An antisense oligonucleotide that inhibits hepatitis B virus expression

Abstract

This invention provides an antisense oligonucleotide that inhibits hepatitis B virus expression, and also relates to pharmaceutical compositions thereof. The antisense oligonucleotide and / or pharmaceutical compositions thereof described in this invention can be used to prepare medicaments for the treatment and / or prevention of hepatitis B.

Description

Technical Field

[0001] This invention belongs to the field of small nucleic acid drugs. The purpose of this invention is to provide a novel antisense oligonucleotide, its conjugates, and their uses. The antisense oligonucleotide and its conjugates described in this invention can be used to prepare drugs for the treatment and / or prevention of hepatitis B. Background Technology

[0002] Hepatitis B (HBV) is a serious infectious disease threatening the world, especially China. The outer envelope proteins of HBV are collectively called hepatitis B surface antigen (HBsAg). HBsAg consists of three related polypeptides, called S, M, and L, encoded by overlapping open reading frames (ORFs). The smallest envelope protein is S, with 226 amino acids, called the S-ORF. M and L are produced from upstream translation initiation sites, adding 55 and 108 amino acids to S, respectively. HBVS, M, and L glycoproteins are present in the viral envelope of the complete infectious HBV viral particle, called Dane particles, and all three are produced and secreted in large quantities, forming non-infectious subviral spherical and filamentous particles (called decoy particles) found in the blood of patients with chronic HBV. The abundant HBsAg on the surface of decoy particles is thought to suppress humoral immunity and spontaneous clearance in patients with chronic HBV infection (CHB).

[0003] The current standard of care for chronic HBV infection is treatment with oral nucleoside (nucleotide) analogs (such as entecavir or tenofovir), which inhibit HBV replication by suppressing HBV DNA synthesis, but do not directly target viral antigens, such as HBsAg. Even with long-term nucleoside (nucleotide) analog treatment, only low levels of HBsAg clearance are observed. In this regard, patients with chronic hepatitis B exhibit very weak HBVT cell responses and lack anti-HB antibodies, which is considered one of the reasons why these patients cannot clear the virus. Therefore, if viral gene expression could be silenced at the gene level, blocking HBV production and replication, thereby fundamentally reducing viral replication and infection of hepatocytes, it would undoubtedly be the most ideal treatment for hepatitis B.

[0004] Antisense technology is an effective means of reducing the expression of specific gene products and has been proven applicable to the treatment, diagnosis, and research of various diseases, with a unique suitability for regulating HBV expression. Unlike nucleoside therapy, antisense therapy directly targets HBV antigen transcripts, thereby reducing serum HBeAg and HBsAg levels. Because multiple overlapping transcripts are produced during HBV infection, a single antisense oligomer may reduce HBV DNA in addition to HBeAg and HBsAg. Therefore, antisense technology, as an effective means of reducing the expression of certain gene products, is uniquely suitable for regulating HBV. Summary of the Invention

[0005] The present invention aims to provide a novel antisense oligonucleotide (ASO), its conjugates, and their uses. The antisense oligonucleotide and its conjugates of the present invention can be used to prepare drugs for the treatment and / or prevention of hepatitis B. The antisense oligonucleotide and its conjugates of the present invention exhibit high gene expression inhibitory activity against HBV and / or low toxicity.

[0006] On one hand, the present invention relates to a modified antisense oligonucleotide comprising one of the nucleotide sequences (i.e., the basic sequence) shown in SEQ ID NO: 1 to SEQ ID NO: 4. The modification is selected from at least one of the following: 2'-O-methoxyethyl modification, 2'-deoxynucleotide modification, 5-methyl modified cytosine, substitution modification of nucleotide D, substitution modification of nucleotide i, InvB modification (i.e., reverse deoxydebasic ribose), locked nucleic acid modification (i.e., the l modification in Table 2 of this invention), thiophosphate modification, and ( S )-cEt modification (i.e., k modification in Table 2 of this invention), The sequence information of SEQ ID NO: 1 to SEQ ID NO: 4 is detailed in Table 1.

[0007] In some embodiments of the antisense oligonucleotide described in this invention, the antisense oligonucleotide is 15 to 30 nucleotides in length.

[0008] In some embodiments of the antisense oligonucleotides described in this invention, the 2'-O-methoxyethyl modification, 2'-deoxynucleotide modification, 5-methyl-modified cytosine, locked nucleic acid modification, and ( S The -cEt modifiers exist independently in one or more of the following locations: The nucleotides at the 5' end of the antisense oligonucleotide are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th positions of the start site.

[0009] In some embodiments of the antisense oligonucleotides described in this invention, the substitution modification of nucleotide D and the substitution modification of nucleotide i each occur independently at one or more of the following positions: The nucleotides at the 5' end of the antisense oligonucleotide are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th positions of the start site.

[0010] In some embodiments of the antisense oligonucleotides described in this invention, the thiophosphate linkage modification is independently present at one or more of the following positions: The 5' end nucleotide is between positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, and 19-20 of the start site. In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is optionally present at the 5' end of the antisense oligonucleotide.

[0011] In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is optionally present at the 3' end of the antisense oligonucleotide.

[0012] In some embodiments of the antisense oligonucleotide of the present invention, the InvB modification is present at the 5' end of the antisense oligonucleotide and / or the InvB modification is present at the 3' end of the antisense oligonucleotide.

[0013] In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is attached to the 5' end of the antisense oligonucleotide via a thiophosphate bond or a phosphate bond.

[0014] In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is attached to the 3' end of the antisense oligonucleotide via a thiophosphate bond or a phosphate bond.

[0015] In some embodiments of the antisense oligonucleotides described in this invention, the InvB modification is attached to the 5' end and / or 5' end of the antisense oligonucleotide via a thiophosphate bond or a phosphate bond.

[0016] In some embodiments of the antisense oligonucleotides described in this invention, all nucleotides in the antisense oligonucleotides are modified nucleotides.

[0017] In some embodiments of the antisense oligonucleotides described in this invention, all nucleosides in the antisense oligonucleotides are phosphate thioester bonds.

[0018] In some embodiments of the antisense oligonucleotides described in this invention, they comprise one of the nucleotide sequences shown in SEQ ID NO: 5 to SEQ ID NO: 243, the sequence information of which is detailed in Table 2.

[0019] In some embodiments of the antisense oligonucleotides described in this invention, the antisense oligonucleotides optionally include a delivery carrier portion.

[0020] In some embodiments of the antisense oligonucleotides described in this invention, the delivery carrier portion comprises a desialyl glycoprotein receptor (ASGP-R) targeting portion or a cholesterol conjugate portion.

[0021] In some embodiments of the antisense oligonucleotide described in this invention, the ASGP-R targeting portion comprises GalNAc or a derivative thereof.

[0022] In some embodiments of the antisense oligonucleotides described in this invention, the ASGP-R targeting portion comprises GalNAc or a derivative thereof linked via a bivalent or trivalent branched linker.

[0023] In some embodiments of the antisense oligonucleotide described in this invention, the ASGP-R targeting portion is L96, DAW40007-4, or their stereoisomers, wherein the structures of L96 and DAW40007-4 are as follows: .

[0024] In some embodiments of the antisense oligonucleotides described in this invention, the 3' or 5' end of the antisense oligonucleotide is linked to the ASGP-R targeting portion via a phosphate ester group, a thiophosphate ester group, or a phosphate group.

[0025] On the other hand, the present invention relates to a pharmaceutical composition comprising the antisense oligonucleotide described herein or a salt thereof, and at least one of pharmaceutically acceptable excipients and diluents.

[0026] In another aspect, the present invention relates to the use of the antisense oligonucleotides or pharmaceutical compositions thereof described herein in the preparation of medicaments for the treatment and / or prevention of HBV-related diseases, conditions or symptoms in a subject (mammal, such as human).

[0027] In some embodiments of the antisense oligonucleotides described in this invention, the disease, condition, or symptom is jaundice, liver fibrosis, hepatitis, cirrhosis, serum hepatitis, liver failure, liver cancer, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, HBV viremia, or transplantation related to liver disease.

[0028] In another aspect, the present invention relates to the use of the antisense oligonucleotide or pharmaceutical composition thereof described herein in the preparation of a medicament for reducing HBsAG levels in a host of HBV infection.

[0029] In another aspect, the present invention also relates to a pharmaceutical composition comprising the antisense oligonucleotides described herein, their conjugates or salts thereof, and pharmaceutically acceptable excipients.

[0030] In another aspect, the present invention also relates to the use of the antisense oligonucleotides, conjugates thereof, and pharmaceutical compositions described herein in the preparation of medicaments for the treatment and / or prevention of hepatitis B.

[0031] Detailed Description of the Invention Definitions and general terms In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0032] In this invention, the term "inhibition of hepatitis B virus gene expression" includes inhibition of hepatitis B virus (HBV) gene expression at any level, such as at least partial inhibition of HBV gene expression, including inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. HBV gene expression can be evaluated based on the level of any variable associated with HBV gene expression, such as HBV mRNA levels or HBV protein levels. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as baseline level before administration, or level measured in similar subjects, cells, or samples that have never been treated or have been treated with a control (e.g., a control with only buffer or a control without active agent).

[0033] In this invention, "pharmaceutical composition" can refer to a drug for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, the composition is prepared by uniformly and sufficiently combining the active ASO with liquid excipients, finely chopped solid excipients, or both.

[0034] In this invention, "2'-deoxynucleotide modification" refers to the deoxygenation of the hydroxyl group (2'-OH) in the pentose nucleotide to hydrogen (2'-H).

[0035] In this invention, "5'-methylated cytosine modification" or "5-methylated cytosine" refers to methylation at the 5th carbon atom of cytosine.

[0036] In this invention, "2'-O-methoxyethyl modification" refers to the substitution of the 2-position of the ribose by a phase-O-methoxyethyl modification, which is represented by e in the sequence of this invention.

[0037] In this invention, "substitution modification of nucleotide D" and "substitution modification of nucleotide i" respectively indicate that the nucleotide in the antisense oligonucleotide is replaced by D or i, such as the basic sequence A, T, G or C being replaced by D or i, wherein the structures of D and i are as follows: and In this invention, "InvB modification" refers to reverse deoxyribose, which is generally modified (or linked) at the 5' end and / or 3' end of the antisense oligonucleotide.

[0038] "Locked nucleoside modification" or "LNA" or "l" indicates a nucleic acid monomer having a bridge connecting two carbon atoms between the 4' and 2' positions of the nucleoside sugar ring, thereby forming a bicyclic sugar. Examples of such bicyclic sugars include, but are not limited to, A) α-L-methyleneoxy(4'-CH2-O-2') LNA; (B) β-D-methyleneoxy(4'-CH2-O-2')-LNA; (C) ethyloxy(4'-(CH2)2-O-2') LNA; (D) aminooxy(4'-CH2-ON(R)-2') LNA; and (E) oxyamine(4'-CH2-N(R)-O-2') LNA, for details of the specific structures, see paragraph

[0098] of patent application TW202340468A, where R can be a methyl group. For example, the structures of Al, Tl, Gl, and Cl are as follows: When connected to the 3' end, the structure is as follows: When connected to the 5' end, the structure is as follows: , where Base represents the corresponding base A, T, G or C.

[0039] "Thiophosphate modification" refers to the substitution of the phosphate group (5'-PO(OH)2) in the pentose of a nucleotide by a thiophosphate group.

[0040] “( S The "-cEt modification" or "k" indicates the presence of a bridge connecting two carbon atoms at the 4' and 2' positions of the nucleotide sugar ring, forming a 4'-CH(CH3)-O-2' bridge. Structures such as Ak, Tk, Gk, or Ck are... , where Base represents the corresponding base A, T, G or C. A "nucleoside bond" refers to a group that can covalently link two entities (such as nucleotides) together. Specific examples include phosphate ester groups and thiophosphate ester groups.

[0041] The term "carrier portion" refers to a molecular carrier intended to carry or transport the oligomers of the present invention to their desired location, such as the desired anatomical location.

[0042] In some embodiments, the carrier portion comprises an ASPG-R targeting moiety with an affinity equal to or greater than that of galactose. The ASPG-R targeting moiety may be selected from galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine (GalNAc), N-propionylgalactosamine, N-butyrylgalactosamine, and N-isobutyrylgalactosamine. In some embodiments, the ASPG-R targeting conjugate moiety is monovalent. In other embodiments, the carrier component comprises a galactose cluster, such as a divalent, trivalent, or tetravalent ASPG-R targeting conjugate moiety (i.e., containing 1, 2, 3, or 4 terminal sugar moieties capable of binding to the ASPG-R). In some embodiments, the carrier component comprises GalNAc (N-acetylgalactosamine), such as monovalent, divalent, trivalent, or tetravalent GalNAc. GalNAc conjugates can be used to target compounds to the liver. A preferred carrier component is an N-acetylgalactosamine trimer. In some embodiments, the carrier portion comprises a glycoconjugate portion (or a conjugate group on the ASO, the glycoconjugate portion including but not limited to galactose, lactose, N-acetylgalactosamine, mannose, and mannose-6-phosphate). Glycoconjugates can be used to enhance delivery or activity in a range of tissues such as the liver and / or muscle.

[0043] In this invention, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated.

[0044] In this invention, the term "pharmaceuticalally acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients that are incompatible with the ASO of this invention, such as any adverse biological effects they produce or interactions that occur in a harmful manner with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0045] As used herein, “chemical modification” or “modification” means a structure that is chemically different from its naturally occurring counterpart, including all alterations made by chemical means, such as the addition or removal of a chemical part, or the substitution of one chemical part for another.

[0046] The compounds of this invention can be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of this invention containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0047] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, 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 desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0048] This invention also includes compounds of the invention that are identical to those described herein, but in which one or more atoms are replaced by isotopes with atomic weights or mass numbers different from those commonly found in nature. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C, 13C, 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, etc.

[0049] Unless otherwise specified, when a position is specifically designated as deuterium (D), that position should be understood as having a deuterium abundance at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium doping). The natural abundance of deuterium in the example compounds can be at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even higher. The invention also includes various deuterated forms of compounds. Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds with reference to relevant literature. Commercially available deuterated starting materials can be used to prepare deuterated compounds, or conventional techniques can be used to synthesize them with deuterated reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0050] The conjugation groups described in this invention can enhance the delivery of therapeutic agents to specific target sites (e.g., specific organs or tissues) within an object, such as a human or animal. In some embodiments of this invention, the conjugation groups can enhance the targeted delivery of expressed repressive oligonucleotides. In some embodiments of this invention, the conjugation groups can enhance the delivery of expressed repressive oligonucleotides to the liver.

[0051] The conjugating groups described in this invention can be directly or indirectly attached to compounds, such as therapeutic agents, for example, expressing repressive oligonucleotides, for example, the 3' or 5' end of the expressing repressive oligonucleotide. In some embodiments of this invention, the expressing repressive oligonucleotide comprises one or more modified nucleotides. In some embodiments of this invention, the expressing repressive oligonucleotide is an ASO. In some embodiments, the conjugating groups disclosed herein are attached to the expressing repressive oligonucleotide reagent at the 3' end via phosphate ester, thiophosphate, or phosphonate groups.

[0052] The definitions and conventions of stereochemistry used in this invention are generally referenced in the following literature: SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of this invention may contain asymmetric or chiral centers, and therefore exist as different stereoisomers. All stereoisomers of the compounds of this invention, including, but not limited to, diastereomers, enantiomers, stenotic isomers, and mixtures thereof, such as racemic mixtures, constitute a part of this invention. Many organic compounds exist in optically active forms, i.e., they are capable of rotating the plane of plane-polarized light. When describing optically active compounds, the prefix... D , L or R , S Used to indicate the absolute configuration of the chiral center of a molecule. Prefix d , l The symbols (+) and (-) are used to name the rotation of plane-polarized light in compounds. l This indicates that the compound is levorotatory, indicated by the prefix (+) or... d This refers to a compound that is dextrorotatory. These stereoisomers have the same chemical structure, but their stereostructures are different. Specific stereoisomers can be enantiomers, and mixtures of isomers are usually called enantiomeric mixtures. A 50:50 enantiomeric mixture is called a racemic mixture or racemate, which may result in a lack of stereoselectivity or stereodirection during chemical reactions. The terms "racemic mixture" and "racemate" refer to a mixture of two equimolar enantiomers that lack optical activity.

[0053] The term "tautomer" or "tautomer form" refers to isomers of structures with different energies that can interconvert through a low energy barrier. For example, proton tautomers (i.e., proton-transfer tautomers) include interconversions via proton transfer, such as isomerization between keto-enols and imine-enamines.

[0054] The term "composition" refers to a mixture of a drug containing one or more of the ASOs described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0055] The term “excipient” means “pharmaceutical-grade excipient” or “pharmaceutical-acceptable excipient”, including but not limited to any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0056] Unless otherwise specified, the "compounds," "ligands," "nucleic acid conjugates," and "nucleic acids" of this invention may exist independently as salts, mixed salts, or non-salts (e.g., free acids or free bases). When present as salts or mixed salts, they may be pharmaceutically acceptable salts.

[0057] The term "acceptable salt" includes acceptable acid addition salts and pharmaceutically acceptable base addition salts. An "acceptable acid addition salt" is a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0058] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that maintain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being the most preferred. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring 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, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0059] Additionally, it should be noted that, unless otherwise explicitly stated, the descriptive phrases “each and each is independently”, “each and each is independently”, and “each and each is independently” used throughout this document are interchangeable and should be interpreted broadly. They can mean either that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0060] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0061] For example, the phrase "in the antisense oligonucleotide, all cytosines are independently and optionally 5-methyl modified" in this invention means that in the antisense oligonucleotide of this invention, all cytosines may be unmodified, or 1, 2, 3, 4, 5 or 6 cytosines may be methylated at the 5-position.

[0062] The term "solid support" or "solid support material" specifically refers to any particle, bead, or surface on which oligonucleotide synthesis can occur. For example, both inorganic and organic solid supports can be selected for use in embodiments of the invention. Inorganic solid supports are preferably selected from silica gel and controlled-pore glass (CPG). Organic solid supports are resins, preferably macroporous resins, more preferably highly cross-linked polystyrene, Tentagel (a graft copolymer of a low-crosslinked polystyrene matrix with polyethylene glycol (PEG or POE) grafted onto it), polyvinyl acetate (PVA), Poros-polystyrene / divinylbenzene copolymers, amino polyethylene glycol, and cellulose, etc. Preferred embodiments of the invention utilize CPG-based solid supports. Many other commercially available solid supports are also included in this invention. Attached Figure Description Appendix Figure 1 The relative residual levels of HBsAg protein in the serum of HBV-Tg transgenic mice treated with ASO compounds 84, 189, and 44 of the present invention, as well as the positive control drug HECN2400791, are shown.

[0063] Appendix Figure 2 The relative residual levels of serum HBsAg protein in HBV-Tg transgenic mice treated with ASO compounds 112, 226, and 84 of the present invention, as well as positive control drugs DAW60487 and HECN2400791, are shown.

[0064] Appendix Figure 3 The relative residual levels of HBsAg protein in the serum of HBV-Tg transgenic mice treated with ASO compounds 227 and 229 of the present invention, as well as positive control drugs DAW60487 and HECN2400791, are shown.

[0065] Appendix Figure 4 The relative residual levels of serum HBsAg protein in HBV-AAV mice treated with ASO compound 189 (10 mpk, 20 mpk, 40 mpk) of the present invention, as well as positive control drugs DAW60487 (20 mpk) and HECN2400791 (20 mpk) are shown.

[0066] Detailed description of the compounds of the present invention The present invention aims to provide a novel antisense oligonucleotide (ASO), its conjugates, and their uses. The antisense oligonucleotide and its conjugates of the present invention can be used to prepare drugs for the treatment and / or prevention of hepatitis B. The antisense oligonucleotide and its conjugates of the present invention exhibit high gene expression inhibitory activity against HBV and / or low toxicity.

[0067] On one hand, the present invention relates to a modified antisense oligonucleotide comprising one of the nucleotide sequences (i.e., the basic sequence) shown in SEQ ID NO: 1 to SEQ ID NO: 4. The modification is selected from at least one of the following: 2'-O-methoxyethyl modification, 2'-deoxynucleotide modification, 5-methyl modified cytosine, substitution modification of nucleotide D, substitution modification of nucleotide i, InvB modification (i.e., reverse deoxydebasic ribose modification), locked nucleic acid modification (i.e., the l modification in Table 2 of this invention), thiophosphate modification, and ( S )-cEt modification (i.e., k modification in Table 2 of this invention), The sequence information of SEQ ID NO: 1 to SEQ ID NO: 4 is detailed in Table 1.

[0068] In some embodiments of the antisense oligonucleotide described in this invention, the antisense oligonucleotide is 15 to 30 nucleotides in length.

[0069] In some embodiments of the present invention, the antisense oligonucleotides of the present invention are composed of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 linked nucleotides.

[0070] In some embodiments of the antisense oligonucleotides described in this invention, the 2'-O-methoxyethyl modification, 2'-deoxynucleotide modification, 5-methyl-modified cytosine, locked nucleic acid modification, and ( S The -cEt modifiers exist independently in one or more of the following locations: The nucleotides at the 5' end of the antisense oligonucleotide are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th positions of the start site.

[0071] In some embodiments of the antisense oligonucleotides described in this invention, the substitution modification of nucleotide D and the substitution modification of nucleotide i each occur independently at one or more of the following positions: The nucleotides at the 5' end of the antisense oligonucleotide are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th positions of the start site.

[0072] In some embodiments of the antisense oligonucleotides described in this invention, the thiophosphate linkage modification is independently present at one or more of the following positions: The nucleotides at the 5' end of the positive strand are between positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, and 19-20 of the start point. In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is optionally present at the 5' end of the antisense oligonucleotide.

[0073] In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is optionally present at the 3' end of the antisense oligonucleotide.

[0074] In some embodiments of the antisense oligonucleotide of the present invention, the InvB modification is present at the 5' end of the antisense oligonucleotide and / or the InvB modification is present at the 3' end of the antisense oligonucleotide.

[0075] In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is attached to the 5' end of the antisense oligonucleotide via a thiophosphate bond or a phosphate bond.

[0076] In some embodiments of the antisense oligonucleotide described in this invention, the InvB modification is attached to the 3' end of the antisense oligonucleotide via a thiophosphate bond or a phosphate bond.

[0077] In some embodiments of the antisense oligonucleotides described in this invention, the InvB modification is attached to the 5' end and / or 5' end of the antisense oligonucleotide via a thiophosphate bond or a phosphate bond.

[0078] In some embodiments of the antisense oligonucleotides described in this invention, all nucleotides in the antisense oligonucleotides are modified nucleotides.

[0079] In some embodiments of the antisense oligonucleotides described in this invention, all nucleosides in the antisense oligonucleotides are phosphate thioester bonds.

[0080] In some embodiments of the antisense oligonucleotides described in this invention, they comprise one of the nucleotide sequences shown in SEQ ID NO: 5 to SEQ ID NO: 243, the sequence information of which is detailed in Table 2.

[0081] It should be specifically noted here that in the event of any conflict or inconsistency between the sequence information in this specification and the sequence information in the sequence list (ST26 sequence list), the sequence information recorded in the specification shall prevail.

[0082] In some embodiments of the antisense oligonucleotides described in this invention, the oligonucleotides are used to inhibit hepatitis B virus expression.

[0083] In some embodiments of the antisense oligonucleotides described in this invention, the antisense oligonucleotides optionally include a delivery carrier portion.

[0084] In some embodiments of the antisense oligonucleotides described in this invention, the delivery carrier portion comprises a desialyl glycoprotein receptor (ASGP-R) targeting portion or a cholesterol conjugate portion.

[0085] In some embodiments of the antisense oligonucleotide described in this invention, the ASGP-R targeting portion comprises GalNAc or a derivative thereof.

[0086] In some embodiments of the antisense oligonucleotides described in this invention, the ASGP-R targeting portion comprises GalNAc or a derivative thereof linked via a bivalent or trivalent branched linker.

[0087] In some embodiments of the antisense oligonucleotide described in this invention, the ASGP-R targeting portion is L-96, DAW40007-4, or their stereoisomers, wherein the structures of L-96 and DAW40007-4 are respectively: .

[0088] In some embodiments of the antisense oligonucleotides described in this invention, the 3' or 5' end of the antisense oligonucleotide is linked to the ASGP-R targeting portion via a phosphate ester group, a thiophosphate ester group, or a phosphate group.

[0089] In some embodiments of the antisense oligonucleotide described in this invention, the antisense oligonucleotide is InvBsGesmCesAesGesAdsGdsGdsTdsGdsAesAdsGdsmCdsGdsAdsAesGesTesGlsmCl (SEQ ID NO: 193) or GesmCesAesGesAesGdsGdsTdsGdsAesAdsGdsmCdsGdsAdsAesGesTesGksmCk (SEQ ID NO: 48).

[0090] On the other hand, the present invention relates to a pharmaceutical composition comprising the antisense oligonucleotide described herein or a salt thereof, and at least one of pharmaceutically acceptable excipients and diluents.

[0091] In another aspect, the present invention relates to the use of the antisense oligonucleotides or pharmaceutical compositions thereof described herein in the preparation of a medicament for the treatment and / or treatment of HBV-related diseases, conditions or symptoms in a subject.

[0092] In some embodiments of the antisense oligonucleotides described in this invention, the disease, condition, or symptom is jaundice, liver fibrosis, hepatitis, cirrhosis, serum hepatitis, liver failure, liver cancer, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, HBV viremia, or transplantation related to liver disease.

[0093] In another aspect, the present invention relates to the use of the antisense oligonucleotide or pharmaceutical composition thereof described herein in the preparation of a medicament for reducing HBsAG levels in subjects infected with HBV (mammals, such as humans).

[0094] Table 1: Basic Sequence Oligonucleotides

[0095] In Table 1, the uppercase letters A, T, G, and C represent the bases of natural nucleotides.

[0096] The present invention relates to antisense oligonucleotides, antisense oligonucleotide conjugates, pharmaceutical compositions, administration methods, and methods for treating diseases. The oligonucleotides and oligonucleotide conjugates of the present invention can be used in pharmaceutical formulations and pharmaceutical compositions. Suitably, such compositions contain pharmaceutically acceptable diluents, carriers, salts, or excipients.

[0097] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, which may contain multiple components that provide a variety of functions, including regulating drug concentration, regulating solubility, chemical stabilization, regulating viscosity, enhancing absorption, and regulating pH.

[0098] Pharmaceutical carriers may include suitable liquid solvents or excipients and optional auxiliary additives or additives. Liquid solvents and excipients are conventional and commercially available. Illustrative pharmaceutical carriers include distilled water, physiological saline, dextran aqueous solution, etc. For water-soluble formulations, the pharmaceutical composition preferably contains a buffer such as phosphate buffer or other organic acid salt, preferably in the pH range of 6.5 to 8. For formulations containing slightly soluble antisense compounds, microemulsions may be used, for example by using a nonionic surfactant such as polysorbate-80 at a concentration of 0.04-0.05% (w / v) to increase solubility. Other components may include antioxidants (such as ascorbic acid), hydrophilic polymers such as monosaccharides, disaccharides and other sugars (including cellulose or its derivatives, dextrin), chelating agents such as EDTA, and similar components well known to those skilled in the art, e.g., Remington's Pharmaceutical Science, latest edition (Mack Publishing Company, Easton, Pa.).

[0099] The oligonucleotides of the present invention include pharmaceutically acceptable salts thereof, including alkaline earth metals (e.g., sodium or magnesium), ammonium, or N×4. + Salts of compounds having a hydroxyl group (where X is a C1-C4 alkyl group). Other pharmaceutically acceptable salts include organic carboxylates such as formates, acetates, lactates, tartrates, malates, hydroxyethyl sulfonates, lactobionates, and succinates; organic sulfonates such as methanesulfonates, ethanesulfonates, toluenesulfonates, and benzenesulfonates; and inorganic acid salts such as hydrochlorides, sulfates, phosphates, and aminosulfonates. Pharmaceutically acceptable salts of compounds having a hydroxyl group include the anions of these compounds, along with suitable cations such as Na+. + NH4 + wait.

[0100] The medication may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral administration and sublingual administration), preferably subcutaneous administration and intravenous administration.

[0101] The pharmaceutical compositions disclosed in this invention comprise formulations suitable for parenteral administration. The formulations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with excipients to prepare a single-dose form is generally the amount of antisense oligonucleotide that produces the therapeutic effect. Generally, this amount, expressed in percent, is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0102] The therapeutically effective amount of one or more drugs present in the pharmaceutical composition of the present invention and used in the method of the present invention applied to mammals (e.g., humans) can be determined by a person skilled in the art, taking into account individual differences in age, weight, and condition of the mammal. The effective amount of the drug of the present invention is administered to a subject (e.g., a mammal, such as a human) in which the drug produces the desired outcome (e.g., slowing or alleviating cancer or neurodegenerative diseases) in the treated subject. The therapeutically effective amount can be determined empirically by a person skilled in the art.

[0103] The patient may also receive the drug once or more weekly (e.g., 2, 3, 4, 5, 6, or 7 times or more per week) at doses of about 0.1 to 3,000 mg per administration, with weekly doses of 0.1 to 2,500 mg (e.g., 2,000, 1,500, 1,000, 500, 100, 10, 1, 0.5, or 0.1 mg). The patient may also receive the composition drug at doses of 0.1 to 3,000 mg every two or three weeks. In certain embodiments, 0.1 to 3,000 mg is administered about once a month to about once a quarter (i.e., about once every three months). In some specific embodiments, 0.1 to 3,000 mg is administered about once a month to about once every six months. Single or multiple administrations containing an effective amount of the composition of the invention may be performed, with the dosage level and pattern chosen by the treating physician. Dosage and administration regimens can be determined and adjusted based on the severity of the disease or symptoms in the patient, wherein the severity can be monitored throughout the treatment process according to methods commonly practiced by clinicians or those described herein.

[0104] In another aspect, the present invention provides a method for inhibiting the expression or activity of hepatitis B virus genes. According to an embodiment of the present invention, the method includes contacting cells with the aforementioned antisense oligonucleotide or the aforementioned pharmaceutical composition. As previously mentioned, the aforementioned antisense oligonucleotide can degrade hepatitis B virus mRNA, inhibiting HBV expression and replication. Therefore, the method of the present invention can be used to inhibit the expression or activity of hepatitis B virus genes. For example, it can inhibit the expression or activity of hepatitis B virus genes in animals; or it can inhibit the expression or activity of hepatitis B virus genes for non-disease treatment purposes, such as inhibiting the expression or activity of hepatitis B virus genes in vitro for subsequent research.

[0105] In another aspect, the present invention provides a method for preventing and / or treating diseases caused by hepatitis B virus. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned antisense oligonucleotide or the aforementioned pharmaceutical composition. According to embodiments of the invention, this method can effectively prevent and / or treat diseases related to hepatitis B virus.

[0106] The general synthetic method of the carrier, antisense oligonucleotide, and antisense oligonucleotide conjugate described in this invention Generally, the carriers, antisense oligonucleotides, and antisense oligonucleotide conjugates of the present invention can be prepared by the methods described in the present invention. The following reaction schemes and examples are used to further illustrate the content of the present invention. Detailed Implementation

[0107] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. In particular, the synthesis of small nucleic acids such as ASO and small nucleic acid conjugates can be obtained by adjusting the synthesis according to the embodiments of the present invention or conventional methods in the art. Where specific techniques, reagent concentrations, reagent dosages, etc., are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in the art, or according to the product instructions, or adjusted according to the technical knowledge in the art. For the synthesis of ASO, even if the dosage and concentration are specified, appropriate adjustments can be made according to the technical knowledge in the art and the needs of the synthesis. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products, and their quality / purity standards are suitable for molecular biology.

[0108] Preparation Examples In the following preparation examples, the inventors have described in detail the preparation process of the compounds of the present invention using some of the compounds as examples, wherein, For CPG.

[0109] Synthesis of compound L96-DMTr-CPG: Compound L96-DMTr-CPG was prepared according to the method described in patent application WO2014025805A1.

[0110]

[0111] Synthesis of compound DAW40007-3: Compound DAW40007-3 can be obtained by referring to the synthesis method in Example 2 of CN 119061009 A.

[0112]

[0113] As is known to those skilled in the art, compound L96-DMTr-CPG, after being conjugated with a small nucleic acid, is deprotected to obtain the L-96-nucleic acid conjugate, and compound DAW40007-3, after being conjugated with a small nucleic acid, is deprotected to obtain the DAW40007-4 nucleic acid conjugate.

[0114] The method for synthesizing antisense oligonucleotides described in this invention The synthesis was performed according to the theoretical yield of 1 μmol. 1 μmol of solid-phase support CPG (purchased from Hebei Dinaxingke), all DNA phosphoramidite monomers, 2'-modified RNA phosphoramidite monomers, and auxiliary reagents were weighed out; all phosphoramidite monomers were provided in 0.1 M anhydrous acetonitrile solution. For thiolated oligonucleotides with a phosphate backbone, 0.1 M DDTT solution was used as the thioating agent. 5-Ethylthio-1 H Tetraazole acetonitrile solution (0.25M) was used as the activator (purchased from Suzhou Kelama), and 3% trichloroacetic acid in dichloromethane solution was used as the deprotection reagent. These were placed in the designated reagent positions on the automated DNA / RNA synthesizer. The synthesis program was set, and the specified oligonucleotide base sequence was entered. After verification, the oligonucleotide synthesis cycle began. Each coupling step lasted 6 minutes, and the sulfurization time was 6 minutes. After automated cycling, oligonucleotides containing the solid-phase support CPG were obtained.

[0115] The nucleotides containing the solid-phase support CPG obtained above were dried by blowing with dry argon gas, then transferred to a 2 mL EP tube, and 0.8 mL of 28% ammonia solution was added. The mixture was heated at 55°C for 5–18 hours. After filtration, the filter cake was washed with water (0.5 mL), the filtrates were combined, and concentrated under reduced pressure to obtain a white or yellow gelatinous solid. After reverse-phase purification, the prepared solution was concentrated, passed through a gel column to remove excess salt, and oligonucleotides were obtained. The concentration of the obtained oligonucleotides was determined by a micro-ultraviolet spectrophotometer (SPECTROstat Nano). Mass spectrometry analysis was performed on an Agilent 6530 LC-MS Q-Tof system. After a single-stage scan, the molecular weight of the nucleic acid was calculated after deconvolution. The above method can be combined with appropriately modified synthetic methods known in the art to synthesize the oligonucleotides of this invention. Some of the synthesized oligonucleotides are shown in Table 2.

[0116] The above oligonucleotide synthesis scheme outlines the general experimental steps for preparing the oligonucleotides of this invention. Those skilled in the art can modify the methods or adjust the raw materials as needed to prepare the antisense oligonucleotides described in this invention. Unless otherwise specified, the oligonucleotides of this invention can be prepared using the methods described in the above synthesis scheme. DAW60487 and HECN2400791 are positive control drugs.

[0117] Table 2: Some oligonucleotides synthesized in this invention

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] Note: In Table 2 and the context, the capital letters A, T, G, and C followed by 'e' indicate nucleosides whose ribose is replaced by 2'-MOE (i.e., 2'-O-methoxyethyl). For example, Ae represents adenine nucleoside modified with 2'-MOE ribose, Te represents thymidine nucleoside modified with 2'-MOE ribose, Ge represents guanine nucleoside modified with 2'-MOE ribose, and mCe represents cytosine nucleoside modified with a 5-methyl group at 2'-MOE ribose. The letter 'd' indicates a 2'-deoxyribonucleotide (i.e., the sugar ring on the nucleotide is hydrogen at 2'), such as Ad representing adenosine modified with 2'-deoxyribose (deoxyadenosine), Td representing thymidine modified with 2'-deoxyribose (deoxythymidine), Gd representing guanine modified with 2'-deoxyribose (deoxyguanosine), and Cd representing cytosine modified with 2'-deoxyribose (deoxycytidine). mC represents cytosine modified with a 5-methyl group; mCd represents cytosine modified with a 5-methyl group in a deoxyribonucleotide. The uppercase letter D indicates... Adding a 'k' after the capital letters A, T, G, and C indicates... Where Base represents the corresponding base A, T, G, or C; the lowercase letter i indicates The 'l' following the capital letters A, T, G, and C indicates locked nucleic acid modification, specifically the β-D-methyleneoxy group (4'-CH2-O-2') on the sugar ring of the nucleoside. The structures of Al, Tl, Gl, and Cl are... Where Base represents the corresponding base A, T, G, or C; s indicates ps modification (thiophosphate group); InvB indicates reverse deoxyribose, and when placed in the middle, InvB represents... When connected to the 3' terminal, InvB represents... When connected to the 5' terminal, InvB represents... .

[0132] Example 4: Cell viability and cytotoxicity test of the oligonucleotides of the present invention HepAD38 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected using transfection reagent. The cell concentration was adjusted to 2.5 × 10⁻⁶ cells / year. 5 / mL, seed 2*10 in each well of a 96-well plate. 4 Cells were transfected with ASO according to the RNAiMAX transfection reagent instructions. After incubation at 37°C and 5% CO2 for 24 hours, the culture medium was replaced with fresh medium, and the cells were cultured for another 72 hours. Cell supernatant was collected, and HBsAg expression was detected using the HBsAg ELISA kit. HBV DNA was detected by qPCR, and cytotoxicity was detected by CCK8 assay. Cell mRNA expression levels were detected using the QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0133] The inhibition rate and cytotoxicity results of the antisense oligonucleotides of this invention against HBsAg are shown in Table A.

[0134] Table A: Results of the inhibition rate of antisense oligonucleotides of the present invention on HBsAg and cell survival rate.

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] Experimental results show that the antisense oligonucleotide of the present invention has good inhibitory activity against HBsAg and low cytotoxicity.

[0146] Example 5: Efficacy test of the oligoantisense nucleic acid of the present invention in HBV-Tg mice for hepatitis B treatment. Experimental methods Experimental animals: HBV-Tg transgenic mice, C57B / 6N-Tg(1.28HBV) / Vst, SPF grade males, 6-8 weeks old, 16-25 g, purchased from Beijing Vitonda Biotechnology Co., Ltd. The animals were fed by Beijing Vitonda Biotechnology Co., Ltd., and were housed individually in cages.

[0147] Animal grouping: Based on the quantitative detection results of HBsAg in mouse serum, the animals were stratified and randomly grouped into groups of 5.

[0148] Preparation and administration of test samples: Calculate the required amount of drug powder based on the purity of each test sample. Add the corresponding physiological saline at a dosage concentration of 12 mg / mL, and shake to mix until a colorless and transparent liquid is obtained. Define the day of the first administration as day 0. Animals in each group were administered the drug on days 0, 3, and 7, with a subcutaneous administration volume of 5 mL / kg. If blood collection is required on the day of administration, the drug should be administered after blood collection.

[0149] Main observation indicators: Weighing and blood collection were performed once each on days 3, 7, 14 and 21, and serum was separated for DNA, HBsAg and ALT level determination.

[0150] Serum HBsAg level determination: After blood collection, serum was separated, diluted with PBS solution, and sent for testing. 10 μL of serum was taken from each sample and diluted to 500 μL (50-fold dilution) with PBS solution. Serum HBsAg levels were measured using a Hepatitis B surface antigen assay kit (Mike Biotech).

[0151] Experiments show that, in Tg / HBV mouse model experiments, the antisense oligonucleotide compound of this invention has excellent anti-HBV activity and can be used for the prevention and treatment of HBV and related diseases.

[0152] The experimental results of ASO (antisense oligonucleotide) compounds 84, 189, and 44, and the positive control drug HECN2400791 of this invention are attached. Figure 1 , attached Figure 1The results show that ASO compounds 84, 189, and 44 of this invention exhibit good HBsAg inhibitory activity, significantly superior to the positive control drug HECN2400791. The experimental results for antisense oligonucleotide compounds 112, 226, and 84 of this invention, as well as the positive control drugs DAW60487 and HECN2400791, are attached. Figure 2 , attached Figure 2 The results show that ASO compounds 112, 226 and 84 of the present invention have good HbsAg inhibitory activity, and are significantly better than the positive control drugs DAW60487 and HECN2400791.

[0153] Example 6: Efficacy test of the oligonucleotide of the present invention in HBV-Tg mice for hepatitis B treatment. Experimental methods Experimental animals: HBV-Tg transgenic mice, C57B / 6N-Tg(1.28HBV) / Vst, SPF grade males, 6-8 weeks old, 16-25 g, purchased from Beijing Vitonda Biotechnology Co., Ltd. The animals were fed by Beijing Vitonda Biotechnology Co., Ltd., and were housed individually in cages.

[0154] Animal grouping: Based on the quantitative detection results of HBsAg in mouse serum, the animals were stratified and randomly grouped into groups of 4.

[0155] Administration: The dosage was 20 mg / kg. Animals in each group were given the drug on days 0, 3, 7, 11, 14 and 21. If blood samples were required on the day of administration, the drug was administered after the blood sample was collected.

[0156] Main observation indicators: Weighing and blood collection were performed once each on days 3, 7, 14 and 21, and serum was separated for DNA, HBsAg and ALT level determination.

[0157] Serum HBsAg level determination: After blood collection, serum was separated, diluted with PBS solution, and sent for testing. 10 μL of serum was taken from each sample and diluted to 500 μL (50-fold dilution) with PBS solution. Serum HBsAg levels were measured using a Hepatitis B surface antigen assay kit (Mike Biotech).

[0158] Results of hepatitis B virus Tg mouse experiment The ASO compound of this invention showed a significant inhibitory effect on HBsAg in Tg / HBV mouse model experiments, and can be used for the prevention and treatment of HBV and related diseases.

[0159] The inhibitory effects of ASO compounds 227 and 229, as well as positive control drugs DAW60487 and HECN2400791, on HBsAg are shown in the appendix. Figure 3 Appendix Figure 3 The experimental results showed that at a dose of 20 mg / Kg, ASO compounds 227 and 229 had a significant inhibitory effect on HBsAg, and were superior to the positive control drugs DAW60487 and HECN2400791.

[0160] Example 7: Efficacy test of the oligonucleotide of the present invention in HBV-AAV mice for hepatitis B treatment. Experimental methods: Preparation and grouping of rAAV8-1.3HBV / C57BL / 6 mouse model: A persistent HBV infection mouse model was established by intravenous injection of rAAV8-1.3HBV (type C) into the tail vein of C57 mice. The AAV virus injection dose was 1E+11vg / mouse. Six weeks after virus injection, blood samples were collected to detect the levels of DNA, HBsAg, and ALT in the serum. Based on the quantitative detection results of HBsAg in mouse serum, 35 animals were stratified and randomly divided into groups of 5.

[0161] Administration: Positive control drugs DAW60487 and HECN2400791 were administered at a dose of 20 mg / kg. ASO compound 189 was administered at doses of 10 mg / kg, 20 mg / kg, and 40 mg / kg. Animals in all groups were administered the drugs on days 0, 3, 7, 11, 14, and 21. If blood samples were required on the day of administration, administration was performed after blood collection.

[0162] Main observation indicators: Weighing and blood collection were performed once each on days 3, 7, 14, 21 and 28, and serum was separated for HBsAg and DNA level determination.

[0163] Serum HBsAg level determination: After blood collection, serum was separated, diluted with PBS solution, and sent for testing. 10 μL of serum was taken from each sample and diluted to 500 μL (50-fold dilution) with PBS solution. Serum HBsAg was measured using a hepatitis B surface antigen assay kit.

[0164] Results of AAV mouse experiment with hepatitis B virus The test compound showed a significant inhibitory effect on HBsAg in AAV / HBV mouse model experiments, and can be used for the prevention and treatment of HBV and related diseases.

[0165] The inhibitory effects of ASO compound 189, as well as positive control drugs DAW60487 and HECN2400791, on HBsAg are shown in the appendix. Figure 4 Appendix Figure 4 The experimental results showed that at a dose of 20 mg / Kg, compound ASO 289 had a significant inhibitory effect on HBsAg, and was superior to the positive control drugs DAW60487 and HECN2400791.

[0166] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A modified antisense oligonucleotide comprising one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4, The modification is selected from at least one of the following: 2'-O-methoxyethyl modification, 2'-deoxynucleotide modification, 5-methyl modified cytosine, substitution modification of nucleotide D, substitution modification of nucleotide i, InvB modification, locked nucleic acid modification, phosphate thioester modification, and ( S Modified with )-cEt; Optionally, the antisense oligonucleotide is 15 to 30 nucleotides in length.

2. The antisense oligonucleotide according to claim 1, wherein the 2'-O-methoxyethyl modification, 2'-deoxynucleotide modification, 5-methyl modification of cytosine, locked nucleic acid modification, and ( S The -cEt modifiers exist independently in one or more of the following locations: The nucleotides at the 5' end of the antisense oligonucleotide are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th positions of the start site; Optionally, the substitution modification of nucleotide D and the substitution modification of nucleotide i each occur independently at one or more of the following positions: The nucleotides at the 5' end of the antisense oligonucleotide are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th positions of the start site; Optionally, the thiophosphate group linkage modification is each independently present at one or more of the following positions: The 5' end nucleotide is between positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, and 19-20 of the start site.

3. The antisense oligonucleotide according to claim 1 or 2, wherein the InvB modification is optionally present at the 5' end of the antisense oligonucleotide; and / or The InvB modification is optionally present at the 3' end of the antisense oligonucleotide; Optionally, the InvB modification is attached to the 5' and / or 3' end of the antisense oligonucleotide via a thiophosphate bond or a phosphate bond.

4. The antisense oligonucleotide according to claim 1, wherein all nucleotides in the antisense oligonucleotide are modified nucleotides; Optionally, all nucleosides in the antisense oligonucleotide are phosphate thioester bonds.

5. The antisense oligonucleotide according to any one of claims 1-4, comprising one of the nucleotide sequences shown in SEQ ID NO: 5 to SEQ ID NO:

243.

6. The antisense oligonucleotide according to any one of claims 1-5, optionally comprising a delivery carrier portion; The delivery carrier portion comprises either an ASGP-R targeting portion or a cholesterol conjugate portion; The ASGP-R targeting portion comprises GalNAc or a derivative thereof; preferably, the ASGP-R targeting portion comprises GalNAc or a derivative thereof connected via a divalent or trivalent branching linker; more preferably, the ASGP-R targeting portion is L-96, DAW40007-4, or a stereoisomer thereof, wherein... The structures of L-96 and DAW40007-4 are as follows: ； Optionally, the 3' or 5' end of the antisense oligonucleotide is linked to the ASGP-R targeting moiety via a phosphate ester group, a thiophosphate ester group, or a phosphate group.

7. A pharmaceutical composition comprising an antisense oligonucleotide or a salt thereof as described in any one of claims 1-6, and at least one of pharmaceutically acceptable excipients and diluents.

8. Use of the antisense oligonucleotide of any one of claims 1-6 or the pharmaceutical composition of claim 7 in the preparation of a medicament for the treatment and / or prevention of HBV-related diseases, conditions or symptoms in a subject.

9. The use according to claim 8, wherein the disease, condition or symptom is jaundice, liver fibrosis, hepatitis, cirrhosis, serum hepatitis, liver failure, liver cancer, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, HBV viremia, or transplantation related to liver disease.

10. Use of the antisense oligonucleotide of any one of claims 1-6 or the pharmaceutical composition of claim 7 in the preparation of a medicament for reducing HBsAG levels in a host of HBV infection.

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