its tautomer or a pharmaceutically acceptable salt thereof.
  • W' represents a nucleotide comprising a chemical modification as shown in formula (I), a tautomer thereof, or a pharmaceutically acceptable salt thereof.
  • the chemical modification represented by formula (I) is selected from:
  • B is selected from guanine, adenine, cytosine and uracil. In some specific embodiments, B is the same as the base of the antisense strand when the 7th nucleotide from the 5' end is not modified.
  • the chemical modification represented by formula (I) is selected from:
  • M is O or S;
  • B is selected from guanine, adenine, cytosine or uracil. In some specific embodiments, B is the same as the base of the antisense strand when the 7th nucleotide from the 5' end is not modified.
  • said M is S.
  • said M is O.
  • At least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modifying group.
  • the modifying group allows the siRNA to have increased stability in a biological sample or environment.
  • the phosphate group with a modifying group is a thiophosphate group.
  • the phosphate group with a modifying group is a thiophosphate diester group.
  • the phosphorothioate diester group is present in at least one of the following positions:
  • the sense strand and/or antisense strand comprises a plurality of phosphorothioate diester groups, wherein the phosphorothioate diester groups are present in:
  • the sense strand comprises a nucleotide sequence as shown in the following formula: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNm-3', or, 5'-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmNmN
  • Nm represents any nucleotide modified with 2'-methoxy, such as C, G, U, A modified with 2'-methoxy
  • Nf represents any nucleotide modified with 2'-fluoro, such as C, G, U, A modified with 2'-fluoro
  • a lowercase letter s indicates that the two nucleotides adjacent to the letter s on the left and right are connected by a thiophosphate diester group; when the lowercase letter s is the first at the 3' end, it indicates that the end of the nucleotide adjacent to the upstream (5' direction) of the letter s is a thiophosphate diester group.
  • the antisense strand comprises a nucleotide sequence as shown in the following formula: 5'-Nm'sNf'sNm'Nf'Nm'Nf'W'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'sNm' sNm'-3';
  • Nm' represents any nucleotide modified with 2'-methoxy, such as C, G, U, A modified with 2'-methoxy
  • Nf' represents any nucleotide modified with 2'-fluoro, such as C, G, U, A modified with 2'-fluoro
  • a lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate diester group. If the lowercase letter s is the first at the 3' end, it indicates that the terminal of the nucleotide adjacent to the upstream of the letter s is a phosphorothioate diester group.
  • W' represents a 2'-methoxy modified nucleotide or a nucleotide modified with the chemical modification represented by formula (I), a tautomer thereof or a pharmaceutically acceptable salt thereof.
  • the dsRNA is selected from any one of the following groups:
  • the dsRNA is selected from any one of the following groups:
  • Af adenine 2'-F ribonucleoside
  • Cf cytosine 2'-F ribonucleoside
  • Uf uracil 2'-F ribonucleoside.
  • Gf guanine 2'-F ribonucleoside
  • Am adenine 2'-OMe ribonucleoside
  • Cm cytosine 2'-OMe ribonucleoside
  • Gm guanine 2'-OMe ribonucleoside
  • Um uracil 2'-OMe ribonucleoside
  • Im hypoxanthine 2'-OMe ribonucleoside
  • nucleosides are linked by phosphodiester groups, and s means that the two nucleosides adjacent to the letter s are linked by thiophosphate diester groups;
  • the dsRNA is selected from the following structures or a pharmaceutically acceptable salt thereof:
  • Af adenine 2'-F ribonucleoside
  • Cf cytosine 2'-F ribonucleoside
  • Gf guanine 2'-F ribonucleoside
  • Uf uracil 2'-F ribonucleoside
  • Am adenine 2'-OMe ribonucleoside
  • Cm cytosine 2'-OMe ribonucleoside
  • Gm guanine 2'-OMe ribonucleoside
  • Um uracil 2'-OMe ribonucleoside
  • Im Inosine 2'-OMe ribonucleoside
  • the pharmaceutically acceptable salt may be a conventional salt in the art, including but not limited to sodium salt, potassium salt, ammonium salt, amine salt, and the like.
  • the dsRNA is selected from TRD007970 and TJR100259.
  • the dsRNA is TRD007970, which has the following structure:
  • the dsRNA is TJR100259, which has the following structure:
  • Af adenine 2'-F ribonucleoside
  • Cf cytosine 2'-F ribonucleoside
  • Gf guanine 2'-F ribonucleoside
  • Uf uracil 2'-F ribonucleoside
  • the pharmaceutically acceptable salt may be a conventional salt in the art, including but not limited to sodium salt, potassium salt, ammonium salt, amine salt, and the like.
  • the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is administered at a fixed dose.
  • the dosage is selected from 0.02mg-50mg, and the specific dosage can be selected from 0.02mg, 0.25mg, 0.50mg, 0.75mg, 1.00mg, 1.25mg, 1.50mg, 1.75mg, 2.00mg, 2.25mg, 2.50mg, 2.75mg, 3.00mg, 3.25mg, 3.50mg, 3.75mg, 4.00mg, 4.25mg, 4.50mg, 4.7 5mg, 5.00mg, 5.25mg, 5.50mg, 5.75mg, 6.00mg, 6.25mg, 6.50mg, 6.75mg, 7.00mg, 7.25mg, 7.50mg, 7.75mg, 8.00mg, 8.25mg, 8.50mg, 8.75mg, 9.00mg, 9.25mg, 9.50mg, 9.75mg or 10.00m
  • the administration frequency of the compound of formula (II) or a pharmaceutically acceptable salt thereof is selected from twice a week, once a week, once every two weeks and once every four weeks.
  • the maintenance time of administration of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is selected from four weeks, eight weeks, twelve weeks, twenty-four weeks, thirty-six weeks, forty-eight weeks, sixty weeks, seventy-two weeks, eighty-four weeks, etc.
  • the dosage of the compound of formula (II) or a pharmaceutically acceptable salt thereof is selected from 0.50 mg, 0.75 mg, 1.00 mg, 1.25 mg, 1.50 mg, 1.75 mg, 2.50 mg or 3 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 0.50 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 0.75 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 1.00 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 1.25 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 1.50 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 1.75 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 2.50 mg, and the administration frequency is once a week.
  • the dosage of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is 3 mg, and the administration frequency is once a week.
  • the dsRNA is administered at a fixed dose, and the dosage is selected from 10 mg to 1500 mg, specifically 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg.
  • the dsRNA is administered according to body weight, and the dosage is selected from about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight, or about 0.5 mg/kg body weight to about 50 mg/kg body weight, about 1 mg/kg to 20 mg/kg.
  • the administration frequency of the dsRNA is selected from once a week and subsequently reduced frequency, specifically once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, or once every twelve weeks.
  • the dsRNA is administered at a frequency selected from once a week, once every two weeks, once every four weeks, once every eight weeks, or once every twelve weeks.
  • the dsRNA is administered at a fixed dose selected from 100 mg, and the administration frequency is once every four weeks.
  • the dsRNA is administered at a fixed dose selected from 200 mg, and the administration frequency is once every four weeks.
  • the dsRNA is administered at a fixed dose selected from 400 mg, and the administration frequency is once every four weeks.
  • the dsRNA is administered at a fixed dose selected from 600 mg, and the administration frequency is once every four weeks.
  • the dsRNA is administered at a fixed dose selected from 900 mg, and the administration frequency is once every four weeks.
  • the duration of administration of the dsRNA is each independently selected from four weeks, eight weeks, twelve weeks, twenty-four weeks, thirty-six weeks, forty-eight weeks, sixty weeks, seventy-two weeks, eighty-four weeks, and the like.
  • dsRNA of the present disclosure such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, constructing the nucleic acid as part of a retroviral or other vector.
  • the administration of the dsRNA and TLR8 modulators described in the present disclosure is conventional, and can be administered locally (e.g., direct injection or implantation) or systemically, or can be administered orally, rectally or parenterally, and the parenteral route includes but is not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (e.g., aerosol), mucosal administration (e.g., sublingual, intranasal administration), intracranial administration, etc.
  • parenteral route includes but is not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (e.g., aerosol), mucosal administration (e.g., sublingual, intranasal administration), intracranial administration, etc.
  • dsRNA provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.
  • the administration of the TLR8 modulators provided by the present disclosure is oral administration.
  • TLR8 modulators provided by the present disclosure require administration on an empty stomach.
  • the dsRNA provided by the present disclosure can be packaged in a kit.
  • the use further comprises combining with one or more other therapeutic agents.
  • the other therapeutic agents are selected from antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, viral entry inhibitors, oligonucleotides that inhibit the secretion or release of HBsAg, capsid inhibitors, and covalently closed circular (ccc) HBV DNA inhibitors.
  • the present disclosure provides a method for preventing and/or treating hepatitis B virus (HBV) infection or a disease associated with hepatitis B virus in a subject, wherein the subject is administered the aforementioned double-stranded ribonucleic acid (dsRNA) and a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.
  • HBV hepatitis B virus
  • dsRNA double-stranded ribonucleic acid
  • the double-stranded ribonucleic acid (dsRNA) and the compound represented by formula (II) or a pharmaceutically acceptable salt thereof are in effective amounts.
  • the disease associated with hepatitis B virus is chronic hepatitis and the subject is HBeAg positive or HBeAg negative.
  • the disease associated with hepatitis B virus is acute hepatitis B, chronic hepatitis B, hepatitis D virus infection, hepatitis D, liver fibrosis, advanced liver disease, and hepatocellular carcinoma.
  • the other therapeutic agent is selected from antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, viral entry inhibitors, oligonucleotides that inhibit HBsAg secretion or release, capsid inhibitors, cccDNA inhibitors, and combinations of any of the foregoing.
  • the present disclosure provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof, which is used in combination with the aforementioned double-stranded ribonucleic acid (dsRNA) for preventing and/or treating hepatitis B virus infection or diseases associated with hepatitis B virus.
  • dsRNA double-stranded ribonucleic acid
  • the present disclosure also provides the aforementioned double-stranded ribonucleic acid (dsRNA), which is used in combination with the compound represented by formula (II) or a pharmaceutically acceptable salt thereof for preventing and/or treating hepatitis B virus infection or diseases associated with hepatitis B virus.
  • dsRNA double-stranded ribonucleic acid
  • the present disclosure provides a method for inhibiting the expression of a target gene or its mRNA, comprising administering to a subject an effective amount or an effective dose of the aforementioned dsRNA and a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.
  • the target gene includes but is not limited to HBV (eg, HBV-S, HBV-X).
  • the subject has been previously identified as having pathological upregulation of the target gene or its mRNA in a targeted cell, cell population, tissue, or subject.
  • the present disclosure also provides a method for inhibiting the replication of hepatitis B virus (HBV) in a cell, the method comprising inducing the cell to be treated with the aforementioned dsRNA of the present disclosure and a compound shown in formula (II) or a pharmaceutically acceptable salt thereof, thereby inhibiting the replication of HBV in the cell.
  • the cell is in a subject.
  • the cell is in vitro.
  • the present disclosure also provides a method for reducing the level of hepatitis B virus (HBV) antigen in a subject infected with HBV, comprising administering to the subject a therapeutically effective amount of the aforementioned dsRNA described in the present disclosure and a compound shown in formula (II) or a pharmaceutically acceptable salt thereof, thereby reducing the level of HBV antigen in the subject.
  • HBV antigen is HBsAg.
  • the HBV antigen is HBeAg.
  • the subject is HBeAg positive.
  • the subject is HBeAg negative.
  • the uses or methods provided by the present disclosure reduce the HBsAg level in the serum of a subject by at least 0.5 log 10 IU/ml.
  • the uses or methods provided by the present disclosure reduce the HBsAg level in the serum of a subject by at least 1 log10 IU/ml.
  • the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms.
  • the present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present disclosure.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present disclosure.
  • the compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or in racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents.
  • Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., a carbamate is generated from an amine).
  • the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or include both
  • the bond No configuration is specified, i.e., the bond
  • the configuration can be E-type or Z-type, or include both E and Z configurations.
  • tautomer or "tautomeric form” refers to structural isomers of different energies that can be interconverted via a low energy barrier.
  • proton tautomers also referred to as prototransfer tautomers
  • prototransfer tautomers include interconversions via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerization.
  • the lactam-lactim equilibrium example is between A and B as shown below.
  • the present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature.
  • isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl , respectively.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to have deuterium (i.e., at least 10% deuterium incorporation) at least 1000 times greater than the natural abundance of deuterium (which is 0.015%).
  • the natural abundance of the compound in the example may be at least 1000 times greater than deuterium, at least 2000 times greater than deuterium, at least 3000 times greater than deuterium, at least 4000 times greater than deuterium, at least 5000 times greater than deuterium, at least 6000 times greater than deuterium or more abundant deuterium.
  • the present disclosure also includes various deuterated forms of formula (I), formula (I'), and formula (II) compounds.
  • Each available hydrogen atom connected to a carbon atom may be independently replaced by a deuterium atom.
  • Those skilled in the art can synthesize deuterated forms of formula (I), formula (I'), and formula (II) compounds with reference to the relevant literature.
  • Commercially available deuterated starting materials can be used to prepare deuterated forms of compounds of formula (I), (I') and (II), or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide.
  • the terms “about” and “approximately” refer to values that are within an acceptable error range for a particular value determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limitations of the measurement system). For example, “about” can mean within a standard deviation. Alternatively, “about” or “substantially comprising” can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 1% and 5%, between 2.5% and 3.5%, between 1% and 2.5%, between 2.5% and 3 ... The range of the present invention is 0.5% to 1%.
  • the "compound”, “chemical modification”, “ligand”, “dsRNA”, “nucleic acid” and “RNAi” of the present disclosure may independently exist in the form of salt, mixed salt or non-salt (e.g., free acid or free base). When it exists in the form of salt or mixed salt, it may be a pharmaceutically acceptable salt.
  • “Pharmaceutically acceptable salt” may be selected from inorganic salts or organic salts, and may also include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to a salt formed with an inorganic acid or organic acid that retains the biological effectiveness of the free base without other side effects.
  • Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, be
  • “Pharmaceutically acceptable base addition salt” refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects.
  • Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like.
  • Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, preferably sodium salts.
  • the salt derived from organic base includes but is not limited to the following salt: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc.
  • Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicycl
  • a "phosphate ester group” may be a phosphate monoester group, a phosphate diester group or a phosphate triester group, preferably a phosphate diester group.
  • a thiophosphate diester group refers to a phosphodiester group modified by replacing a non-bridging oxygen atom with a sulfur atom.
  • M is an S atom
  • the group middle It can be replaced by any group that can achieve linkage with adjacent nucleotides.
  • linked when referring to the connection between two molecules, means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond), including direct connection and indirect connection.
  • a non-covalent bond eg, a hydrogen bond or an ionic bond
  • directly linked means that a first compound or group is linked to a second compound or group without any intervening atoms or groups of atoms.
  • directly linked means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
  • “Pharmaceutically acceptable excipients” include, but are not limited to, any adjuvant, carrier, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, buffer, solvent or emulsifier that has been approved for use by humans or domestic animals.
  • the term “inhibit” can be used interchangeably with “reduce”, “silence”, “downregulate”, “suppress” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by the reduction of one or more of these variables in absolute or relative levels compared to the control level.
  • the control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from a similar untreated or controlled subject, cell, or sample (e.g., only a buffer control or an inert agent control).
  • the remaining expression of mRNA can be used to characterize the degree of inhibition of siRNA (or dsRNA) on target gene expression, such as the remaining expression of mRNA being no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%.
  • an “effective amount” or “effective dose” encompasses an amount sufficient to ameliorate or prevent the symptoms or symptoms of a medical condition. Amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects.
  • the effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • subject As used herein, “subject,” “patient,” “subject,” or “individual” are used interchangeably and include humans or non-human animals, such as mammals, such as humans or monkeys.
  • the sense strand (also referred to as SS, SS strand or positive strand) refers to a strand comprising a sequence identical or substantially identical to a target mRNA sequence
  • the antisense strand (also referred to as AS or AS strand) refers to a strand having a sequence complementary to a target mRNA sequence.
  • the "5' region" of the sense strand or antisense strand i.e., the "5' end” or “5' terminal end”
  • the nucleotides from positions 2 to 8 in the 5' region of the antisense strand can also be replaced by the nucleotides from positions 2 to 8 from the 5' terminal end of the antisense strand.
  • the "3' region", "3' terminal end” and “3' terminal” of the sense strand or antisense strand can also be used interchangeably.
  • dsRNA refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.
  • the term "differences from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotide sequences and comprises at least 15 consecutive nucleotides” is intended to mean that the sense strand of the siRNA described herein comprises at least 15 consecutive nucleotides of the sense strand of SEQ ID NO: 1, or differs from at least 15 consecutive nucleotides in the sense strand of SEQ ID NO: 1 by no more than 3 nucleotide sequences (optionally, differs from no more than 2 nucleotide sequences, optionally, differs from 1 nucleotide sequence).
  • the sense strand of the siRNA described herein comprises at least 16 consecutive nucleotides of the sense strand of SEQ ID NO: 1, or differs from at least 16 consecutive nucleotides in the sense strand of SEQ ID NO: 1 by no more than 3 nucleotide sequences (optionally, differs from no more than 2 nucleotide sequences, optionally, differs from 1 nucleotide sequence).
  • the term "differences from the antisense strand of either SEQ ID NO: 2 or SEQ ID NO: 3 by no more than 3 nucleotide sequences, and comprises at least 15 consecutive nucleotides” is intended to mean that the siRNA antisense strand described herein comprises at least 15 consecutive nucleotides of the antisense strand of either SEQ ID NO: 2 or SEQ ID NO: 3, or differs from at least 15 consecutive nucleotides of the antisense strand of either SEQ ID NO: 2 or SEQ ID NO: 3 by no more than 3 nucleotide sequences (optionally, differs from no more than 2 nucleotide sequences, optionally, differs from 1 nucleotide sequence).
  • G", “C”, “A”, “T” and “U” represent nucleotides, respectively, comprising the bases of guanine, cytosine, adenine, thymidine and uracil, respectively.
  • I is equivalent to a nucleotide comprising the nucleobase hypoxanthine.
  • the term inosine used in the present disclosure is equivalent to a nucleoside comprising hypoxanthine and a sugar or modified sugar.
  • the term "2'-fluoro (2'-F) modified nucleotide” refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine
  • non-fluorinated modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group.
  • 2'-methoxy (2'-OMe) modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • nucleotide difference between a nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position in the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is considered that there is a nucleotide difference at that position between the two nucleotide sequences. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference occurs at that position.
  • the terms "complementary" or “reverse complement” are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner.
  • the purine base adenine is always paired with the pyrimidine base thymine (or uracil in RNA); the purine base guanine is always paired with the pyrimidine base cytosine.
  • Each base pair includes a purine and a pyrimidine.
  • mismatch means in the art that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary form.
  • chemical modification or “modification” includes all changes in the nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.
  • base includes any known DNA and RNA base, base analogs, such as purine or pyrimidine, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs. Base analogs can also be universal bases.
  • blunt end or blunt end are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of the siRNA, i.e., no nucleotide overhangs. In most cases, a siRNA with both ends blunt-ended will be double-stranded throughout its entire length.
  • the siRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services.
  • the modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.
  • combination use refers to a method of administration of a drug within a certain period of time. At least one dose of dsRNA, and at least one dose of another therapeutic agent, wherein the drugs administered all show pharmacological effects.
  • the dsRNA and another therapeutic agent can be administered simultaneously or sequentially. This period includes such treatment, wherein the dsRNA and another therapeutic agent are administered by the same administration route or different administration routes.
  • the combined administration mode of the present disclosure is selected from simultaneous administration, independently formulated and co-administered, or independently formulated and administered sequentially.
  • the starting material compound 1 was purchased from Jiangsu Beida Pharmaceutical Technology Co., Ltd.
  • the combined organic phase was washed with saturated brine (100mL), dried over Na 2 SO 4 , filtered, and the residue obtained by concentration was separated by silica gel column chromatography to obtain the target compound 2 (20.0g, 51.8mmol, yield 51%).
  • TMSCN 13.5 mL, 101 mmol was added to a solution of compound 2 (13.0 g, 33.6 mmol) in DCM (300 mL) at once, followed by dropwise addition of a solution of TMSOTf (9.14 mL, 50.5 mmol) in DCM (30 mL).
  • the reaction solution was stirred at 20°C for 15 hours. After the reaction was completed, the system was quenched with saturated aqueous NaHCO 3 solution (80 mL) and extracted with DCM (150 mL ⁇ 2).
  • the combined organic phase was washed with saturated brine (80 mL), dried over Na 2 SO 4 , filtered and concentrated, and separated by silica gel column chromatography to obtain the target compound 3 (3.30 g, 9.18 mmol, yield 27%) and a light yellow oily liquid compound 4 (8.50 g, 9.18 mmol, yield 70%).
  • compound NAG0024 (271 mg, 0.151 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (4 mL), 3A molecular sieves were added, and compound 12 (100 mg, 0.151 mmol), HOBt (25 mg, 0.181 mmol), DCC (38 mg, 0.181 mmol) and DIEA (39 mg, 0.302 mmol) were added in sequence.
  • the reaction solution was reacted at 45°C for 16 h. After LC-MS showed that the reaction was complete, water was added to quench the reaction and the mixture was filtered. After the filtrate was concentrated, it was purified by C18 reverse phase column (H 2 O/MeCN) to obtain compound 13 (210 mg, yield 57%).
  • the compound NAG0052 was connected to the sequence through solid phase synthesis, and then after aminolysis, some functional groups of the NAG0052 structure were removed to become NAG0052'.
  • each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization.
  • the prepared dsRNA had the sense strand and antisense strand shown in Tables 2 and 3.
  • NAG0052' The structure of NAG0052' is:
  • Table 4 shows the positive control compounds.
  • AD81890 was prepared according to CN201980053789.8; the structure of L96 is
  • the target sequence of dsRNA was constructed with HBV gene and inserted into psiCHECK-2 plasmid.
  • the plasmid contains Renilla luciferase gene and firefly luciferase gene.
  • the target sequence of dsRNA was inserted into the 3'UTR region of Renilla luciferase gene.
  • the activity of dsRNA against the target sequence can be The expression of Renilla luciferase was detected after calibration with firefly luciferase using Dual-Luciferase Reporter Assay System (Promega, E2940).
  • HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO 2. 24 h before transfection, HEK293A cells were seeded in 96-well plates at a seeding density of 8 ⁇ 10 3 cells per well with 100 ⁇ L of culture medium per well.
  • dsRNA and corresponding plasmids were co-transfected into cells using Lipofectamine 2000 (ThermoFisher, 11668019), and 0.2 ⁇ L of Lipofectamine 2000 was used per well. The amount of plasmid transfection was 20 ng per well.
  • the target sequence plasmid 11 concentration points of dsRNA were set, and the final concentration of the highest concentration point was 20 nM, 3-fold gradient dilution, 20 nM, 6.6667 nM, 2.2222 nM, 0.7407 nM, 0.2469 nM, 0.0823 nM, 0.0274 nM, 0.0091 nM, 0.0030 nM, 0.0010 nM and 0.0003 nM. 24 h after transfection, the Dual-Luciferase Reporter Assay System (Promega, E2940) was used to detect the target level.
  • Psi-CHECK plasmid was purchased from Sangon Biotech (Shanghai) Co., Ltd.
  • DMEM complete medium (DMEM + 10% FBS) was stored at 4°C and taken out to equilibrate to room temperature before the experiment.
  • HEK293A cells were seeded in 96-well plates at a seeding density of 8 ⁇ 10 3 cells/well in 100 ⁇ L of culture medium/well;
  • dsRNA sample was quantified to 20 ⁇ M, and its concentration was determined by psi-CHECK plasmid. It was stored at -20°C for future use and required to be centrifuged briefly before use.
  • Tube A Dispense the prepared plasmid into 8 corresponding tubes, 22 ⁇ L/tube, named: Tube A;
  • Dilute dsRNA Take out the dsRNA from -20°C and thaw it, mix it evenly, and dilute it to different concentrations according to different experimental requirements as working solution according to Table 8, and prepare it before use.
  • Dual luciferase reporter gene assay kit (Promega, cat. E2940) components and preparation method: Buffer and via Luciferase Substrate (lyophilized) was pre-mixed and then dispensed into 15 mL centrifuge tubes, 7.5 mL per tube. Stop& Buffer was dispensed in advance, 12 mL per tube. After re-thawing, add 7.5 mL of DMEM to each tube after equilibration to room temperature. Stop& Buffer was reconstituted and then equilibrated to room temperature. Stop& Prepare at a ratio of 100:1 and use immediately.
  • Ratio Ren/Fir (Ratio of Renilla to Firefly);
  • HepG2.2.15 cells were seeded into 96-well plates, with 20,000 cells per well. At the same time, different concentrations of dsRNA were transferred into HepG2.2.15 cells using RNAiMax.
  • the cell culture supernatant was collected and HBsAg was detected by ELISA (the remaining supernatant was frozen for later use).
  • the cells were collected, the intracellular RNA was extracted, and the total HBV RNA (including 3.5kb+2.4kb+2.1kb+0.7kb RNA) and 3.5kb HBV RNA (including pgRNA+preCore RNA) were detected by RT-PCR, and the GAPDH gene RNA was detected as an internal reference. The compound was measured at 8 concentrations, and the assay was performed in duplicate wells. The final concentration of DMSO in the culture medium was 0.5%.
  • the inhibition percentage was calculated as follows:
  • % HBsAg inhibition rate (1-HBsAg content in sample/HBsAg content in DMSO control group) ⁇ 100
  • % HBV RNA inhibition rate (1-HBV RNA content in sample/HBV RNA content in DMSO control group) ⁇ 100
  • % cell viability (absorbance of sample - absorbance of culture medium control)/(absorbance of DMSO control - absorbance of culture medium control) ⁇ 100.
  • Graphpad Prism software was used to analyze the data (four parameter logistic equations) and calculate the EC50 values.
  • Example 5 Evaluation of dsRNA anti-HBV activity in vitro using PHH cells (free uptake)
  • TRD007970, TJR100259 and AD81890 The in vitro anti-HBV activity of TRD007970, TJR100259 and AD81890 was evaluated respectively.
  • dsRNA was diluted with PBS to 7 concentrations (100, 25, 6.25, 1.563, 0.391, 0.098, 0.024 nM) and added to a 48-well plate. Frozen PHH was resuscitated and then plated into a 48-well plate; while the test compound was freely taken up (free uptake) into the cells.
  • Example 6 Exonuclease stability assay to evaluate dsRNA stability
  • the exonuclease stability was evaluated for naked siRNA sequences TJR100381 and TJR100382 (see Table 13) of TRD007970 and TJR100259, respectively.
  • the corresponding reaction solutions were prepared according to the experimental reaction system for the experiment, see Tables 14-15.
  • the final concentration of 5' exonuclease (PDII, Worthington, cat#LS003602) is 500U/mL
  • the final concentration of 3' exonuclease (SVPD, Worthington, cat#LS003926) is 0.5U/mL.
  • the mixture was divided into 8 different strip tubes (16ul per well) at five time points: 0h, 1.5h, 2h, 3h, and 4h, and incubated at 37°C. After reaching the time point, the reaction solution was immediately taken out and a loading buffer containing 9M urea (32ul per well) was added, and the mixture was placed in a -80°C refrigerator for storage.
  • the metabolic stability of dsRNA was analyzed by using cynomolgus monkey liver S9 (cynomolgus monkey liver S9, male, supplier Xenotech, batch number 1510192) for TRD007970, TJR100259 and AD81890, respectively.
  • the experimental process is as follows:
  • TJR100259 and AD81890 samples were incubated in a 37°C water bath.
  • Remaining % peak area ratio of analyte to internal standard at each time point / peak area ratio of analyte to internal standard at T0 ⁇ 100%
  • Clint (liver) Clint (S9) * (mg S9) / g liver * g liver / (kg body weight).
  • the experimental results are shown in Table 16.
  • the results show that after 48 hours of incubation with cynomolgus monkey liver S9, 93.8% of the antisense strand of TJR100259, 70.2% of the antisense strand of AD81890, and 61.0% of the antisense strand of TRD007970 remained.
  • the data show that after 48 hours of incubation with cynomolgus monkey liver S9, the antisense strand in TJR100259 is more stable than the antisense strand in AD81890 and the antisense strand in TRD007970.
  • TJR100259 is more stable than AD81890 and TRD007970 under the metabolic stability test conditions of cynomolgus monkey liver S9.
  • TRD007970, TJR100410 and TJR100259 were subjected to tissue distribution experiments.
  • the experimental process was as follows: 72 male C56BL/6J mice (7-8w, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were adapted for about 1 week and divided into 3 groups, 24 in each group.
  • the collected organs were rinsed with physiological saline. After pretreatment of liver and kidney samples, high-resolution mass spectrometry was used to analyze the concentration of AS chains in the liver and kidneys to characterize the concentrations of TRD007970, TJR100259 and TJR100410.
  • the experimental results are shown in Table 17.
  • the results showed that in the tissue distribution experiment of C56BL/6J mice, the liver and kidney exposure ratios of TRD007970, TJR100259 and TJR100410 antisense chains were 9.44, 29.38 and 3.49, respectively.
  • the large liver and kidney exposure ratios indicate that the concentration in the target organ (liver) is higher and the concentration in the non-target organ (kidney) is lower.
  • TJR100259 showed a higher liver-kidney ratio than TRD007970, and TRD007970 showed a higher liver-kidney ratio than TJR100410, indicating that the risk of renal toxicity of TJR100259 is lower than that of TRD007970, and the risk of renal toxicity of TRD007970 is lower than that of TJR100410.
  • TJR100410 and TJR100259 were used to analyze the stability of liver homogenate.
  • the experimental process is as follows:
  • wash 1 Rinse 600 ⁇ L of wash buffer 1 (25 mM NH4Ac pH 5.5) through the SPE plate, and then repeat the above steps again.
  • wash 2 Rinse 600 ⁇ L of wash buffer 2 (25 mM NH4Ac pH 5.5, 50% ACN) through the SPE plate and repeat the above steps again.
  • TJR100410 is the control dsRNA, and its sense strand is:
  • the antisense strand is:
  • NAG1 The structure of NAG1 is
  • TJR100259 is more stable than TJR100410 under the stability test conditions of cynomolgus monkey liver homogenate.
  • Example 10 Study on the efficacy of TRD007970 and the compound of formula (II) in the TLR8 humanized mouse AAV-HBV (adeno-associated virus-hepatitis B virus) infection model
  • mice Female human TLR8 mice (Shanghai South Model Organisms Technology Co., Ltd., Animal Health Certificate No. 20190002028579) were injected with 200 ⁇ L rAAV8-1.3HBV virus suspension (containing 1 ⁇ 1011 rAAV8-1.3HBV virus copies, Wuhan Shumi Brain Science Technology Co., Ltd., batch number ayw1-p4-220412) through the tail vein on Predose day 0.
  • Predose days 14 and 25 14 and 25 days after AAV-HBV injection
  • blood was collected from each mouse through the mandibular vein to prepare (10+15) ⁇ L serum for HBV DNA quantitative detection.
  • mice were selected and randomly divided into 4 groups, as shown in Table 20.
  • Vehicle (Group 1), compound of formula (II) (refer to WO2020007275A
  • the test samples were prepared by the method in Example 6 of the present invention (Groups 2 and 4) and were administered orally twice on days 0 to 7 (days 0 and 3).
  • TRD007970 (Groups 3 and 4) was administered subcutaneously once on days 0 to 7 (day 0).
  • the administration volume of the test samples was 5 mL/kg. Blood was collected on days 0 and 7 to collect plasma, 50 ⁇ L per animal each time, and stored at 2 to 8°C for quantitative detection of HBV DNA.

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Abstract

Toll样受体调节剂与dsRNA的联合治疗。具体而言，涉及Toll样受体调节剂与dsRNA联合在制备预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病的药物中的用途。

Description

Toll样受体调节剂与dsRNA的联合治疗 技术领域
本公开涉及Toll样受体调节剂与dsRNA联合在制备预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病的药物中的用途，属于医药领域。
技术背景
Toll样受体(TLR)家族在病原体识别和先天免疫激活中起着重要作用，Toll样受体8(TLR-8)主要由骨髓免疫细胞表达，该受体的激活会刺激广泛的免疫反应。TLR-8的激动剂激活髓样树突细胞、单核细胞、单核细胞衍生的树突细胞和Kupffer细胞，导致促炎细胞因子和趋化因子的产生，例如白细胞介素18(IL-18)、白细胞介素12(IL-12)、肿瘤坏死因子-α(TNF-a)和干扰素-γ(IFN-g)。此类激动剂还促进共刺激分子如CD8+细胞、主要组织相容性复合物分子(MAIT、NK细胞)和趋化因子受体的表达增加。TLR8调节化合物包括WO2020007275A中描述的那些。总的来说，这些先天性和适应性免疫反应的激活会诱导免疫反应，并在涉及自身免疫、炎症、过敏、哮喘、移植物排斥、移植物抗宿主病(GvHD)、感染、癌症和免疫缺陷的各种病症中提供治疗益处。例如，对于乙型肝炎，专职抗原呈递细胞(pAPC)和其他肝内免疫细胞上TLR8的激活与IL-12和促炎细胞因子的诱导有关，这有望增强HBV特异性T细胞反应，激活肝内NK细胞并驱动抗病毒免疫的重建。
RNA干扰(RNAi)是一种有效的沉默基因表达的方式，利用RNA干扰技术，可以根据编码这些蛋白的mRNA，设计合适的siRNA，特异性靶向目标mRNA并降解目标mRNA，从而达到抑制相关的蛋白生成的目的。采用靶向配体缀合siRNA，利用靶向配体与细胞膜表面的受体分子结构，从而内吞进入到细胞内，是一种有效的药物递送方式。例如，去唾液酸糖蛋白受体(ASGPR)是肝细胞特异性表达的受体，在肝细胞表面具有高丰度，胞内外转换快速的特点。半乳糖、半乳糖胺、N-乙酰半乳糖胺等单糖和多糖分子对ASGPR有高亲和性。文献报道(10.16476/j.pibb.2015.0028)使用氨基半乳糖分子簇(GalNAc)可以有效递送siRNA到肝细胞，GalNAc分子被设计成三价或四价的分子簇可以显著提高单价或二价的GalNAc分子靶向肝细胞的能力。
目前患有慢性HBV感染的患者使用的主要药物是核苷/核苷酸逆转录酶抑制剂(NRTI)，NRTI抑制感染性病毒粒子的产生，并且通常将血清HBV DNA减少到不可检测。然而，NRTI不直接消除cccDNA，所以病毒蛋白质继续转录和翻译。因此，肝细胞上病毒表位的表达、亚病毒粒子的分泌和免疫功能异常在很大程度上保持不受NRTI疗法的影响。导致的结果是需要长时间的甚至终身的治疗。因此，临床上对有效、耐受良好并且不需要终身服药的新HBV疗法仍有需求。 WO2022241134A公开了一种TLR8调节剂联合dsRNA，以及PD-1/PD-L1抑制剂治疗和/或预防受试者的乙型肝炎病毒感染的方法，WO2020232024A公开一种用于治疗HBV感染的组合物及方法，具体涉及dsRNA与PEG-INF。
发明内容
本公开涉及一种双链核糖核酸(dsRNA)联合Toll样受体8(TLR8)调节剂在制备预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病的药物中的用途。
本公开还提供了一种预防和/或治疗受试者的乙型肝炎病毒(HBV)感染或与乙型肝炎病毒相关的疾病的方法，给与受试者dsRNA和TLR8调节剂。
本公开还提供了一种TLR8调节剂，其与dsRNA联合，用于预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病。
本公开还提供一种dsRNA，其与TLR8调节剂或其可药用盐联合，用于预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病。
本公开还提供一种抑制靶基因或其mRNA表达的方法，其包括向受试者给予有效量或有效剂量的本公开所述的dsRNA和TLR8调节剂。
一些实施方案中，所述与乙型肝炎病毒相关的疾病选自慢性肝炎、急性乙型肝炎、慢性乙型肝炎、丁型肝炎病毒感染、丁型肝炎、肝纤维化、晚期肝病或肝细胞癌。
一些实施方案中，所述与乙型肝炎病毒相关的疾病是慢性肝炎，所述受试者是HBeAg阳性或HBeAg阴性。
一些实施方案中，所述Toll样受体8(TLR8)调节剂是如式(II)所示的化合物或其可药用盐，
一些实施方案中，所述dsRNA，其包含：
siRNA和一个或多个与其缀合的配体；
所述siRNA包含有义链和反义链，
所述反义链在其5’端起第7位核苷酸位置处包含式(II)所示的化学修饰、其互变异构体或其药学上可接受的盐：
所述式(I)所示的化学修饰选自以下任一结构：
B与所述反义链5’端起第7位核苷酸未被修饰时的碱基相同；
所述配体是以下结构所示或其药学上可接受的盐：
所述有义链和反义链选自以下任一组：
有义链包含SEQ ID NO：1所示的核苷酸序列，反义链包含SEQ ID NO：2所示的核苷酸序列；
有义链包含SEQ ID NO：1所示的核苷酸序列，反义链包含SEQ ID NO：3所示的核苷酸序列；
所述有义链的3’端与所述配体缀合；
所述B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
本公开中，按照5’-3’方向，
SEQ ID NO:1是GUGUGCACUUCGCUUCACC；
SEQ ID NO:2是AGUGAAGCGAAGUGCACACGG；
SEQ ID NO:3是IGUGAAGCGAAGUGCACACGG。
一些实施方案中，所述dsRNA中的配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端连接。
一些实施方案中，所述dsRNA中的配体通过磷酸二酯基团或硫代磷酸二酯基团与所述siRNA末端连接。
一些实施方案中，所述dsRNA中的配体通过磷酸二酯基团与所述siRNA末端连接。
一些实施方案中，所述dsRNA中的配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端间接连接。
一些实施方案中，所述dsRNA中的配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端直接连接。
一些实施方案中，所述dsRNA中的所述配体通过磷酸酯基团或硫代磷酸酯基 团与所述siRNA的有义链3’末端直接连接。
一些实施方案中，所述dsRNA中的磷酸酯基团为磷酸一酯基团或磷酸二酯基团。在一些实施方案中，所述磷酸酯基团为磷酸二酯基团。
一些实施方案中，所述dsRNA中的硫代磷酸酯基团为硫代磷酸一酯基团或硫代磷酸二酯基团。
一些实施方案中，所述dsRNA中的硫代磷酸酯基团为硫代磷酸二酯基团。
一些实施方案中，所述dsRNA可为以下结构或其药学上可接受的盐，
其中，Z为siRNA，所述siRNA的有义链的3’末端通过磷酸二酯基团与配体直接连接，所述siRNA如本公开所定义。
一些实施方案中，所述dsRNA在包含式(I)所示化学修饰以外的其余位置处，所述的有义链和/或反义链中至少一个另外的核苷酸为修饰的核苷酸。
一些实施方案中，所述dsRNA中的修饰的核苷酸选自：2'-甲氧基修饰的核苷酸、2'-经取代的烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-氟代修饰的核苷酸、2'-脱氧核苷酸、2'-脱氧-2'-氟代修饰的核苷酸、3'-脱氧-胸腺嘧啶核苷酸、异核苷酸、LNA、ENA、cET、UNA、GNA。
一些实施方案中，所述dsRNA中的修饰的核苷酸相互独立地选自：2'-甲氧基修饰的核苷酸或2'-氟代修饰的核苷酸。
一些实施方案中，所述dsRNA的有义链含有连续三个具有相同修饰的核苷酸。
一些实施方案中，所述的三个具有相同修饰的核苷酸为2'-氟代修饰的核苷酸。
一些实施方案中，按照5'端到3'端的方向，所述反义链的第2、4、6、10、12、14、16和18位的核苷酸各自独立地为2'-氟代修饰的核苷酸。
一些实施方案中，所述siRNA的反义链与靶序列至少部分地反向互补。在一些实施方案中，所述反义链与靶序列之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配。在一些实施方案中，所述反义链与靶序列完全反向互补。
一些实施方案中，所述siRNA的有义链与反义链至少部分地反向互补以形成双链区。在一些实施方案中，所述有义链与反义链之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配。在一些实施方案中，所述有义链与反义链完全反向互补。
一些实施方案中，所述siRNA的有义链和反义链各自独立地具有16至35个、16至34个、17至34个、17至33个、18至33个、18至32个、18至31个、18至30个、18至29个、18至28个、18至27个、18至26个、18至25个、18至24个、18至23个、19至25个、19至24个或19至23个核苷酸(例如19、20、21、22、23个核苷酸)。
一些实施方案中，所述siRNA的有义链和反义链长度相同或不同，所述有义链的长度为19-23个核苷酸，所述反义链的长度为19-26个核苷酸。本公开提供的dsRNA中的有义链和反义链的长度比可以是19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/19、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25或23/26。
一些实施方案中，所述siRNA有义链和反义链的长度比为19/21、21/23或23/25。在一些实施方案中，所述有义链和反义链的长度比为19/21。
一些实施方案中，所述siRNA包含一个或两个平端。
一些具体的实施方案中，siRNA的每条链各自独立地包含具有1至2个未配对的核苷酸，形成突出端。
一些实施方案中，所述siRNA包含位于所述反义链3’端的突出端。
一些实施方案中，siRNA有义链中位于5’端第7-9位的三个连续核苷酸为2'-氟代修饰的核苷酸。
在一些实施方案中，所述siRNA的有义链含有如下式所示的核苷酸序列(5’-3’)：
NaNaNaNaXNaNbNbNbNaNaNaNaNaNaNaNaNaNa
其中，每个X独立地为Na或Nb；Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸。
在一些实施方案中，所述siRNA有义链含有如下式所示的核苷酸序列：
5’-NaNaNaNaNaNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；或，
5’-NaNaNaNaNbNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
其中，Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸。
在一些实施方案中，所述siRNA反义链含有如下式所示的核苷酸序列：
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
其中，Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；W’表示2'-甲氧基修饰的核苷酸或包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰的核苷酸。
在一些具体的实施方案中，W’表示包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些具体的实施方案中，式(I)所示的化学修饰选自：
其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶。在一些具体的实施方案中，B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
在一些具体的实施方案中，式(I)所示的化学修饰选自：
其中：M为O或S；其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶或尿嘧啶。在一些具体的实施方案中，B与所述反义链在其5’端起第7位核苷酸未被修饰时的碱基相同。
在一些具体的实施方案中，所述M为S。
一些具体的实施方案中，所述M为O。
在一些实施方案中，所述有义链和/或反义链中至少一个磷酸酯基为具有修饰基团的磷酸酯基。所述修饰基团使得所述siRNA在生物样品或环境中具有增加的稳定性。在一些实施方案中，所述具有修饰基团的磷酸酯基为硫代磷酸酯基。在一些实施方案中，所述具有修饰基团的磷酸酯基为硫代磷酸二酯基。
在一些实施方案中，所述硫代磷酸二酯基存在于以下位置中的至少一处：
所述有义链的5'端第1个核苷酸和第2个核苷酸之间；
所述有义链的5'端第2个核苷酸和第3个核苷酸之间；
所述反义链的5'端第1个核苷酸和第2个核苷酸之间；
所述反义链的5'端第2个核苷酸和第3个核苷酸之间；
所述反义链的3'端第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述有义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
所述有义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述有义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述反义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述反义链的3'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述有义链包含如下式所示的核苷酸序列：
5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，或，
5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，
其中，Nm表示2'-甲氧基修饰的任意核苷酸，例如2'-甲氧基修饰的C、G、U、A；Nf表示2'-氟代修饰的任意核苷酸，例如2'-氟代修饰的C、G、U、A；
小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；小写字母s在3’端第一个时表示与该字母s上游(5'方向)相邻的一个核苷酸末端为硫代磷酸二酯基。
在一些实施方案中，所述反义链包含如下式所示的核苷酸序列：
5’-Nm’sNf’sNm’Nf’Nm’Nf’W’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’sNm’
sNm’-3’；
其中，Nm’表示2'-甲氧基修饰的任意核苷酸，例如2'-甲氧基修饰的C、G、U、A；Nf’表示2'-氟代修饰的任意核苷酸，例如2'-氟代修饰的C、G、U、A；
小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接，小写字母s在3’端第一个时表示与该字母s上游相邻的一个核苷酸末端为硫代磷酸二酯基；
W’表示2'-甲氧基修饰的核苷酸或包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰的核苷酸。
在一些实施方案中，所述dsRNA选自以下任一组：
包含SEQ ID NO：4和SEQ ID NO：5；
包含SEQ ID NO：4和SEQ ID NO：6。
在一些实施方案中，所述dsRNA选自以下任一组：
包含或选自SEQ ID NO：4所示的有义链和SEQ ID NO：5所示的反义链；
包含或选自SEQ ID NO：4所示的有义链和SEQ ID NO：5所示的反义链；
本公开中，按照5’-3’方向，
SEQ ID NO:4是
GmsUmsGmUmGmCmAfCfUfUmCmGmCmUmUmCmAmCmCm-NAG0052’；
SEQ ID NO:5是
AmsGfsUmGfAmAf(-)hmpNA(G)CmGmAfAmGfUmGfCmAfCmAfCmsGmsGm；
SEQ ID NO:6是
ImsGfsUmGfAmAf(-)hmpNA(G)CmGmAfAmGfUmGfCmAfCmAfCmsGmsGm；
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F  ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；Im＝次黄嘌呤2'-OMe核糖核苷(hypoxanthine 2'-OMe ribonucleoside)
如无特别说明，核苷通过之间为磷酸二酯基连接，s表示与该字母s左右相邻的两个核苷之间为硫代磷酸二酯基连接；
NAG0052’表示
(-)hmpNA(G)表示
在一些实施方案中，所述dsRNA选自如下结构或其药学上可接受的盐：

其中，
Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；
Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；
Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；
Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；
Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；
Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；
Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；
Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；
Im＝次黄嘌呤2'-OMe核糖核苷(Inosine 2'-OMe ribonucleoside)；
表示硫代磷酸二酯基阴离子形式，表示磷酸二酯基阴离子形式，
表示硫代磷酸二酯基，表示磷酸二酯基；
表示；
表示
表示
表示
在一些实施方案中，所述药学上可接受的盐可为本领域常规的盐，包括但不限于：钠盐、钾盐、铵盐、胺盐等。
在一些具体的实施方案中，所述dsRNA选自TRD007970、TJR100259。
在一些实施方案中，所述dsRNA为TRD007970，其为如下结构：
在一些实施方案中，所述dsRNA为TJR100259，其为如下结构：
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；
Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；Im＝次黄嘌呤2'-OMe核糖核苷(Inosine 2'-OMe ribonucleoside)。
表示硫代磷酸二酯基阴离子形式，表示磷酸二酯基阴离子形式，表示
表示
在一些实施方案中，所述药学上可接受的盐可为本领域常规的盐，包括但不限于：钠盐、钾盐、铵盐、胺盐等。
在一些实施方案中，所述式(II)所示化合物或其可药用盐按照固定剂量给药， 给药剂量选自0.02mg-50mg，具体的给药剂量可选自0.02mg、0.25mg、0.50mg、0.75mg、1.00mg、1.25mg、1.50mg、1.75mg、2.00mg、2.25mg、2.50mg、2.75mg、3.00mg、3.25mg、3.50mg、3.75mg、4.00mg、4.25mg、4.50mg、4.75mg、5.00mg、5.25mg、5.50mg、5.75mg、6.00mg、6.25mg、6.50mg、6.75mg、7.00mg、7.25mg、7.50mg、7.75mg、8.00mg、8.25mg、8.50mg、8.75mg、9.00mg、9.25mg、9.50mg、9.75mg或10.00mg，或者任意两点之间的点值。
一些实施方案中，所述式(II)所示化合物或其可药用盐的给药频次选自每周两次，每周一次，每两周一次和每四周一次。
一些实施方案中，所述式(II)所示化合物或其可药用盐给药的维持时间选自四周、八周、十二周、二十四周、三十六周、四十八周、六十周、七十二周、八十四周等。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量选自0.50mg、0.75mg、1.00mg、1.25mg、1.50mg、1.75mg、2.50mg或3mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为0.50mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为0.75mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为1.00mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为1.25mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为1.50mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为1.75mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为2.50mg，给药频次为每周一次。
在一些实施方案中，所述式(II)所示化合物或其可药用盐的给药剂量为3mg，给药频次为每周一次。
在一些实施方案中，所述dsRNA按照固定剂量给药，给药剂量选自10mg至1500mg，具体可选50mg、75mg、100mg、125mg、150mg、175mg、200mg、225mg、250mg、275mg、300mg、325mg、350mg、375mg、400mg、425mg、450mg、475mg、500mg、525mg、550mg、575mg、600mg、625mg、650mg、675mg、700mg、725mg、750mg、775mg、800mg、825mg、850mg、875mg、900mg、925mg、950mg、975mg、1000mg、1025mg、1050mg、1075mg、1100mg、1125mg、1150mg、1175mg、1200mg、 1225mg、1250mg、1275mg、1300mg、1325mg、1350mg、1375mg、1400mg、1425mg、1450mg、1475mg或1500mg，或者任意两点之间的点值。
在一些实施方案中，所述dsRNA按照体重给药，给药剂量选自约0.001mg/kg体重至约200mg/kg体重、约0.01mg/kg体重至约100mg/kg体重或约0.5mg/kg体重至约50mg/kg体重，约1mg/kg至20mg/kg。
在一些实施方案中，所述dsRNA的给药频次选自每周一次及后续减少频次，具体可以是每周一次、每两周一次、每三周一次、每四周一次、每五周一次、每六周一次、每七周一次，每八周一次、每九周一次、每十周一次、每十一周一次或每十二周一次。
在一些实施方案中，所述dsRNA的给药频次选自每周一次、每两周一次、每四周一次、每八周一次或每十二周一次。
在一些实施方案中，所述dsRNA按照固定剂量给药，给药剂量选自100mg，给药频次为每四周一次。
在一些实施方案中，所述dsRNA按照固定剂量给药，给药剂量选自200mg，给药频次为每四周一次。
在一些实施方案中，所述dsRNA按照固定剂量给药，给药剂量选自400mg，给药频次为每四周一次。
在一些实施方案中，所述dsRNA按照固定剂量给药，给药剂量选自600mg，给药频次为每四周一次。
在一些实施方案中，所述dsRNA按照固定剂量给药，给药剂量选自900mg，给药频次为每四周一次。
在一些实施方案中，所述dsRNA的给药的维持时间各自独立地选自四周、八周、十二周、二十四周、三十六周、四十八周、六十周、七十二周、八十四周等。
各种递药系统是已知的，并且可以用于本公开中的dsRNA，例如封装在脂质体中、微粒、微囊、能够表达该化合物的重组细胞、受体介导的细胞内吞作用、构建核酸作为逆转录病毒或其他载体的一部分。
在一些实施方案中，本公开所述的dsRNA、TLR8调节剂的给药方式是常规的，可通过局部给药(例如，直接注射或植入)或全身给药，也可通过口服、直肠或胃肠外途径进行给药，所述肠胃外途径包括但不限于皮下注射、静脉注射、肌肉注射、腹腔注射、透皮给药、吸入给药(如气溶胶)、粘膜给药(如舌下、鼻内给药)、颅内给药等。
在一些实施方案中，本公开提供的dsRNA可以通过注射给予，例如，静脉内、肌内、皮内、皮下、十二指肠内或腹膜内注射。
在一些实施方案中，本公开提供的TLR8调节剂给药方式为口服给药。
在一些实施方案中，本公开提供的TLR8调节剂需要空腹给药。
在一些实施方案中，本公开提供的dsRNA可被包装在试剂盒中。
一些实施方案中，所述用途还包含联合一种或多种其他治疗剂。
一些具体的实施方案中，所述其他治疗剂选自治疗选自抗病毒剂、反转录酶抑制剂、免疫刺激剂、治疗性疫苗、病毒侵入抑制剂、抑制HbsAg的分泌或释出的寡核苷酸、壳体抑制剂、共价闭合环状(ccc)HBV DNA抑制剂。
本公开提供了一种预防和/或治疗受试者的乙型肝炎病毒(HBV)感染或与乙型肝炎病毒相关的疾病的方法，给与受试者前述的双链核糖核酸(dsRNA)和式(II)所示化合物或其可药用盐。
在一些实施方案中，所述的双链核糖核酸(dsRNA)和式(II)所示化合物或其可药用盐是有效量的。
在一些实施方案中，所述与乙型肝炎病毒相关的疾病是慢性肝炎，所述受试者是HBeAg阳性或HBeAg阴性。
在一些实施方案中，所述与乙型肝炎病毒相关的疾病是急性乙型肝炎、慢性乙型肝炎、丁型肝炎病毒感染、丁型肝炎、肝纤维化、晚期肝病和肝细胞癌。
在一些实施方案中，所述另一种治疗剂选自抗病毒剂、反转录酶抑制剂、免疫刺激剂、治疗性疫苗、病毒侵入抑制剂、抑制HbsAg分泌或释出的寡核苷酸、壳体抑制剂、cccDNA抑制剂，及上述任一者的组合。
本公开提供一种式(II)所示化合物或其可药用盐，其与前述的双链核糖核酸(dsRNA)联合，用于预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病。
本公开还提供一种前述双链核糖核酸(dsRNA)，其与式(II)所示化合物或其可药用盐联合，用于预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病。
另一方面，本公开提供了一种抑制靶基因或其mRNA表达的方法，其包括向受试者给予有效量或有效剂量的前述dsRNA和式(II)所示化合物或其可药用盐。
在一些实施方案中，所述的靶基因包括但不限于HBV(例如HBV-S、HBV-X)。
在一些实施方案中，所述对象已在先前被鉴定为在靶向的细胞、细胞群、组织或受试者中具有靶基因或其mRNA的病理性上调。
本公开还提供了抑制细胞中乙型肝炎病毒(HBV)复制的方法，该方法包括使细胞与本公开的前述dsRNA和式(II)所示化合物或其可药用盐，从而抑制细胞中HBV的复制。在一些实施方案中，细胞在受试者体内。在一些实施方案中，细胞是体外的。
本公开还提供了降低感染了HBV的受试者中乙型肝炎病毒(HBV)抗原水平的方法，其包括向受试者给与治疗有效量的本公开所述的前述dsRNA和式(II)所示化合物或其可药用盐，从而降低受试者中HBV抗原的水平。在一些实施方案中，HBV抗原是HBsAg。在一些实施方案中，HBV抗原是HBeAg。在一些实施方案中，所述受试者是HBeAg阳性。在一些实施方案中，所述受试者是HBeAg阴性。
一些实施方案中，本公开提供的用途或方法，使得受试者的血清中的HBsAg水平降低至少0.5log 10IU/ml。
一些实施方案中，本公开提供的用途或方法，使得受试者的血清中的HBsAg水平降低至少1log10IU/ml。
本公开引证WO2022028462A1、WO2022206946A1、PCT/CN2022/103275全文。
术语解释
为了更容易理解本公开，以下具体定义了一些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开化合物可以存在特定的几何或立体异构体形式。本公开设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-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公开的范围之内。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，均包括在本公开的范围之内。本公开的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
可以通过的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。如果想得到本公开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)。
本公开所述化合物的化学结构中，键表示未指定构型，即如果化学结构中存在手性异构体，键可以为或者同时包含两种构型。本公开所述化合物的化学结构中，键并未指定构型，即键的构型可以为E型或Z型，或者同时包含E和Z两种构型。
在本公开的化学结构式中，可以根据本文所述发明范围连接一个或多个任何基团；星号“*”表示手性中心。
在不指明构型的情况下，本公开的化合物和中间体还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本公开的范围内。术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。例如，质子互变异构体(也称为质子转移互变异构体)包括经由质子迁移的互变，如酮-烯醇及亚胺-烯胺、内酰胺-内酰亚胺异构化。内酰胺-内酰亚胺平衡实例是在如下所示的A和B之间。
本公开中的所有化合物可以被画成A型或B型。所有的互变异构形式在本发明的范围内。化合物的命名不排除任何互变异构体。
本公开还包括一些与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本公开化合物。可结合到本公开化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、31P、32P、35S、18F、123I、125I和36Cl等。
除另有说明，当一个位置被特别地指定为氘(D)时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少1000倍的丰度的氘(即，至少10％的氘掺入)。示例中化合物的具有大于氘的天然丰度可以是至少1000倍的丰度的氘、至少2000倍的丰度的氘、至少3000倍的丰度的氘、至少4000倍的丰度的氘、至少5000倍的丰度的氘、至少6000倍的丰度的氘或更高丰度的氘。本公开还包括各种氘化形式的式(I)、式(I’)、式(II)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(I)、式(I’)、式(II)化合物。在制备氘代形式的式(I)、式(I’)、式(II)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
除另有说明，“任选地”、“任选”、“可选的”或“可选”是指意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如“任选地，R1和R2直接相连成环”是指R1和R2直接相连成环可以发生但不必须存在，该说明包括R1和R2直接相连成环的情形和R1和R2不成环的情形。
术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”可意味着在标准差范围内。或者，“约”或“基本上包含”可意味着至多20％的范围，例如1％至15％之间、在1％至10％之间、在1％至5％之间、在0.5％至5％之间、 在0.5％至1％之间变化，本公开中，数字或数值范围之前有术语“约”的每种情况也包括给定数的实施方案。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开中，术语“包含”可替换为“由……组成”。
如无特殊说明，本公开的“化合物”、“化学修饰”、“配体”、“dsRNA”、“核酸”和“RNAi”均可独立地以盐、混合盐或非盐(例如游离酸或游离碱)的形式存在。当以盐或混合盐的形式存在时，其可为药学上可接受的盐。
“药学上可接受的盐”可选自无机盐或有机盐，也可包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其它副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、2,2-二氯乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、辛酸盐、癸酸盐、十一碳烯酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、癸二酸盐、己二酸盐、戊二酸盐、丙二酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、棕榈酸盐、硬脂酸盐、油酸盐、肉桂酸盐、月桂酸盐、苹果酸盐、谷氨酸盐、焦谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、海藻酸盐、抗坏血酸盐、水杨酸盐、4-氨基水杨酸盐、萘二磺酸盐等。这些盐可通过本领域已知的方法制备。
“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它副作用的、与无机碱或有机碱所形成的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等。优选的无机盐为铵盐、钠盐、钾盐、钙盐及镁盐，优选钠盐。衍生自有机碱的盐包括但不限于以下的盐：伯胺类、仲胺类及叔胺类，被取代的胺类，包括天然的被取代胺类、环状胺类及碱性离子交换树脂，例如氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡萄糖胺、甲基葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺树脂等。优选的有机碱包括异丙胺、二乙胺、乙醇胺、三甲胺、二环己基胺、胆碱及咖啡因。这些盐可通过本领域已知的方法制备。
本公开中，“磷酸酯基团”可为磷酸一酯基团、磷酸二酯基团或磷酸三酯基团，优选磷酸二酯基团。
本公开中，硫代磷酸二酯基是指一个非桥接氧原子被硫原子替代而修饰的磷酸二酯基，可用(M为S原子)互换使用。
本公开上下文中，基团中的可以替换为能够与相邻核苷酸实现连接的任意基团。
术语“连接”，当表示两个分子之间的联系时，指两个分子通过共价键连接或者两个分子经由非共价键(例如，氢键或离子键)关联，包括直接连接、间接连接。
术语“直接连接”指第一化合物或基团与第二化合物或基团在没有任何间插原子或原子基团的情况下连接。
术语“间接连接”指第一化合物或基团与第二化合物或基团通过中间基团、化合物或分子(例如，连接基团)连接。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“药学上可接受的赋形剂”包括但不限于任何已批准对于人类或家畜动物使用可接受的任何助剂、载体、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增香剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、缓冲剂、溶剂或乳化剂。
如本文所使用的，术语“抑制”，可以与“减少”、“沉默”、“下调”、“阻抑”和其他类似术语交替使用，并且包括任何水平的抑制。抑制可通过这些变量中的一个或多个与对照水平相比的绝对或相对水平的减少来评估。该对照水平可以是本领域中使用的任何类型的对照水平，例如给药前基线水平或从类似的未经处理或经对照(例如仅缓冲液对照或惰性剂对照)处理的受试者、细胞、或样品确定的水平。例如，可以采用mRNA剩余表达量来表征siRNA(或dsRNA)对靶基因表达的抑制程度，如mRNA剩余表达量为不高于99％、不高于95％、不高于90％、不高于85％、不高于80％、不高于75％、不高于70％、不高于65％、不高于60％、不高于55％、不高于50％、不高于45％、不高于40％、不高于35％、不高于30％、不高于25％、不高于20％、不高于15％、或不高于10％。靶基因表达的抑制率可以采用Luciferase Assay System检测，分别读取萤火虫(Firefly)化学发光值和海肾(Renilla)化学发光值，计算相对值Ratio＝Ren/Fir，抑制率(％)＝1-(Ratio+siRNA/仅报告基因)*100％；本公开中，剩余mRNA表达量比例(或剩余活性％)＝100％-抑制率(％)。
“有效量”或“有效剂量”包含足以改善或预防医学病症的症状或病症的量。有效 量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
如本文所使用的，“对象”、“患者”、“受试者”或“个体”可互换使用，包括人类或者非人类动物，例如哺乳动物，例如人或猴。
如本文所使用的，有义链(又称SS、SS链或正义链)是指包含与靶mRNA序列相同或基本上相同的序列的链；反义链(又称AS或AS链)是指具有与靶mRNA序列互补的序列的链。
本公开中，有义链或反义链的“5’区域”也即“5’端”、“5’末端”，可替换使用。例如反义链5’区域的第2位至第8位的核苷酸，也可替换为反义链5’端起第2位至第8位的核苷酸。同理，有义链或反义链的“3’区域”、“3’末端”和“3’端”也可替换使用。
术语“dsRNA”是指能够进行RNA干扰的双链RNA分子，包含有义链和反义链。
在描述本文所述的siRNA有义链的上下文中，术语“与SEQ ID NO：1的核苷酸序列相差不超过3个核苷酸序列，且包含至少15个连续核苷酸”旨在表示本文所述的siRNA有义链包含SEQ ID NO：1有义链的至少15个连续核苷酸，或与SEQ ID NO：1有义链中至少15个连续核苷酸相差不超过3个核苷酸序列(任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列)。任选地，本文所述的siRNA有义链包含SEQ ID NO：1有义链的至少16个连续核苷酸，或与SEQ ID NO：1有义链的至少16个连续核苷酸相差不超过3个核苷酸序列(任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列)。
在描述本文所述的siRNA反义链的上下文中，术语“与SEQ ID NO：2或SEQ ID NO：3任一反义链相差不超过3个核苷酸序列，且包含至少15个连续核苷酸”旨在表示本文所述的siRNA反义链包含SEQ ID NO：2或SEQ ID NO：3中任一反义链的至少15个连续核苷酸，或与SEQ ID NO：2或SEQ ID NO：3中任一反义链的至少15个连续核苷酸相差不超过3个核苷酸序列(任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列)。
如无特别说明，“G”、“C”、“A”、“T”与“U”分别代表核苷酸，其分别包含鸟嘌呤、胞嘧啶、腺嘌呤、胸苷与尿嘧啶的碱基。本公开上下文中，I等同于包含核碱基次黄嘌呤(nucleobase hypoxanthine)的核苷酸。在一些实施方案中，本公开所用的术语肌苷等同于包含次黄嘌呤和糖或修饰的糖的核苷(nucleoside comprising hypoxanthine and a sugar or modified sugar)。
如无特别说明，在本公开上下文中，小写字母d表示该字母d下游相邻的一个核苷酸为脱氧核糖核苷酸；小写字母m表示该字母m上游相邻的一个核苷酸为 2’-甲氧基修饰的核苷酸；小写字母f表示该字母f上游相邻的一个核苷酸为2’-氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接。
如本公开所使用的，术语“2'-氟代(2’-F)修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。
如本公开所使用的，术语“2'-甲氧基(2’-OMe)修饰的核苷酸”指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在本公开的上下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方案中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
如本文所使用的，术语“互补”或“反向互补”一词可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤始终与嘧啶碱基胸腺嘧啶(或者在RNA中为尿嘧啶)相配对；嘌呤碱基鸟嘌呤始终与嘧啶碱基胞嘧啶相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
术语“化学修饰”或“修饰”包括核苷酸经化学手段的所有改变，例如化学部分的添加或去除、或以一个化学部分取代另一个化学部分。
术语“碱基”包含任何已知的DNA和RNA碱基、碱基类似物，例如嘌呤或嘧啶，其还包括天然化合物腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、次黄苷和天然类似物。碱基类似物还可以是通用碱基。
术语“平端”或“平末端”可互换使用，是指在siRNA的给定的末端没有未配对的核苷酸或核苷酸类似物，即，没有核苷酸突出。大多数情况下，两个末端都是平末端的siRNA将在其整个长度范围内是双链的。
本公开提供的siRNA可以通过本领域常规的制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
本公开所述的“联用”、“联合使用”是一种给药方式，是指一定时间期限内给予 至少一种剂量的dsRNA，以及至少一种剂量的另一种治疗剂，其中给予的药物都显示药理学作用。可以同时或依次给予dsRNA与另一种治疗剂。这种期限包括这样的治疗，其中通过相同给药途径或不同给药途径给予dsRNA与另一种治疗剂。本公开所述联合的给药方式选自同时给药、独立地配制并共给药或独立地配制并相继给药。
附图说明
图1.为核酸外切酶稳定性凝胶电泳实验结果。
图2.为5’核酸外切酶稳定性实验定量结果。
图3.为3’核酸外切酶稳定性实验定量结果。
图4.受试物对TLR8AAV-HBV小鼠第7日血浆HBVDNA水平的影响(n＝6，单因素方差分析，****P<0.0001,***P<0.001vs.组1；###P<0.001vs.组2；&P<0.05vs.组3)。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非限制本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，则该试剂可自任意分子生物学试剂的供应商以用于分子生物学应用的质量/纯度而获得。
实施例1：配体的制备(NAG0052、L96)
化合物NAG0024、NAG0026购买自天津药明康德新药开发有限公司。除非特别说明，以下实施例中所用的试剂均为市售商品。
化合物NAG0052的合成
起始原料化合物1采购自江苏倍达医药科技有限公司。

化合物2
在0℃以及氮气保护下，往化合物1(12.3mL,101mmol)的THF(300mL)溶液中分批加入NaH(12.2g,304mmol，纯度60％)。该混合物在20℃下搅拌1小时之后再次冷却到0℃，接着往体系中逐滴加入苄溴(36.3mL,304mmol)，并且在20℃搅拌12小时。将该反应液用H2O(100mL)淬灭后，用EtOAc(200mL×2)萃取。合并后的有机相用饱和食盐水(100mL)洗涤,Na2SO4干燥，过滤，浓缩得到的残留物经过硅胶柱层析分离后得到目标化合物2(20.0g,51.8mmol,产率51％)。
LCMS:tR＝2.615and 2.820min in 30-90AB_7min_220&254_Shimadzu.lcm(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝351.2[M+Na]+。
1H NMR:(400MHz,CDCl3)δppm 7.35-7.12(m,10H),5.06-4.95(m,1H),4.51-4.39(m,4H),4.24-3.87(m,2H),3.50-3.40(m,2H),3.38-3.20(m,3H),2.20-1.91(m,2H)。
化合物3和化合物4
在20℃以及氮气保护下，往化合物2(13.0g,33.6mmol)的DCM(300mL)溶液中一次性加入TMSCN(13.5mL,101mmol)，接着逐滴加入TMSOTf(9.14mL,50.5mmol)的DCM(30mL)溶液。该反应液在20℃下搅拌15小时。反应结束之后用饱和NaHCO3水溶液(80mL)淬灭该体系，并且用DCM(150mL×2)萃取，合并后的有机相用饱和食盐水(80mL)洗涤，Na2SO4干燥，过滤以及浓缩后通过硅胶柱层析分离后得到目标化合物3(3.30g,9.18mmol,产率27％)以及淡黄色油状液体化合物4(8.50g,9.18mmol,产率70％)。
化合物3
1H NMR:(400MHz,CDCl3)δppm 7.42-7.29(m,10H),4.81(t,J＝7.8Hz,1H),4.65-4.49(m,4H),4.30-4.21(m,2H),3.65-3.57(m,1H),3.57-3.49(m,1H),2.49-2.40(m,2H)。
化合物4
1H NMR:(400MHz,CDCl3)δppm 7.42-7.26(m,10H),4.93-4.87(m,1H),4.65-4.48(m,4H),4.43-4.38(m,1H),4.21-4.17(m,1H),3.79-3.70(m,1H),3.54(d,J＝4.0Hz,1H),2.45-2.37(m,2H)。
化合物5
在0℃及氮气保护下将化合物4(3.00g,9.28mmol)的THF(15mL)溶液，滴加到LiAlH4(0.79g,20.9mmol)的THF(15mL)溶液中，滴加完后体系在0℃反应1小时。TLC(PE:EtOAc＝3:1)监测到原料完全消失。向反应液中缓慢加入十水硫酸钠，加至不冒泡为止。之后将反应液过滤，滤饼用二氯甲烷(60mL)洗涤三次后，收集滤液旋干，得目标化合物5(3.00g,产率90％)
1H NMR:(400MHz,DMSO-d6)δppm 7.40-7.14(m,10H),4.54-4.38(m,4H),4.06-3.99(m,2H),3.91(q,J＝6.4Hz,1H),3.48-3.37(m,2H),2.67-2.52(m,2H),2.21-2.18(m,1H),1.77-1.73(m,1H)。
化合物6
在氮气保护下，将化合物5(3.00g,8.25mmol)溶于DCM(30mL)，加入TEA(3.44mL,24.7mmol)和CbzCl(1.76mL,12.4mmol)，20℃反应2小时。LCMS显示反应完成。将反应液加入二氯甲烷(30mL)和水(60mL)萃取。有机相用水(60mL×3)洗涤三次，无水硫酸钠干燥，浓缩用正向柱纯化(PE:EtOAc＝1:1)，得到目标化合物6(2.5g,产率90％)。
LCMS:tR＝0.810min in 5-95AB_1min,MS(ESI)m/z＝462.2[M+H]+。
1H NMR:(400MHz,CDCl3)δppm 7.39-7.29(m,15H),5.35(s,1H),5.15-5.01(m,2H),4.72(d,J＝6.0Hz,1H),4.54-4.40(m,3H),4.26(s,1H),4.23-4.18(m,1H),4.11-4.04(m,1H),3.54-3.41(m,3H),3.37-3.25(m,1H),2.34-2.23(m,1H),1.85-1.79(m,1H)。
化合物7
在氮气保护下，将化合物6(2.00g,3.90mmol)溶于DCM(5mL)，在-78℃下 加入BCl3的THF溶液(1M,27.3mL)，反应1小时。TLC(DCM:MeOH＝10:1)监测到原料完全消失。将反应液在-78℃下加入甲醇(20mL)淬灭，浓缩，用正向柱纯化(DCM:MeOH＝10:1)，得到目标化合物7(2.00g,产率60％)。
1H NMR:(400MHz,CD3OD)δppm 7.41-7.23(m,5H),5.08(s,2H),4.25-4.07(m,2H),3.85-3.75(m,1H),3.63-3.56(m,1H),3.54-3.48(m,1H),3.30-3.27(m,2H),2.34-2.21(m,1H),1.71-1.64(m,1H)。
化合物8
在氮气保护下，将化合物7(0.50g,1.78mmol)溶于吡啶(5mL)中，在0℃下加入4A分子筛(500mg)和DMTrCl(0.66mL,2.13mmol)，之后升温至20℃反应1.5小时。TLC(PE:EtOAc＝2:1)监测到原料完全消失。将反应液加入乙酸乙酯(60mL)和水(60mL)萃取，有机相用水(60mL×3)洗涤三次后用无水硫酸钠干燥，浓缩，用正向柱纯化(PE:EtOAc＝1:1)，得到目标化合物8(800mg,产率90％)。
1H NMR:(400MHz,CDCl3)δppm 7.44(d,J＝7.6Hz,2H),7.37-7.23(m,11H),7.22-7.15(m,1H),6.84(d,J＝8.8Hz,4H),5.09(s,2H),4.31-4.17(m,2H),4.02-3.91(m,1H),3.84-3.73(m,6H),3.33(s,1H),3.28(s,1H),3.19-3.01(m,2H),2.34-2.25(m,1H),1.70-1.62(m,1H)。
化合物9
将化合物8(800mg,1.234mmol)溶于EtOAc(5mL)，加入Pd/C 10％(800mg,7.517mmol)，反应在H2条件(15Psi)，20℃下反应1小时。LCMS显示反应已经完成。反应液过滤，滤饼用二氯甲烷(100mL)和甲醇(100mL)洗涤三次，浓缩，经过反相柱分离得到化合物9(300mg,54％)。
LCMS:tR＝2.586min in 10-80CD_3min MS(ESI)m/z＝450.2[M+H]+。
化合物11
将化合物10(435mg,1.780mmol)溶于DCM(10mL)，加入DIEA(0.441mL,2.67mmol)和HATU(677mg,1.78mmol)后,再加入化合物9(400mg,0.890mmol)，20℃反应1小时。TLC(DCM:MeOH＝10:1)监测反应完成。将反应液加入二氯甲烷(60mL)和水(60mL)萃取，有机相用水(60mL×3)洗涤三次,无水硫酸钠干燥，浓缩用正向柱纯化(PE:EtOAc＝0:1过柱，在100％处出产品峰),得到目标化合物11(600mg,产率90％)。
LCMS:tR＝2.745min in 30-90CD_3min,MS(ESI)m/z＝698.4[M+Na]+。
1H NMR:(400MHz,CD3OD)δppm 7.46-7.38(m,2H),7.35-7.24(m,6H),7.22-7.16(m,1H),6.90-6.78(m,4H),4.29-4.21(m,2H),4.02-3.95(m,1H),3.77(s,6H),3.66-3.62(m,3H),3.41(s,1H),3.18-3.04(m,2H),2.36-2.17(m,5H),1.71-1.50(m,5H),1.39-1.25(m,14H)。
化合物12
将化合物11(600mg,0.799mmol)溶于THF(3mL)和H2O(1mL)，加入LiOH.H2O(134mg,3.20mmol)，20℃反应12小时。TLC(DCM:MeOH＝10:1)显示 反应完成。将反应液旋干，用水(5mL)和甲醇(5mL)溶解，用反向柱纯化(H2O:CH3CN＝1:1,在35％左右出峰)，得到目标化合物12(460mg,产率100％，锂盐)。
LCMS:tR＝1.346min in 10-80CD_3min,MS(ESI)m/z＝684.3[M+Na]+。
HPLC:tR＝1.879min in 10-80CD_6min。
1H NMR:(400MHz,CD3OD)δppm 7.47-7.39(m,2H),7.35-7.24(m,6H),7.22-7.15(m,1H),6.91-6.79(m,4H),4.31-4.18(m,2H),4.02-3.95(m,1H),3.78(s,6H),3.44-3.33(m,2H),3.18-3.04(m,2H),2.35-2.27(m,1H),2.24-2.10(m,4H),1.70-1.51(m,5H),1.31-1.23(m,12H)。
化合物13
室温环境，氮气保护下，将化合物NAG0024(271mg,0.151mmol)溶解于无水THF(2mL)和无水DMF(4mL)，加入3A分子筛，再依次加入化合物12(100mg,0.151mmol),HOBt(25mg,0.181mmol),DCC(38mg,0.181mmol)和DIEA(39mg,0.302mmol)。反应液45℃反应16h.LC-MS显示反应完全后，加水淬灭，过滤。滤液浓缩后，经C18反相柱纯化(H2O/MeCN)，得到化合物13(210mg，产率57％)。
化合物NAG0052
室温环境下，化合物13(230mg，0.094mmol)溶于吡啶(5mL)，加入分子筛，加入DMAP(12mg，0.283mmol)，丁二酸酐(28mg，0.283mmol)。氮气保护，50℃搅拌16小时。LCMS检测反应完全，过滤旋干。过C18反相柱纯化后，由制备HPLC二次纯化，得到目标化合物NAG0052(123mg，0.048mmol，产率51％)。
MS(ESI)m/z＝2535.3[M-1]-.理论：2536.2。
1H NMR(400MHz,乙腈-d3)δ7.48-7.43(m,2H),7.37-7.12(m,11H),7.00-6.85(m,10H),6.66(s,1H),5.31(dd,J＝3.4,1.1Hz,3H),5.20-5.13(m,1H),5.05(dd,J＝11.3,3.4Hz,3H),4.56(d,J＝8.5Hz,3H),4.30(dd,J＝7.7,5.3Hz,1H),4.18-3.93(m,14H),3.79(s,10H),3.65(q,J＝4.7,3.6Hz,13H),3.56-3.07(m,24H),2.56(s,6H),2.37(t,J＝5.8Hz,10H),2.17(t,J＝7.5Hz,9H),2.02-1.96(m,20H),1.88(s,8H),1.82-1.73(m,2H),1.60(dt,J＝15.0,7.3Hz,16H),1.27(s,13H)。
化合物NAG0052经过固相合成连接到序列上，再经过胺解后，NAG0052结构脱去一部分官能团成为NAG0052’。
L96的合成
按照专利申请WO2014025805A1记载的方法制备获得。
实施例2：合成dsRNA
1.自制带有载体的树脂
具体操作同实施例1。
2.使用带有NAG0052的树脂作为起始，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。
制得的dsRNA具有表2和表3中所示的有义链和反义链。
表1.dsRNA列表
表2.dsRNA的有义链和反义链的对应的裸序列
表3.dsRNA的有义链和反义链
其中，(-)hmpNA(G)的结构
NAG0052’的结构为：
实施例3：dsRNA对HBV的在靶活性
表4中为阳性对照化合物。
表4.阳性对照化合物序列信息
其中，AD81890参照CN201980053789.8制备获得；L96结构为
在HEK293A细胞中采用11个浓度梯度对表3及表4中的dsRNA序列进行体外分子水平模拟在靶活性筛选。
以HBV基因构建dsRNA对应的在靶序列，插入到psiCHECK-2质粒中。该质粒包含海肾荧光素酶基因及萤火虫荧光素酶基因。作为双报告基因系统，dsRNA的靶序列插入到海肾荧光素酶基因的3’UTR区域，dsRNA对于靶标序列的活性可 以通过经萤火虫荧光素酶校准后的海肾荧光素酶表达情况的检测来反映，检测使用Dual-Luciferase Reporter Assay System(Promega，E2940)。
HEK293A细胞培养于含10％胎牛血清的DMEM高糖培养基中，在37℃，5％CO2条件下培养。转染前24h，将HEK293A细胞接种于96孔板，接种密度为每孔8×103个细胞，每孔100μL培养基。
按照说明书，使用Lipofectamine2000(ThermoFisher，11668019)对细胞共转染dsRNA及对应质粒，Lipofectamine2000每孔使用0.2μL。质粒转染量为20ng每孔。对于在靶序列质粒，dsRNA共设置11个浓度点，最高浓度点终浓度为20nM，3倍梯度稀释，20nM，6.6667nM，2.2222nM，0.7407nM，0.2469nM，0.0823nM，0.0274nM，0.0091nM，0.0030nM，0.0010nM和0.0003nM。转染后24h，采用Dual-Luciferase Reporter Assay System(Promega，E2940)检测在靶水平。
psiCHECK活性筛选实验步骤具体如下：
在HEK293A细胞系通过进行psi-CHECK筛选4条dsRNA序列的在靶活性。实验材料和仪器详见表5和表6。
Psi-CHECK质粒购自于生工生物工程(上海)股份有限公司。
表5.psi-CHECK实验耗材和试剂
表6.psi-CHECK实验仪器
psiCHECK实验步骤：
(一)细胞铺板
1.实验准备：
1.1 HEK293A细胞准备：
购买于南京科佰，贴壁细胞消化完全后需要计数，若细胞活率大于等于95％即可用。
1.2 DMEM完全培养基(DMEM+10％FBS)储存于4℃，实验前取出平衡到室温。
1.3 96孔板细胞板。
2.细胞铺板
转染前18h，计数完毕后将HEK293A细胞接种于96孔板中，接种密度为8×103个细胞/孔，100μL培养基/孔；
2.1将培养基放于37℃水浴锅孵育20min备用；
2.2吸取100μL均匀的细胞悬液与5μL细胞计数染料混匀，静止染色1min，吸取15μL混悬液注入到细胞计数板上计算活细胞量(绿色)，细胞活率98.7％；
2.3根据细胞计数结果，加入合适体积的培养基，取100μL铺到96孔板中，保证细胞量为8×103个细胞/孔，细胞板置于37℃，5％CO2培养箱中进行培养。
(二)细胞转染实验
1.实验准备：
1.1 dsRNA样品和质粒准备：dsRNA样品定量至20μM，psi-CHECK质粒测定其浓度，于-20℃储存备用，使用前需要短暂离心；
1.2转染试剂Lipofectamine 2000储存于4℃；
1.3 PCR 96孔板管和八连PCR管；
1.4 Opti-MEM培养基。
2.细胞转染实验
2.1转染前，预热Opti-MEM培养基，细胞板中更换为Opti-MEM培养基，80μL培养基/孔。
2.2配制转染复合物：每个浓度设2个复孔，转染复合物具体配制量如表18所示；
转染复合物成分：
表7.孔板每孔所需转染复合物用量
将配好的质粒分装到相应8连管中，22μL/管，命名为：Tube A；
2.3换液：将孔中的含10％FBS的H-DMEM完全培养基吸弃，换成80μL Opti-MEM，饥饿处理1.5h。
2.4稀释dsRNA：将dsRNA从-20℃拿出解冻，混匀，依照表8根据不同的实验需求稀释至不同浓度作为工作液备用，现用现配。
表8.dsRNA样品多浓度稀释方案
2.5将稀释好的dsRNA加入到相对应Tube A的8连管中，2.2μL/管，现配现用；
2.6 Lipofectamine 2000Mix的配制：用Opti-MEM稀释Lipofectamine 2000，静置5min，Lipo Mix具体配制量如表19；
2.7再将配制好的Lipo Mix分装到对应Tube A的8连管中，22μL/管，吹打混匀后(不产生气泡)，室温孵育20min。
2.8将上述Tube A混合物加入每孔细胞中，20μL/孔，加上原有80μL Opti-MEM，每孔终体积为100μL。4h温箱培养后，每孔补加100μL含20％FBS的H-DMEM培养基。
2.9 37℃CO2培养箱培养24h。
(三)Luciferase Assay System检测
1.实验准备：
1.1Dual luciferase reporter gene assay kit(Promega,cat.E2940)组分及配制方法：Buffer和vialLuciferase Substrate(lyophilized)预先进行混合，然后分装到15mL的离心管中，每管7.5mL。Stop&Buffer提前进行分装，每管12mL。在实验前先将混合好的进行复融，等平衡到室温后每管加入7.5mL DMEM进行配制，现配现用。将Stop&Buffer进行复融，等平衡到室温后与Stop&100:1进行配制，现配现用。
2.信号采集
2.1吸液：吸去96孔培养板中原有的培养基；
2.2加底物每孔加入150μL LARII底物，摇床上摇10min；
2.3移液：取120μL底物(Mix)，转移到96孔酶标板上，读取Firefly化学发光值；
2.4加底物(Stop&Glo)：再向每孔加入60μLStop&Glo底物，摇床上摇10min，读取Renilla化学发光值；
2.5计算相对值Ratio＝Ren/Fir(海肾/萤火虫比值)；
2.6计算抑制率1-(Ratio+dsRNA/仅报告基因)*100％＝抑制率(％)；
本公开中，剩余活性％(也称为mRNA剩余表达量％或mRNA剩余表达比例)＝100％-抑制率(％)。
2.7利用GraphPad Prism5作图。
结果如表9所示。
表9.dsRNA的psi-CHECK在靶活性筛选结果
以上结果表明，参比对照化合物AD81890，TRD007970在psiCHECK系统针对HBV基因具有高水平的在靶抑制活性。
另外一批次结果如表10所示。
表10.dsRNA的psi-CHECK在靶活性筛选结果
以上结果表明，参比对照AD81890，本公开的TRD007970、TJR100259在psiCHECK系统针对HBV基因具有更高水平的在靶抑制活性。
实施例4：应用HepG2.2.15细胞评价dsRNA体外抗HBV活性
在HepG2.2.15细胞中采用8个浓度梯度对dsRNA进行体外抗HBV活性评估。
第1天种HepG2.2.15细胞到96孔板，每孔2万个细胞。种细胞同时用RNAiMax将不同浓度的dsRNA转入HepG2.2.15细胞；第4天收集细胞培养上清，ELISA检测HBsAg(剩余上清冻存备用)。最后收集细胞，提取细胞内RNA，RT-PCR分别检测总HBV RNA(包括3.5kb+2.4kb+2.1kb+0.7kb RNA)和3.5kb HBV RNA(包括pgRNA+preCore RNA)，同时检测GAPDH基因RNA作为内参。待测 化合物为8个浓度点，平行测定2复孔。培养液中DMSO的终浓度为0.5％。
抑制百分比计算公式如下：
％HBsAg抑制率＝(1-样品中HBsAg含量/DMSO对照组中HBsAg含量)×100
％HBV RNA抑制率＝(1-样品中HBV的RNA含量/DMSO对照组中HBV的RNA含量)×100
％细胞活力＝(样品吸光值–培养液对照的吸光值)/(DMSO对照的吸光值-培养液对照的吸光值)×100。
应用Graphpad Prism软件分析(four parameter logistic equations)计算EC50值。
结果如表11所示，参比对照AD81890，综合抗病毒活性检测指标，测试TRD007970在HepG2.2.15细胞上展现出优秀的抗病毒活性。
表11.dsRNA在HepG2.2.15的抗病毒活性
实施例5：应用PHH细胞评价dsRNA体外抗HBV活性(自由摄取)
分别针对TRD007970、TJR100259和AD81890进行体外抗HBV活性评价。
第0天，先dsRNA用PBS梯度稀释7个浓度(100,25,6.25,1.563,0.391,0.098,0.024nM)，加入48孔板中。复苏冻存的PHH，再将PHH铺种到48孔板中；铺板同时将测试化合物自由摄取(free uptake)进入细胞。
第1天，更换不含dsRNA的培养基，加入HBV感染PHH。
第2、4和6天，更换不含dsRNA的新鲜培养基。
第8天，收集上清，将收集的细胞上清用ELISA法检测HBsAg和HBeAg，qPCR法检测HBV DNA水平。实验结果见表12。
表12.dsRNA在PHH的抗病毒活性
结果显示，与对照AD81890相比，TRD007970和TJR100259在PHH上抗病毒活性更好。
实施例6：核酸外切酶稳定性实验评价dsRNA的稳定性
分别针对TRD007970和TJR100259的siRNA裸序列TJR100381和TJR100382(见表13)进行外切酶稳定性评价，按照实验的反应体系配制相应的反应液进行实验，见表14-15。
表13.裸序列TJR100381和TJR100382

表14. 5’核酸外切酶(PDII)稳定性反应体系(100ul)
表15. 3’核酸外切酶(SVPD)稳定性反应体系(100ul)
5’核酸外切酶(PDII,Worthington，cat#LS003602)的终浓度为500U/mL,3’核酸外切酶(SVPD，Worthington,cat#LS003926)的终浓度为0.5U/mL。配制完成后，按时间0h、1.5h、2h、3h、4h五个时间点分装到不同的8联排管中(每孔16ul)，置于37℃中进行孵育，到时间点后立即取出反应液并加入含9M尿素的上样缓冲液(每孔32ul)，放于-80℃冰箱备。后续通过20％(7M尿素)PAGE胶进行电泳。电泳结束猴将胶浸于gelred染料中，摇床染色10min，凝胶成像(312nm的UV)观察并照相。实验结果见图1(凝胶电泳结果)，图2(5’核酸外切酶定量结果)和图3(3’核酸外切酶定量结果)，结果表明TJR100382的稳定性显著优于TJR100381。
实施例7：肝脏S9代谢稳定性分析
分别针对TRD007970、TJR100259和AD81890进行食蟹猴的肝脏S9(食蟹猴肝S9，雄性，供应商Xenotech，批号1510192)，对dsRNA进行代谢稳定性分析，实验过程如下：
1、制备8个96孔样品板，命名为T0、T60、T120、T240、T360、T1440、T2880、空白。
2、将190μL/孔S9悬浮液(或空白缓冲液)加到每块板，然后37℃孵育约10分钟。
3、除基质孔外，每个板(T0,T60,T120,T240,T360,T1440,T2880)每孔加入10μL样品或空白缓冲液。
4、除T0外，各时间点(60、120、240、360、1440、2880min)的TRD007970、 TJR100259和AD81890样品均在37℃水浴中孵育。
5、在每个时间点结束时，加入200μL(100mM NH4Ac pH 10.0、1mM EDTA和750ng/mL内标于水中)，在旋涡混合器上震荡60秒。
6、每孔加入200μL PCI(苯酚/氯仿/异戊醇(25:24:1))试剂和400μL二氯甲烷，在旋涡混合器上震荡10min后离心(4℃，3220g，20min)，获得上清液。
7、将上清液转移至新板上，在进行LC-MS分析前放4℃保存。
8、分别检测AS链和SS链的单链的剩余百分比，以表征TRD007970、TJR100259和AD81890的剩余含量。
9、使用以下公式计算：
剩余％＝各时间点分析物与内标的峰面积比/T0分析物与内标物的峰面积比×100％
Ct＝C0*e-ke*t
T1/2＝Ln2/ke＝0.693/keClint(s9)＝0.693/体外T1/2*1/(mg/mL反应体系中S9蛋白)
Clint(liver)＝Clint(S9)*(mgS9)/g肝*g肝/(kg体重)。
实验结果见表16，结果表明，食蟹猴肝S9孵育48h，TJR100259反义链剩余93.8％，AD81890反义链剩余70.2％，TRD007970反义链剩余61.0％。数据显示，食蟹猴肝S9孵育48h，TJR100259中的反义链较AD81890中的反义链和TRD007970中的反义链更稳定。
食蟹猴肝S9孵育48h，TJR100259正义链剩余21.0％，AD81890正义链剩余12.2％，TRD007970正义链剩余8.9％。数据显示，食蟹猴肝S9孵育48h，TJR100259中的正义链较AD81890中的正义链和TRD007970中的正义链更稳定。
单链的剩余率可以反映dsRNA的稳定性，剩余率越高，稳定性越好。因此，TJR100259较AD81890和TRD007970在食蟹猴肝S9代谢稳定性实验条件下稳定性更好。
表16.食蟹猴肝S9代谢稳定性分析

实施例8：组织分布实验
TRD007970、TJR100410和TJR100259进行组织分布实验，实验过程如下：72只雄性C56BL/6J小鼠(7-8w，北京维通利华实验动物技术有限公司)适应约1周，分成3组，每组24只。TRD007970、TJR100259和TJR100410剂量均为10mg/kg，给药后0.5h，1h，2h，4h，8h，24h，72h，168h(n＝3)采集样品。安乐死后心脏采血，采集左肾和肝左侧最大叶，收集的脏器用生理盐水冲洗干净。肝肾样品经前处理后，使用高分辨质谱分析肝脏和肾脏中AS链浓度，以表征TRD007970、TJR100259和TJR100410的浓度。
实验结果见表17。结果表明，C56BL/6J小鼠组织分布实验中，TRD007970、TJR100259和TJR100410反义链肝肾暴露量比分别为：9.44，29.38和3.49。肝肾暴露量比值大提示在靶器官(肝脏)浓度较高，非靶器官(肾脏)浓度较低。因此，同等剂量下，TJR100259展现出高于TRD007970的肝肾比，TRD007970展现出高于TJR100410的肝肾比，提示TJR100259的出现肾脏毒性的风险低于TRD007970，TRD007970的出现肾脏毒性的风险低于TJR100410。
表17.组织分布结果
实施例9：肝匀浆稳定性
TJR100410和TJR100259进行肝脏匀浆稳定性分析，实验过程如下：
1.用Apricot把食蟹猴肝匀浆(由实验单位药明康德提供)加入190μL/孔，并将板子于37℃孵育约30分钟。
2.除基质孔，每块板每孔(T0，T1，T2，T4，T6，T24，T48，表32)加入10μL dsRNA或对照缓冲溶液，开始计时。
表18.各时间点表

2.1、样品准备
(1)向指定孔中加入200μL的dsRNA。
(2)加入1000μL Clarify OTX裂解缓冲液。
(3)以800rpm的速度振荡5分钟。
2.2、SPE
(1)条件：通过飞诺美Clarity OTX SPE板(8e-s103-cga)用600μL的MeOH冲洗。
(2)平衡：通过SPE板用600μL平衡缓冲液(pH 5.5的50mM NH4Ac含有0.0025％Triton X-100和0.01mg/mL半胱氨酸)冲洗。
(3)加载：通过SPE板冲洗2.1中的样品。
(4)清洗1：将600μL的清洗缓冲液1(25mM NH4Ac pH 5.5)通过固相萃取板冲洗，然后再次重复上述步骤。
(5)清洗2：将600μL的清洗缓冲液2(25mM NH4Ac pH 5.5,50％CAN)通过固相萃取板冲洗，并再次重复上述步骤。
(6)洗脱：用150μL洗脱缓冲液(100mM NH4HCO3，1mM TCEP pH 9.5含有40％ACN和10％THF)洗脱样品，并重复此步骤。
(7)使用N2蒸发器在45℃干燥样品(约2小时)。
(8)用70μL的流动相A重组样品。
(9)LC-MS/MS分析前，以800rpm的速度轻轻振荡0.5小时。
(10)使用以下公式计算。
Ct＝C0*e-ke*t Ct＝1/2C0T1/2＝Ln2/(-ke)＝0.693/(-ke)。
其中，TJR100410为对照dsRNA，其有义链为：
GmsUmsGmUmGmCmAfCfUfUmCmGmCmUmUmCmAmCmCm-NAG1(SEQ ID NO：9)；
反义链为：
AmsGfsUmGfAmAf(-)hmpNA(G)CmGmAfAmGfUmGfCmAfCmAfCmsGmsGm(SEQ ID NO：10)；
NAG1的结构为
实验结果见表19。
结果表明，食蟹猴肝匀浆孵育48h，TJR100259中的反义链剩余94.6％，TJR100410中的反义链剩余42.7％；TJR100259中的正义链剩余18.3％，TJR100410中的正义链剩余4.3％。数据显示，食蟹猴肝匀浆孵育48h，TJR100259中的反义链较TJR100410中的反义链更稳定；TJR100259中的正义链较TJR100410中的正义链更稳定。
单链的剩余率可以反映dsRNA的稳定性，剩余率越高，稳定性越好。因此，TJR100259较TJR100410在食蟹猴肝匀浆稳定性实验条件下稳定性更好。
表19.食蟹猴肝匀浆稳定性分析
实施例10：TRD007970和式(II)化合物在TLR8人源化小鼠AAV-HBV(腺相关病毒-乙型肝炎病毒)感染模型中的药效评价研究
50只9周龄雌性human TLR8小鼠(上海南方模式生物科技股份有限公司，动物健康合格证编号20190002028579)经过5天的适应后，在Predose第0天由尾静脉注射200μL rAAV8-1.3HBV病毒悬液(含1×1011rAAV8-1.3HBV病毒拷贝，武汉枢密脑科学技术有限公司，批号ayw1-p4-220412)。在Predose第14天和25天(AAV-HBV注射14和25天后)，每只小鼠通过下颌静脉取血制备(10+15)μL血清，用于HBV DNA定量检测。
根据Predose第25天指标挑选24只human TLR8小鼠，随机分成4组，如表20所示。溶媒(Vehicle，组1)、式(II)化合物(可参考WO2020007275A说明 书实施例6中的方法制备)(组2、4)在第0～7天灌胃给药两次(第0、3天)。TRD007970(组3、4)在第0-7天皮下给药一次(第0天)。受试物的给药体积均为5mL/kg。于第0、7天采血收集血浆，每只动物每次50μL，储存于2～8℃，用于HBVDNA定量检测。
表20.实验分组及给药方案
结果如图4所示，第7天血浆中HBVDNA水平，与溶媒组比较，TRD0079709mg/kg组和式(II)化合物6mg/kg+TRD007970 9mg/kg组均有非常显著的降低；与式(II)化合物6mg/kg组比较，式(II)化合物6mg/kg+TRD007970 9mg/kg组有非常显著的降低；与TRD007970 9mg/kg组比较，式(II)化合物6mg/kg+TRD007970 9mg/kg组有明显的降低。
上述结果显示，式(II)化合物6mg/kg+TRD007970 9mg/kg组与单药比较，第7天血浆中HBVDNA水平降低具有显著性。因此，式(II)化合物6mg/kg与TRD007970 9mg/kg联用在第7天小鼠血浆的HBV DNA水平降低上具有明显的联合治疗效果。

Claims (13)

  1. 一种双链核糖核酸(dsRNA)联合式(II)所示化合物或其可药用盐在制备预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病的药物中的用途，
    所述dsRNA，其包含：
    siRNA和一个或多个与其缀合的配体；
    所述siRNA包含有义链和反义链，
    所述反义链在其5’端起第7位核苷酸位置处包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐：
    所述式(I)所示的化学修饰选自以下任一结构：
    B与所述反义链5’端起第7位核苷酸未被修饰时的碱基相同；
    所述配体如以下结构所示或是其药学上可接受的盐：
    所述有义链和反义链选自以下任一组：
    有义链包含SEQ ID NO：1所示的核苷酸序列，反义链包含SEQ ID NO：2所示的核苷酸序列；
    有义链包含SEQ ID NO：1所示的核苷酸序列，反义链包含SEQ ID NO：3所示的核苷酸序列；
    所述有义链的3’端与所述配体缀合；
    所述B选自鸟嘌呤。
  2. 如权利要求1所述的用途，所述的dsRNA，其中，
    所述配体通过磷酸酯基团或硫代磷酸酯基团与所述siRNA末端连接；优选通过磷酸二酯基团或硫代磷酸二酯基团连接，更优选通过磷酸二酯基团连接。
  3. 如权利要求1至2任一项所述的用途，所述的dsRNA，其中，所述有义链含有如下式所示的核苷酸：
    5’-NaNaNaNaNaNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
    其中，Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸；
    所述反义链含有如下式所示的核苷酸：
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
    Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；
    W’表示包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
  4. 如权利要求1至3中任一项所述的用途，所述的dsRNA，其中，
    所述有义链和/或反义链中至少一个磷酸酯基为具有修饰基团的磷酸酯基；优选地，所述具有修饰基团的磷酸酯基为硫代磷酸二酯基。
  5. 如权利要求1至4中任一项所述的用途，所述的dsRNA，其中：
    所述dsRNA选自以下任一组：
    包含SEQ ID NO：4所示的有义链和SEQ ID NO：5所示的反义链；
    包含SEQ ID NO：4所示的有义链和SEQ ID NO：6所示的反义链。
  6. 如权利要求1至5中任一项所述的用途，所述的dsRNA，其中所述dsRNA选自如下结构或其药学上可接受的盐：
    其中，
    Af＝腺嘌呤2'-F核糖核苷；Cf＝胞嘧啶2'-F核糖核苷；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷；
    Am＝腺嘌呤2'-OMe核糖核苷；Cm＝胞嘧啶2'-OMe核糖核苷；Gm＝鸟嘌呤2'-OMe核糖核苷；Um＝尿嘧啶2'-OMe核糖核苷；Im＝次黄嘌呤2'-OMe核糖核苷；
    表示硫代磷酸二酯基阴离子形式，表示磷酸二酯基阴离子形式，表示 表示
  7. 如权利要求1至6任一项所述的用途，所述式(II)所示化合物或其可药用盐的给药方式为固定剂量给药，给药剂量选自0.02mg-50mg，给药频次选自每周两次，每周一次，每两周一次和每四周一次。
  8. 如权利要求1至7任一项所述的用途，所述dsRNA的给药方式为固定剂量给药，给药剂量选自10mg至1500mg，给药频次选自每周一次，每周两次，每四周一次、每六周一次、每八周一次或每十二周一次。
  9. 如权利要求1至8任一项所述的用途，所述与乙型肝炎病毒相关的疾病选自：慢性肝炎、急性乙型肝炎、慢性乙型肝炎、丁型肝炎病毒感染、丁型肝炎、 肝纤维化、晚期肝病、肝细胞癌。
  10. 一种抑制HBV靶基因或其mRNA表达的方法，其包括：向受试者给与有效量或有效剂量的如权利要求1至9任一项所述的dsRNA和有效量或有效剂量的式(II)所示化合物或其可药用盐。
  11. 一种预防和/或治疗受试者乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病的方法，其包括：给与受试者权利要求1至9任一项所述的dsRNA和式(II)所示化合物或其可药用盐。
  12. 一种式(II)所示化合物或其可药用盐，其与权利要求1至9任一项所述的dsRNA联合，用于预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病。
  13. 一种根据权利要求1至9任一项所述的dsRNA，其与式(II)所示化合物或其可药用盐联合，用于预防和/或治疗乙型肝炎病毒感染或与乙型肝炎病毒相关的疾病。
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