Double-gap modified oligonucleotide and use thereof in resisting hepatitis b viruses and hepatitis d viruses

Abstract

Provided are a double-gap modified oligonucleotide and the use thereof in resisting hepatitis B viruses and hepatitis D viruses. In particular, provided is a compound, or pharmaceutically acceptable salts, hydrates or solvates thereof, wherein (a) the compound is a double-gap modified oligonucleotide, the oligonucleotide having a sequence as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5; (b) the double-gap modification refers to that each nucleoside portion in the oligonucleotide is modified in the manner as shown in formula (I); and (c) internucleoside linkages of the oligonucleotide are unmodified or modified internucleoside linkages. The double-gap modified oligonucleotide has remarkably improved antiviral activity. Pl＞SI-P2-S2-P3 (I)

Description

Double-Gap modified oligonucleotides and their applications in anti-hepatitis B and hepatitis D viruses Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a double-Gap modified oligonucleotide and its application in resisting hepatitis B and hepatitis D viruses. Background Art

[0002] Hepatitis B is a viral disease caused by the hepatitis B virus (HBV). It is primarily transmitted through blood, sexual intercourse, and mother-to-child transmission. The World Health Organization estimates that in 2015, over 200 million people worldwide were chronically infected with HBV, and 887,000 people died from complications related to HBV infection. While 90% of adults infected with HBV recover on their own, 90% of infants and young children infected with HBV will develop chronic hepatitis. Chronic HBV infection can lead to liver fibrosis, which can progress to cirrhosis and hepatocellular carcinoma (HCC). Furthermore, studies have shown that hepatitis B increases the risk of pancreatic cancer.

[0003] Hepatitis D virus (HDV) is a satellite virus of HBV. It relies on hepatitis B surface antigen (HBsAg) to form its complete, infectious HDV virion. HDV infection can only occur in patients with concomitant HBV infection. HDV / HBV co-infection can cause significant complications and significantly increases the rate of progression from liver fibrosis to cirrhosis. For patients with chronic HDV infection, interferon therapy is the only available treatment option, with no marketed drugs that directly target HDV. Existing treatments are ineffective and have significant side effects.

[0004] HBV attaches to the surface of hepatocytes via low-affinity receptors and then enters the cell via endocytosis, using specific receptors on the hepatocyte membrane. The nucleocapsid disintegrates and imports the rcDNA into the host cell nucleus. Within the nucleus, the rcDNA is converted into covalently closed circular DNA (cccDNA) by the cell's DNA repair machinery. cccDNA is the intracellular storage form of HBV genetic material and serves as the primary template for HBV transcription. Host cells recognize promoters and enhancers on the cccDNA and transcribe mRNAs of 3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb, of which the 3.5 kb is the pregenomic RNA (pgRNA). The mRNA enters the cytoplasm and is translated into viral proteins, including core antigen (HBcAg), e antigen (HBeAg), surface antigen (HBsAg), x protein (HBx), and polymerase. Polymerase is used as a template for reverse transcription to synthesize the negative-sense strand of HBV DNA using the pgRNA. The negative-sense strand then serves as a template for further synthesis of a partial positive-sense strand, forming rcDNA. At the same time, HBcAg assembles into a nucleocapsid that encapsulates the rcDNA to form the viral core particle. After HBsAg synthesis, it polymerizes in the endoplasmic reticulum and is transported to the Golgi apparatus to package the viral core particle. The assembled viral particles are finally secreted out of the cell by budding.

[0005] When HBV infects human hepatocytes, it primarily produces two distinct types of particles. One is the Dane particle, which is the complete HBV virus itself. It includes a viral nucleocapsid composed of hepatitis B core antigen (HBcAg) and viral nucleic acid (rcDNA), and a viral envelope composed of hepatitis B surface antigen (HBsAg). The other is the subviral particle (SVP), a non-infectious particle composed of lipids, cholesterol, cholesterol esters, and hepatitis B surface antigen (HBsAg). The HBsAg contained in the SVP accounts for the vast majority (>99.9%) of the HBsAg in a patient's blood. HBV-infected hepatocytes also secrete an e-antigen (HBeAg) into the blood. Hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), hepatitis B core antibody (HBcAb), hepatitis B e antigen (HBeAg), and hepatitis B e antibody (HBeAb) are important molecular markers for evaluating drug interventions against the virus.

[0006] In patients with chronic HBV infection, a large amount of hepatitis B surface antigen (HBsAg) in the form of subviral particles (SVPs) in the blood can neutralize specific hepatitis B surface antibodies (HBsAbs) secreted by B lymphocytes, leading to immune tolerance. However, the small number of HBV virions can evade immune detection, which may be a key factor in the maintenance of chronic HBV infection. Hepatitis B surface antigen (HBsAg) seroconversion (the clearance of HBsAg from the blood and the appearance of free HBsAbs) is a recognized prognostic indicator of functional control of viral infection with treatment. Another key factor in the maintenance of HBV chronic infection is the synthesis of a stable circular DNA reservoir, HBV covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes with the help of host DNA repair enzymes. cccDNA is stable and continuously replenished in hepatocytes, producing HBV viral nucleic acid (rcDNA) and mRNA encoding all viral antigens through transcription and reverse transcription. Transcriptional inhibition or elimination of cccDNA is crucial for the cure or functional cure of HBV infection. Long-term treatment with nucleoside (acid) analogs cannot completely eliminate cccDNA or inhibit its transcription. Therefore, hepatitis B surface antigen (HBsAg) expression levels are almost unaffected by nucleoside (acid) drugs. Immune regulation can mediate humoral and cellular immunity, thereby inhibiting cccDNA transcription or clearing infected cells. However, a high antigen load can greatly inhibit this immune process. Therefore, significantly reducing antigen, especially hepatitis B surface antigen (HBsAg), combined with immune regulation is an effective means to help patients achieve lasting immune control.

[0007] The drugs used clinically to treat hepatitis B mainly include interferons and nucleoside (acid) drugs. Interferon drugs include ordinary interferon and polyethylene glycol-modified long-acting interferon, the latter including PEG-IFNα-2a and PegIntron (PEG-IFNα-2b). Nucleoside (acid) drugs include lamivudine, telbivudine, adefovir dipivoxil, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), entecavir, etc. These nucleoside drugs can effectively control the replication of the virus and improve liver function, and are therefore the most widely used. Interferon needs to be administered by injection, individual reactions vary greatly, adverse reactions are obvious, and the efficacy is poor. Nucleoside drugs only act on the viral replication process from pgRNA to rcDNA, and have no inhibitory effect on other links in the hepatitis B virus life cycle. Even with long-term treatment, the HBeAg conversion rate remains low, and very few patients achieve HBsAg conversion. After 48 weeks of treatment with entecavir (354 patients) and tenofovir (176 patients), the HBsAg conversion rates were 2% and 3.2% in HBeAg-positive patients, respectively, and 0.3% and 0% in HBeAg-negative patients, respectively. Because existing treatment options cannot cure hepatitis B, they require long-term medication, potentially exposing patients to significant side effects. For example, long-term use of adefovir dipivoxil and tenofovir disoproxil fumarate can cause nephrotoxicity and bone toxicity. Existing drug therapies or combination therapies fail to induce an effective immune response or HBsAg seroconversion that can provide sustained control of infection or a functional cure, except in a small percentage of patients (<3%). Curing or functionally curing chronic HBV infection is a huge unmet clinical need.

[0008] In summary, there is an urgent need to discover and develop new antiviral treatments in this field, especially new therapies that can effectively inhibit the production of hepatitis B virus antigens HBsAg and / or HBeAg and improve their seroconversion rate.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a new compound for anti-hepatitis B virus / hepatitis D virus treatment.

[0011] In a first aspect of the present invention, there is provided a compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound is a double-Gap modified oligonucleotide;

[0012] in,

[0013] (a) The oligonucleotide has a sequence as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5;

[0014] (b) The double-Gap modification means that each nucleoside portion in the oligonucleotide is modified in the manner shown in Formula I;

[0015] P1-S1-P2-S2-P3 (I)

[0016] In Formula I,

[0017] P1 represents a modified region located at the 5′ end consisting of at least one consecutive nucleotide;

[0018] P2 represents a modified region consisting of at least one consecutive nucleotide starting from the 3′ end of the S1 region;

[0019] P3 represents a modified region located at the 3′ end consisting of at least one consecutive nucleotide;

[0020] S1 represents a spacer region consisting of at least one consecutive nucleotide; and

[0021] S2 represents a spacer region consisting of at least one consecutive nucleotide starting from the 3′ end of the P2 region;

[0022] and,

[0023] In the modified region, all nucleoside moieties have sugar modifications, and all nucleoside moieties optionally have base modifications; and

[0024] In the spacer region, all nucleoside moieties are free of sugar modifications and all nucleoside moieties are optionally base modified;

[0025] as well as,

[0026] (c) the internucleoside bonds of the oligonucleotide are unmodified or modified internucleoside bonds; and the modified internucleoside bonds refer to that some or all of the phosphodiester internucleoside bonds in the oligonucleotide are independently replaced by internucleoside bonds selected from the group consisting of phosphorothioate internucleoside bonds, phosphorodithioate internucleoside bonds, or a combination thereof.

[0027] In another preferred embodiment, each nucleoside in P1, P2 and P3 is a modified nucleoside (preferably, the modified nucleoside comprises a modified sugar and a modified or unmodified base); and each nucleoside in S1 and S2 is an unmodified nucleoside or a sugar in an unmodified nucleoside.

[0028] In another preferred embodiment, the glycosyl modification is selected from: 2'-O-methylated glycosyl modification, 2'-O-methoxyethylated glycosyl modification, or a combination thereof; preferably, it is 2'-O-methoxyethylated glycosyl modification (moe).

[0029] In another preferred embodiment, the base modification is a methyl modification (5-methyl, 5Me) at the 5-position of cytosine (C) base.

[0030] In another preferred embodiment, the internucleoside bond of the oligonucleotide is a phosphorothioate internucleoside bond, a phosphorodithioate internucleoside bond, or a combination thereof.

[0031] In another preferred embodiment, the internucleoside bond of the oligonucleotide is a phosphorothioate bond.

[0032] In another preferred embodiment, P1 represents a modified region located at the 5' end consisting of 2 to 6 (preferably 3 to 4) consecutive nucleotides.

[0033] In another preferred embodiment, P2 represents a modified region consisting of 1 to 5 (preferably 1, 2, 3 or 4) consecutive nucleotides.

[0034] In another preferred embodiment, P3 represents a modified region consisting of 2 to 6 (preferably 3 or 4) consecutive nucleotides.

[0035] In another preferred embodiment, S1 represents a spacer region consisting of 3 to 7 (preferably 4, 5, 6 or 7, more preferably 4, 5 or 6, and most preferably 5 or 6) consecutive nucleotides.

[0036] In another preferred embodiment, S2 represents a spacer region consisting of 3 to 7 (preferably 4, 5, 6 or 7, more preferably 4, 5 or 6, most preferably 5 or 6) consecutive nucleotides.

[0037] In another preferred embodiment, the oligonucleotide described in (a) has a sequence as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; (b) the double-Gap modification refers to that each nucleoside portion in the oligonucleotide is modified in a manner as shown in Formula I; P1-S1-P2-S2-P3 (I); in Formula I, P1 represents a modified region located at the 5' end consisting of 2 to 6 consecutive nucleotides; P2 represents a modified region consisting of 1 to 5 (preferably 1, 2, 3 or 4; more preferably, 2, 3 or 4, most preferably, 3 or 4) consecutive nucleotides; P3 represents a modified region consisting of 2 to 6 consecutive nucleotides; S1 represents a spacer region consisting of 4, 5 or 6 consecutive nucleotides; S2 represents a spacer region consisting of 4, 5 or 6 consecutive nucleotides; and in the modified region, all nucleosides (c) the internucleoside bonds of the oligonucleotide are modified internucleoside bonds; and the modified internucleoside bonds refer to that some or all of the phosphodiester internucleoside bonds in the oligonucleotide are independently replaced by internucleoside bonds selected from the group consisting of phosphorothioate internucleoside bonds, phosphorodithioate internucleoside bonds, or combinations thereof.

[0038] In another preferred embodiment, the oligonucleotide described in (a) has a sequence as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; (b) the double-Gap modification refers to that each nucleoside portion in the oligonucleotide is modified in a manner as shown in Formula I; P1-S1-P2-S2-P3 (I); in Formula I, P1 represents a modified region located at the 5' end consisting of 3 to 4 consecutive nucleotides; P2 represents a modified region consisting of 1, 2, 3 or 4 (preferably, 2, 3 or 4; more preferably, 3 or 4) consecutive nucleotides; P3 represents a modified region consisting of 3 or 4 consecutive nucleotides; S1 represents a spacer region consisting of 5 or 6 consecutive nucleotides; S2 represents a spacer region consisting of 5 or a spacer region consisting of 6 consecutive nucleotides; and, in the modified region, all nucleoside moieties are sugar-modified, and all nucleoside moieties are optionally base-modified; and in the spacer region, all nucleoside moieties are not sugar-modified, and all nucleoside moieties are optionally base-modified; wherein the sugar modification is selected from: 2'-O-methylated sugar modification, 2'-O-methoxyethylated sugar modification, or a combination thereof; the base modification is a methyl modification at position 5 of cytosine (C); and, (c) the internucleoside bonds of the oligonucleotide are all phosphorothioate internucleoside bonds.

[0039] In another preferred embodiment, the oligonucleotide has a sequence as shown in SEQ ID NO.2 or SEQ ID NO.5.

[0040] In another preferred embodiment, the oligonucleotide has the sequence shown in SEQ ID NO.2.

[0041] In another preferred embodiment, when the oligonucleotide has the sequence shown in SEQ ID NO.2, the sugar modification is a 2'-O-methoxyethyl sugar modification; and / or, when the oligonucleotide has the sequence shown in SEQ ID NO.5, the sugar modification is a 2'-O-methyl sugar modification.

[0042] In another preferred embodiment, the oligonucleotide has a sequence as shown in SEQ ID NO. 2, and the sugar modification is a 2'-O-methoxyethylated sugar modification.

[0043] In another preferred embodiment, the oligonucleotide described in (a) has a sequence as shown in SEQ ID NO.2; (b) the double-Gap modification refers to that each nucleoside portion in the oligonucleotide is modified in a manner as shown in Formula I; P1-S1-P2-S2-P3 (I); in Formula I, P1 represents a modified region located at the 5' end consisting of 3 to 4 consecutive nucleotides; P2 represents a modified region consisting of 1, 2, 3 or 4 (preferably 2, 3 or 4; more preferably, 3 or 4) consecutive nucleotides; P3 represents a modified region consisting of 3 or 4 consecutive nucleotides; S1 represents a spacer consisting of 5 or 6 consecutive nucleotides domain; S2 represents a spacer region consisting of 5 or 6 consecutive nucleotides; and, in the modified region, all nucleoside moieties are sugar-modified, and all nucleoside moieties are optionally base-modified; and in the spacer region, all nucleoside moieties are not sugar-modified, and all nucleoside moieties are optionally base-modified; wherein the sugar group is a 2'-O-methoxyethylated sugar group modification, or a combination thereof; the base modification is a methyl modification at position 5 of the cytosine (C) base; and, (c) the internucleoside bonds of the oligonucleotide are all phosphorothioate internucleoside bonds.

[0044] In another preferred embodiment, the compound is selected from Table A and Table B

[0045] Table A

[0046] In Table A, A / T / G / C represent conventional unmodified deoxyribonucleotide residues; A / T / G / C represent 2'-O-methyl modified nucleobases; and the internucleoside bonds of each oligonucleotide are phosphorothioate internucleoside bonds;

[0047] Table B

[0048] In Table B, A / T / G represent conventional unmodified deoxyribonucleotide residues and C represent 5-methyl modified deoxyribonucleotide residues; A / T / G / C represent 2'-O-methoxyethyl modified nucleotide bases; and the internucleoside bonds of each oligonucleotide are phosphorothioate internucleoside bonds.

[0049] In another preferred embodiment, the compound is selected from: PA0088, PA0089, PA0114, PA0090, PA0115, PA0091, PA0116, PA0092, PA0093, PA0094 and PA0095.

[0050] In another preferred embodiment, the compound is PA0093 or PA0094.

[0051] In the second aspect of the present invention, a pharmaceutical composition is provided, comprising the compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable carrier.

[0052] In the third aspect of the present invention, there is provided a compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as described in the third aspect for use in the preparation of a drug for treating and / or preventing diseases associated with hepatitis B virus or hepatitis D virus.

[0053] In another preferred embodiment, the disease includes one or more of the following diseases: diseases related to hepatitis B virus infection, and diseases related to co-infection of hepatitis B virus and hepatitis D virus.

[0054] In another preferred embodiment, the disease can be an acute disease or a chronic disease.

[0055] In another preferred embodiment, the disease includes hepatitis B, hepatitis D, liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC), or a combination thereof.

[0056] In another preferred embodiment, the disease includes acute or chronic liver disease.

[0057] In the fourth aspect of the present invention, a method for treating and / or preventing diseases related to hepatitis B virus or hepatitis D virus is provided, the method comprising the steps of administering a safe and effective amount of the compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition as described in the second aspect to a subject in need.

[0058] In another preferred embodiment, the disease is as defined in the third aspect.

[0059] In another preferred embodiment, the method comprises administering a safe and effective amount of the compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition as described in the second aspect to a subject in need thereof by intravenous injection and / or subcutaneous injection.

[0060] In another preferred embodiment, the subject is a mammal; preferably, the subject is selected from humans, rats, mice, or a combination thereof.

[0061] In the fifth aspect of the present invention, a method for regulating the expression of HBV DNA / RNA, HBsAg and / or HBeAg is provided, the method comprising the steps of: contacting a subject with the compound as described in the first aspect, or a pharmaceutically acceptable salt, hydrate or solvate thereof, thereby regulating the expression of HBV DNA / RNA, HBsAg and / or HBeAg.

[0062] In another preferred embodiment, the method is non-therapeutic in vitro.

[0063] In another preferred embodiment, the object is a cell.

[0064] In another preferred embodiment, the regulation refers to inhibiting the expression of HBV DNA / RNA, HBsAg and / or HBeAg, or reducing the level of HBV RNA and / or HBeAg outside the cell (such as the cell culture medium).

[0065] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figures 1 and 2 show the relative inhibition rates of antisense oligonucleotides with different Gap modification patterns on the expression and secretion of HBsAg by HepG2.2.1.5.

[0067] FIG3 shows the antiviral effects of antisense oligonucleotides with different Gap modification patterns in HepG2.2.15 cell lines.

[0068] FIG4 shows the efficacy of antisense oligonucleotides with Gap modification patterns in animals.

[0069] FIG5 shows the body weight gain of animals administered with antisense oligonucleotides with Gap modification pattern. DETAILED DESCRIPTION

[0070] After extensive and in-depth research, the inventors unexpectedly discovered that modifying an oligonucleotide having a specific sequence (particularly the sequence shown in SEQ ID NO. 2) with a double-Gap modification can enhance, and even significantly enhance, the antiviral activity of the antisense oligonucleotide and the HBsAg concentration at the cellular level. Based on this discovery, the inventors completed the present invention.

[0071] the term

[0072] The term "oligonucleotide" refers to an oligomer of nucleotides in ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA). The term includes oligonucleotides consisting of modified or unmodified core bases, modified or unmodified sugars (ribose or deoxyribose) and modified or unmodified internucleoside bonds (phosphodiester bonds), and one or more bases in the oligonucleotides may be optionally replaced (e.g., T is replaced with a modified or unmodified U), as well as functionally similar oligonucleotides with non-naturally occurring parts. In particular, modified or substituted oligonucleotides may be superior to natural forms due to desirable properties such as: for example, reduced immunoreactivity, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and / or improved stability to nuclease-mediated degradation. In the present invention, oligonucleotides may be single-stranded or double-stranded, including single-stranded molecules such as antisense oligonucleotides (ASOs), aptamers, miRNAs, and double-stranded molecules such as small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs). Oligonucleotides can include various modifications, such as stabilizing modifications, and thus can have at least one modification or at least one modifying group on the phosphodiester linkage (partially or fully) and / or on the sugar and / or base. For example, an oligonucleotide can include, but is not limited to, one or more modifications, or can be fully modified so as to contain all linkages, sugars, or bases with the modifications described (that is, each phosphodiester linkage, sugar, and base comprising the oligonucleotide is unmodified, partially modified, or fully modified). In the present invention, the modified internucleoside linkage can be a phosphorothioate and / or phosphorodithioate linkage. Other modifications useful in the present invention include, but are not limited to, modifications at the 2' position of the sugar, including 2'-O-alkyl modifications (such as 2'O-methyl modifications, 2'-O-methoxyethyl modifications (2'MOE)), 2'-amino modifications, 2'-halogen modifications (such as 2'-fluoro substitutions); acyclic nucleotide analogs. Other 2'-position modifications are also well known in the art and can be used, such as locked nucleic acids. Specifically, the oligonucleotide has modified bonds throughout or has each modified bond, for example, phosphorothioate; with 3'-cap and / or 5'-cap: including terminal 3'-5' bonds. Base modification can include 5' methylation (5' methylcytosine) of cytosine bases and / or 4' thiolation (4' thiouracil) of uracil bases. When the synthesis conditions are chemically compatible, different chemically compatible modified bonds can be combined, for example, there are oligonucleotides with phosphorothioate bonds, 2' ribose modifications (e.g., 2' O-methylation) and modified bases (e.g., 5' methylcytosine). All of these different modifications (e.g., each phosphorothioate bond, each 2' modified ribose and each modified base) can be utilized to further fully modify the oligonucleotide.

[0073] For the sake of brevity, unless otherwise specified, the oligonucleotides represented herein in the form of DNA / RNA sequences or defined in the manner shown in, for example, SEQ ID NO. 2 include modified or unmodified oligonucleotides having the sequence.

[0074] The term "antisense oligonucleotide" refers to a single-stranded oligonucleotide having a nucleic acid base sequence that allows hybridization to the corresponding segment of a target nucleic acid.

[0075] As used herein, the term "nt" refers to a nucleotide.

[0076] The term "complementary" refers to the ability of the nucleic acid base sequence of the antisense oligonucleotide to accurately base pair with the corresponding nucleic acid base sequence in the target nucleic acid, and is mediated by hydrogen bonding between the corresponding nucleic acid bases, such as adenine base pairing with thymine (or uracil) and guanine pairing with cytosine.

[0077] In the present invention, the terms "comprising", "including" or "comprising" indicate that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the term "comprising".

[0078] In the present invention, the term "pharmaceutically acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, has a reasonable benefit / risk ratio.

[0079] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. The precise effective amount for a given subject depends on the subject's size and health, the nature and severity of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation and is within the judgment of the clinician.

[0080] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed between a compound of the present invention and a base that is suitable for use as a pharmaceutical.

[0081] Unless otherwise specified, all compounds mentioned in the present invention are intended to include all possible optical isomers, such as single chiral compounds or mixtures of various chiral compounds (i.e., racemates). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0082] Some compounds of the present invention may be crystallized or recrystallized with water or various organic solvents, in which case various solvates may be formed. The solvates of the present invention include stoichiometric solvates such as hydrates, and also compounds containing variable amounts of water formed when prepared by low-pressure sublimation drying.

[0083] The present invention provides methods, compounds and compositions for regulating the expression of HBV DNA / RNA and HBsAg and HBeAg. In an embodiment, the compound suitable for regulating the expression of HBV DNA / RNA and HBsAg and HBeAg is an antisense oligonucleotide.

[0084] In certain embodiments, the regulation can be performed in cells. In certain embodiments, the regulation is performed in an animal. In certain embodiments, the animal is a human. In certain embodiments, HBV RNA levels are reduced. In certain embodiments, HBV DNA levels are reduced. In certain embodiments, HBsAg levels are reduced. In certain embodiments, HBeAg levels are reduced. The reduction occurs in a dose- and time-dependent manner.

[0085] Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, the HBV-related diseases, disorders, and conditions are acute or chronic liver diseases. In certain embodiments, the liver diseases, disorders, and conditions include hepatitis B, hepatitis D, liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and the like.

[0086] In certain embodiments, the treatment method comprises administering an HBV antisense oligonucleotide to an individual in need thereof by intravenous or subcutaneous injection.

[0087] Oligonucleotides (antisense oligonucleotides)

[0088] The present invention unexpectedly discovered oligonucleotides (or antisense oligonucleotides) that can inhibit the expression of viral proteins such as hepatitis B virus (HBV) DNA / RNA and hepatitis B virus (HBV) surface antigen (HBsAg) and e antigen (HBeAg). Typically, the antisense oligonucleotides provided by the present invention can complement a pan-genotypic conserved sequence in the HBV genome. For example, the antisense oligonucleotides provided by the present invention can complement a pan-genotypic conserved sequence such as SEQ ID NO. 6 in the HBV genome (genotype D) as shown in SEQ ID NO. 1, or a sequence having greater than 96% or greater than 98% homology to the sequence.

[0089] Preferably, the sequence of the oligonucleotide of the present invention is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, more preferably, as shown as SEQ ID NO.2.

[0090] In another preferred embodiment, the oligonucleotide is complementary to at least a portion of the fragment in SEQ ID NO.6.

[0091] In another preferred embodiment, the oligonucleotide is complementary to at least a portion of SEQ ID NO. 6 and at least a portion of SEQ ID NO. 1.

[0092] In another preferred embodiment, the oligonucleotide is at least 96% complementary to SEQ ID NO. 6.

[0093] Double-Gap modified oligonucleotides

[0094] The present invention also provides modified antisense oligonucleotides. The preferred antisense oligonucleotides provided by the present invention are modified by a double-Gap design, i.e., the ribose of the 5' and 3' terminal nucleotides of the oligonucleotide is modified, thereby increasing affinity with the target RNA, enhancing efficacy, reducing the effective dose, and increasing the safety window; at the same time, the ribose modification at both ends further increases the stability of the oligonucleotide. The more preferred modified antisense oligonucleotides provided by the present invention are phosphorothioate-modified oligonucleotides, wherein the phosphorothioate modification increases the stability of the oligonucleotide in the body, enhances plasma protein binding, and facilitates the distribution of the oligonucleotide to target tissues such as the liver. The most preferred antisense oligonucleotides provided by the present invention are double-Gap antisense oligonucleotides, which are modified at the 5' and 3' ends of the oligonucleotide and in the middle of the sequence, and the bases of the cytosine nucleotides in the CpG sequence (i.e., cytosine (C)-phosphate (P)-guanine (G)) are modified, thereby further increasing the affinity of the oligonucleotide and reducing the off-target toxicity caused by its activation of innate immune Toll-like receptors.

[0095] In a specific embodiment, a compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof is provided, wherein the compound is a modified or unmodified oligonucleotide, and the length of the oligonucleotide is 14 to 26 nt (e.g., 16 to 24 nt, preferably, 18 to 22 nt, most preferably, 20 nt); and the oligonucleotide has the structural characteristics of formula I

[0096] P1-S1-P2-S2-P3 Formula I

[0097] In the formula, P1, P2 and P3 are one or more modified nucleotides, S1 and S2 are deoxyribonucleosides, the spacer region represented by S1 is Gap1, and the spacer region represented by S2 is Gap2.

[0098] Wherein, the modification is one or more modifications selected from the following group:

[0099] (i) modification of nucleosides; the modification of nucleosides includes 2'-O-methylation of sugars, 2'-O-methoxyethylation of sugars, and / or methylation of the 5-position of cytosine;

[0100] (ii) modification of internucleoside bonds; the modification of internucleoside bonds is that some or all of the internucleoside bonds in the oligonucleotide are replaced by phosphorothioate internucleoside bonds and / or phosphorodithioate internucleoside bonds.

[0101] In another preferred embodiment, each nucleoside in P1, P2 and P3 is a modified nucleoside (preferably, the modified nucleoside comprises a modified sugar and a modified or unmodified base);

[0102] Each nucleoside in S1 and S2 is an unmodified nucleoside or a sugar in an unmodified nucleoside;

[0103] The nucleoside moieties of the oligonucleotide are independently linked by phosphodiester bonds or phosphorothioate bonds (preferably, both are linked by phosphorothioate bonds).

[0104] In another preferred embodiment, the internucleoside bonds in the modified oligonucleotide are at least partially modified internucleoside bonds (i.e., phosphorothioate internucleoside bonds).

[0105] In another preferred embodiment, all the internucleoside bonds in the modified oligonucleotide are modified internucleoside bonds (i.e., phosphorothioate internucleoside bonds).

[0106] In another preferred embodiment, the P1 corresponds to a region of 2-6 nt at the 5' end where the modification occurs.

[0107] In another preferred embodiment, the S1 corresponds to L1 consecutive nucleotides without nucleoside modification in the spacer region Gap1, wherein L1 is a positive integer of 3-7, preferably 4, 5 or 6, and more preferably 5 or 6.

[0108] In another preferred embodiment, the P2 corresponds to a modified region of 2-5 nt.

[0109] In another preferred embodiment, the S2 corresponds to L2 consecutive nucleotides without nucleoside modification in the spacer region Gap2, wherein L1 is a positive integer of 3-7, preferably 4, 5 or 6, and more preferably 5 or 6.

[0110] In another preferred embodiment, the P3 corresponds to a region of 2-6 nt at the 3' end.

[0111] In another preferred embodiment, P1 is 3, 4 or 5 nt in length, S1 is 4, 5 or 6 nt in length, P2 is 2, 3 or 4 nt in length, S2 is 4, 5 or 6 nt in length, and / or P3 is 3, 4 or 5 nt in length.

[0112] In another preferred embodiment, in P1, P2 and P3, the modified nucleosides are each independently a sugar-modified nucleoside, or a sugar-modified nucleoside and a base-modified nucleoside.

[0113] In another preferred embodiment, the sugar-modified nucleoside refers to a nucleoside in which the sugar group contains a 2'-O-methyl group or a 2'-O-methoxyethyl group.

[0114] In another preferred embodiment, the nucleoside in which both the sugar group and the base are modified refers to a nucleoside in which the sugar group contains a 2'-O-methyl group or a 2'-O-methoxyethyl group and the base is 5-methylcytosine.

[0115] In another preferred embodiment, in P1, P2 and P3, the modified nucleosides are each independently a sugar-modified nucleoside, or a nucleoside moiety in which both the sugar and the base are modified when the base is cytosine;

[0116] The sugar-modified nucleoside refers to a nucleoside whose sugar group contains a 2'-O-methyl group or a 2'-O-methoxyethyl group, and the sugar-and-base-modified nucleoside refers to a nucleoside whose sugar group contains a 2'-O-methyl group or a 2'-O-methoxyethyl group and whose base is 5-methylcytosine.

[0117] In another specific embodiment, the present invention provides an oligonucleotide, or an optical isomer, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the antisense oligonucleotide sequence is selected from: SEQ ID NO. 2-5, and wherein one or more Ts are optionally substituted by U.

[0118] In another preferred embodiment, the compound is PA0093 or PA0094.

[0119] In another preferred embodiment, the modified oligonucleotide is PA0088, the sequence of which is SEQ ID No. 2, and the sugar modification and base modification are: 5'-Gmoe* 5Me Cmoe*Amoe*Gmoe*dA*dG*dG*dT*dG*Amoe*dA*dG* 5Me dC*dG*dA*dA*Gmoe*Tmoe*Gmoe* 5MeCmoe-3', where *: phosphorothioate; Amoe, Gmoe, 5Me Cmoe, Tmoe: 2'-O-methoxyethyl modified monomers; dA, dG, 5Me dC, dT: natural DNA monomers; 5Me : Methyl modification at position 5 of the base.

[0120] In another preferred embodiment, the modified oligonucleotide is PA0089, the sequence of which is SEQ ID No. 2, and the sugar modification and base modification are: 5'-Gmoe* 5Me Cmoe*Amoe*dG*dA*dG*dG*dT*dG*Amoe*dA*dG* 5Me dC*dG*dA*dA*dG*Tmoe*Gmoe* 5Me Cmoe-3', where *: phosphorothioate; Amoe, Gmoe, 5Me Cmoe, Tmoe: 2'-O-methoxyethyl modified monomers; dA, dG, 5Me dC, dT: natural DNA monomers; 5Me : Methyl modification at position 5 of the base.

[0121] Preparation of antisense oligonucleotides

[0122] The antisense oligonucleotides of the present invention can be prepared and synthesized using conventional synthesis methods in the oligonucleotide industry. For example, phosphorothioate bonds can be synthesized using standard phosphoramidite chemical synthesis on equipment such as the GE OP100, using 1,2-benzodithiol-3-one-1,1-dioxide as the oxidizing agent instead of iodine.

[0123] Pharmaceutical compositions and methods of administration

[0124] Because the compounds of the present invention (or modified or unmodified oligonucleotides of the present invention) have excellent ability to inhibit hepatitis B virus DNA replication, the compounds of the present invention and their isomers (such as optical isomers), crystal forms, solvates, pharmaceutically acceptable inorganic salts, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent, and alleviate diseases associated with or caused by hepatitis B virus (i.e., hepatitis B virus) infection or diseases associated with or caused by co-infection of hepatitis B virus and hepatitis D virus. These diseases can be acute or chronic. According to the prior art, the compounds of the present invention can be used to treat the following diseases: hepatitis B; pancreatic cancer, cirrhosis, and hepatocellular carcinoma (such as pancreatic cancer, cirrhosis, and hepatocellular carcinoma caused by chronic hepatitis B).

[0125] The pharmaceutical composition provided by the present invention comprises a safe and effective amount of a compound of the present invention or other pharmacologically acceptable forms thereof (such as optical isomers, pharmaceutically acceptable salts, hydrates or solvates thereof), and a pharmaceutically acceptable adjuvant, diluent or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-500 mg of the compound of the present invention per dose. Preferably, the "one dose" is an ampoule or a vial.

[0126] "Pharmaceutically acceptable carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0127] Application method

[0128] The compounds of the present invention and compositions containing the same can be administered by any suitable means, for example, oral ingestion; oral inhalation; subcutaneous, intravenous injection or infusion. The compounds of the present invention or compositions containing the same can be administered in the form of dosage unit formulations containing non-toxic pharmaceutically acceptable carriers or diluents; or in the form of immediate release or sustained release formulations.

[0129] The antisense oligonucleotides of the present invention are administered to humans in a suggestive and effective dosage regimen, following the dosage regimen commonly used for other antisense oligonucleotides; the conventional use of 100-500 mg of the compound administered parenterally weekly is well established in the art.

[0130] In light of the disclosure presented herein, it is useful to treat subjects having HBV infection or HBV / HDV co-infection using pharmaceutically acceptable antisense oligonucleotide formulations.

[0131] The main advantages of the present invention include:

[0132] (a) The double-Gap modified compounds or oligonucleotides of the present invention have significantly improved antiviral ability compared to unmodified or non-double-Gap modified oligonucleotides

[0133] (b) The compounds or oligonucleotides provided by the present invention can effectively inhibit viral gene products at the transcriptional level in vitro, thereby significantly inhibiting hepatitis B virus antigens (such as HBsAg).

[0134] (c) The modified oligonucleotides provided by the present invention have improved in vivo stability through modification and have an ability to inhibit hepatitis B virus antigens (such as HBsAg) that is comparable to or even significantly superior to that of unmodified oligonucleotides.

[0135] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0136] It should be understood that those skilled in the art can obtain the oligonucleotides used in the examples based on the sequences and corresponding modifications described in the examples, according to conventional techniques in the prior art (such as standard solid-phase synthesis methods) and using raw materials that are commercially available or synthesized according to prior art methods (such as modified or unmodified nucleosides).

[0137] Example 1 Antiviral effects of antisense oligonucleotides with different Gap modification patterns in HepG2.2.15 cell lines

[0138] The HepG2.2.1.5 cell line stably expresses and replicates HBV and secretes HBV particles, HBsAg, and HBeAg into the cell supernatant. HepG2.2.1.5 cells were cultured in DMEM:F12 medium (Hyclone) supplemented with 10% FBS (ExCell Bio) and 400 μg / ml G418 and were used after three passages.

[0139] On day 0, siRNA was serially diluted with PBS to two concentrations (final concentrations of 1 nM and 10 nM). HepG2.2.15 cells were washed with DPBS, digested with trypsin, and adjusted to an appropriate cell density using 10% FBS DMEM:F12 medium (without G418). Simultaneously, siRNA was transfected into HepG2.2.15 cells using Lipofectamine RNAiMax and seeded into 96-well plates at a density of 225,000 cells per well in 135 μl of culture medium per well. The cells were cultured in a 5% CO2, 37°C incubator for 3 days. The collected cell supernatant was assayed for HBsAg using ELISA. Cell viability was also assessed using CellTiter-Glo.

[0140] ELISA is briefly described as follows: 50 μl of sample and standard are added to the reaction plate respectively, then 50 μl of enzyme conjugate is added to each well, shaken to mix, incubate at 37°C for 60 minutes, then wash the plate 5 times with washing solution, then add 50 μl of luminescent substrate to each well, mix, react at room temperature in the dark for 10 minutes, and finally detect the chemiluminescence intensity with a microplate reader.

[0141] The CellTiter-Glo assay is briefly described as follows: 100 μl of CTG was added to each well and incubated in the dark for 10 minutes. Chemiluminescence intensity was measured using a microplate reader.

[0142] The antisense oligonucleotides used are shown in Table 1.

[0143] Table 1 Description of oligonucleotides used in Example 1

[0144] A / T / G / C represents conventional unmodified deoxyribonucleotide residues; A / T / G / C represents 2'-O-methyl modified nucleobases; and the internucleoside bonds of each oligonucleotide are phosphorothioate internucleoside bonds.

[0145] The relative inhibition rate of transfected antisense oligonucleotide on the expression and secretion of HBsAg by HepG2.2.1.5 was calculated as follows:

[0146] Relative inhibition rate = 100% * [1-(HBsAg concentration in the treatment group / HBsAg concentration in the blank group)]

[0147] The relative inhibition rates of each antisense oligonucleotide treatment group are shown in Table 2

[0148] Table 2 Relative inhibition rate of oligonucleotides in Example 1 on the expression and secretion of HBsAg by HepG2.2.1.5

[0149] As shown in Table 2 and Figure 1, the addition of double-Gap modification helps improve the relative inhibition rate of HBsAg compared to the single-Gap modified antisense oligonucleotide PA0087. The combined results of the two experiments show that the weighted average of PA0075, PA0077, PA0079, and PA0081 significantly outperforms the single-Gap modified antisense oligonucleotide.

[0150] Example 2 Antiviral effects of antisense oligonucleotides with different Gap modification patterns in HepG2.2.15 cell lines

[0151] The HepG2.2.1.5 cell line stably expresses and replicates HBV and secretes HBV particles, HBsAg, and HBeAg into the cell supernatant. HepG2.2.1.5 cells were cultured in DMEM:F12 medium (Hyclone) supplemented with 10% FBS (ExCell Bio) and 400 μg / ml G418 and were used after three passages.

[0152] On day 0, siRNA was serially diluted with PBS to two concentrations (final concentrations of 10 nM and 30 nM). HepG2.2.15 cells were washed with DPBS, digested with trypsin, and adjusted to an appropriate cell density using 10% FBS DMEM:F12 medium (without G418). Simultaneously, siRNA was transfected into HepG2.2.15 cells using Lipofectamine RNAiMax and seeded into 96-well plates at a density of 225,000 cells per well in 135 μl of culture medium per well. The cells were cultured in a 5% CO2, 37°C incubator for 3 days. The collected cell supernatant was assayed for HBsAg using ELISA. Cell viability was also assessed using CellTiter-Glo.

[0153] ELISA is briefly described as follows: 50 μl of sample and standard are added to the reaction plate respectively, then 50 μl of enzyme conjugate is added to each well, shaken to mix, incubate at 37°C for 60 minutes, then wash the plate 5 times with washing solution, then add 50 μl of luminescent substrate to each well, mix, react at room temperature in the dark for 10 minutes, and finally detect the chemiluminescence intensity with a microplate reader.

[0154] The CellTiter-Glo assay is briefly described as follows: 100 μl of CTG was added to each well and incubated in the dark for 10 minutes. Chemiluminescence intensity was measured using a microplate reader.

[0155] The antisense oligonucleotides used are shown in Table 3.

[0156] Table 3 Description of oligonucleotides used in Example 2

[0157] A / T / G represents conventional unmodified deoxyribonucleotide residues and C represents 5-methyl modified deoxyribonucleotide residues; A / T / G / C represents 2'-O-methoxyethyl modified nucleotide bases; and the internucleoside linkages of each oligonucleotide are phosphorothioate internucleoside linkages.

[0158] The relative inhibition rate of transfected antisense oligonucleotide on the expression and secretion of HBsAg by HepG2.2.1.5 was calculated as follows:

[0159] Relative inhibition rate = 100% * [1-(HBsAg concentration in the treatment group / HBsAg concentration in the blank group)]

[0160] The relative inhibition rates of each antisense oligonucleotide treatment group are shown in Table 4

[0161] Table 4 Relative inhibition rate of oligonucleotides in Example 2 on the expression and secretion of HBsAg by HepG2.2.1.5

[0162] As shown in Table 4 and Figure 2, compared to the single-Gap modified antisense oligonucleotide Bepirovirsen, currently in Phase III clinical trials, the double-Gap modified antisense oligonucleotides significantly improved the relative inhibition rate of HBsAg. The combined results of the two experiments showed that the weighted average of PA0088 and PA0089 significantly outperformed the single-Gap modified antisense oligonucleotide.

[0163] Example 3 Antiviral EC of antisense oligonucleotides with different Gap modification patterns in HepG2.2.15 cell line 50 Value and Security CC 50 value

[0164] The HepG2.2.1.5 cell line stably expresses and replicates HBV and secretes HBV particles, HBsAg, and HBeAg into the cell supernatant. HepG2.2.1.5 cells were cultured in DMEM:F12 medium (Hyclone) supplemented with 10% FBS (ExCell Bio) and 400 μg / ml G418 and were used after three passages.

[0165] On day 0, siRNA was gradiently diluted with PBS to concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.05 nM (Note: GSK-3228836 compound concentrations are: 40 nM, 13.33 nM, 4.44 nM, 1.48 nM, 0.49 nM, 0.16 nM, 0.05 nM and 0.02 nM). HepG2.2.15 cells were washed with DPBS and trypsinized. The cells were then adjusted to an appropriate cell density using 10% FBS DMEM / F12 medium (without G418). Simultaneously, siRNA was transfected into HepG2.2.15 cells using Lipofectamine RNAiMax. The cells were seeded at a density of 225 cells per well in a 96-well plate, with 135 μl of culture medium per well. The cells were incubated in a 5% CO2, 37°C incubator for 3 days. The collected cell supernatant was assayed for HBsAg by ELISA. Cell viability was also assessed using CellTiter-Glo.

[0166] ELISA is briefly described as follows: 50 μl of sample and standard are added to the reaction plate respectively, then 50 μl of enzyme conjugate is added to each well, shaken to mix, incubate at 37°C for 60 minutes, then wash the plate 5 times with washing solution, then add 50 μl of luminescent substrate to each well, mix, react at room temperature in the dark for 10 minutes, and finally detect the chemiluminescence intensity with a microplate reader.

[0167] The CellTiter-Glo assay is briefly described as follows: 100 μl of CTG was added to each well and incubated in the dark for 10 minutes. Chemiluminescence intensity was measured using a microplate reader.

[0168] The antisense oligonucleotides used are shown in Table 5.

[0169] Table 5 Description of oligonucleotides used in Example 3

[0170] A / T / G represents conventional unmodified deoxyribonucleotide residues and C represents 5-methyl modified deoxyribonucleotide residues; A / T / G / C represents 2'-O-methoxyethyl modified nucleotide bases; and the internucleoside linkages of each oligonucleotide are phosphorothioate internucleoside linkages.

[0171] The relative inhibition rate of transfected antisense oligonucleotide on the expression and secretion of HBsAg by HepG2.2.1.5 was calculated as follows:

[0172] Relative inhibition rate = 100% * [1-(HBsAg concentration in the treatment group / HBsAg concentration in the blank group)]

[0173] The relative inhibition rates of each antisense oligonucleotide treatment group are shown in Table 6

[0174] Table 6 Relative inhibition rate and cell viability of the oligonucleotides in Example 3 on the expression and secretion of HBsAg by HepG2.2.1.5

[0175] As shown in Table 6 and Figure 3, the double-Gap modified compounds help to improve the relative inhibition rate of HBsAg. The combined results of the two experiments show that the weighted average of PA0088 and PA0089 is significantly better than the antisense oligonucleotide with a single Gap modification pattern. 50 The EC values ​​of PA0093 and PA0094 were 10.5 and 9.6 times higher than those of Bepirovirsen (GSK-3228836), respectively. 50 The results were 2.78 and 3.59 times higher than those of Bepirovirsen (GSK-3228836), respectively. 50 Significantly higher, with better safety.

[0176] Example 4 Antiviral Effects of Double Gap Antisense Oligonucleotides PA0088, PA0089, and Bepirovirsen in an AAV-HBV Mouse Model

[0177] The dose-dependent antiviral activity of the antisense oligonucleotide PA0020 was evaluated in C57 mice infected with adeno-associated virus carrying HBV 1.3 fold (AAV-HBV, Acanthopanax spp.), which continuously replicates HBV DNA and expresses HBV antigens. The oligonucleotides PA0020 used are shown in Table 7.

[0178] Use 5X10 10 A rAAV8-1.3HBV (Euphorbia acutiloba) was injected into male C57BL / 6 mice via the tail vein to establish a persistent hepatitis B infection mouse model. After confirming stable HBV viral replication, the mice were randomly divided into four groups (5 mice per group) based on body weight. The first group, the control group (Vehicle), was gavaged with normal saline daily. Groups two to four received intraperitoneal injections of Bepirovirsen, PA0088, and PA0089, respectively, with a weekly dosing frequency of 30 mg / kg, 60 mg / kg, 60 mg / kg, and 90 mg / kg*5. Blood was collected 1-2 times a week, and the hepatitis B virus nucleic acid (HBV-DNA) load in the serum was analyzed by qPCR. The HBsAg and HBeAg concentrations in the serum were analyzed by ELISA. The curves are plotted in Figure 4.

[0179] The test results, as shown in Figure 4, showed that the HBsAg and HBV-DNA in the serum of the animals in the control group showed a stable fluctuation; the HBV-DNA in the serum of the animals in the Bepirovirsen, PA0088 and PA0089 groups continued to decline, and the reduction at the end of the administration was >3 log compared with the 0th day. 10 , which is a decrease of >99.9%. HBsAg and HBeAg levels in the serum of animals in the Bepirovirsen, PA0088, and PA0089 groups also continued to decrease, with a decrease of >2 log compared to day 0 at the end of dosing. 10 PA0088 achieved a slightly greater reduction than the control drug, bepirovirsen. Therefore, PA0088 and PA0089 not only reduce HBV DNA but also surface antigen, offering hope for achieving a functional cure for hepatitis B in the future clinical setting. Furthermore, as shown in Figure 5, animals in all groups continued to gain weight. While the control group, bepirovirsen, showed no further weight gain after 42 days of dosing, PA0088 and PA0089 showed greater weight gain, demonstrating that PA0088 and PA0089 are safer than bepirovirsen.

[0180] In particular, the cytosine base of the CpG sequence was further methylated, that is, 2'-O-methoxyethylated-5-methylcytosine was used to replace cytosine to reduce its possibility of activating Toll-like receptors (TLRs), especially TLR9, thereby reducing its risk of immune off-target toxicity.

[0181] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that: The compound is a double-Gap modified oligonucleotide; in, (a) The oligonucleotide has a sequence as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; (b) The double-Gap modification means that each nucleoside portion in the oligonucleotide is modified in the manner shown in Formula I; P1-S1-P2-S2-P3 (I) In Formula I, P1 represents a modified region located at the 5′ end consisting of at least one consecutive nucleotide; P2 represents a modified region consisting of at least one consecutive nucleotide starting from the 3′ end of the S1 region; P3 represents a modified region located at the 3′ end consisting of at least one consecutive nucleotide; S1 represents a spacer region consisting of at least 1 consecutive nucleotide; and S2 represents a spacer region consisting of at least one continuous nucleotide starting from the 3′ end of the P2 region; and, In the modified region, all nucleoside moieties have sugar modifications, and all nucleoside moieties optionally have base modifications; and In the spacer region, all nucleoside moieties are free of sugar modification, and all nucleoside moieties are optionally free of base modification; as well as, (c) the internucleoside bonds of the oligonucleotide are unmodified or modified internucleoside bonds; and the modified internucleoside bonds refer to that some or all of the phosphodiester internucleoside bonds in the oligonucleotide are independently replaced by internucleoside bonds selected from the following groups: phosphorothioate internucleoside bonds, phosphorodithioate internucleoside bonds, or a combination thereof.

2. The compound according to claim 1, characterized in that (a) The oligonucleotide has SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID The sequence shown in NO.5; (b) The double-Gap modification means that each nucleoside portion in the oligonucleotide is modified in the manner shown in Formula I; P1-S1-P2-S2-P3 (I) In Formula I, P1 represents the modified region consisting of 2 to 6 consecutive nucleotides located at the 5′ end; P2 represents a modified region consisting of 2, 3, or 4 consecutive nucleotides; P3 represents a modified region consisting of 2 to 6 consecutive nucleotides; S1 represents a spacer region consisting of 4, 5, or 6 consecutive nucleotides; S2 represents a spacer region consisting of 4, 5, or 6 consecutive nucleotides; and, In the modified region, all nucleoside moieties have sugar modifications, and all nucleoside moieties optionally have base modifications; and In the spacer region, all nucleoside moieties are free of sugar modifications and all nucleoside moieties are optionally free of base modifications; Wherein, the sugar modification is selected from: 2'-O-methylated sugar modification, 2'-O-methoxyethylated sugar modification, or a combination thereof; the base modification is methyl modification of the 5-position of cytosine (C) base; as well as, (c) the internucleoside bonds of the oligonucleotide are modified internucleoside bonds; and the modified internucleoside bonds refer to that some or all of the phosphodiester internucleoside bonds in the oligonucleotide are independently replaced by internucleoside bonds selected from the following groups: phosphorothioate internucleoside bonds, phosphorodithioate internucleoside bonds or a combination thereof.

3. The compound according to claim 1, characterized in that (a) The oligonucleotide has a sequence as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; (b) The double-Gap modification means that each nucleoside portion in the oligonucleotide is modified in the manner shown in Formula I; P1-S1-P2-S2-P3 (I) In Formula I, P1 represents the modified region consisting of 3 to 4 consecutive nucleotides located at the 5′ end; P2 represents a modified region consisting of 3 or 4 consecutive nucleotides; P3 represents a modified region consisting of 3 or 4 consecutive nucleotides; S1 represents a spacer region consisting of 5 or 6 consecutive nucleotides; S2 represents a spacer region consisting of 5 or 6 consecutive nucleotides; and, In the modified region, all nucleoside moieties have sugar modifications, and all nucleoside moieties optionally have base modifications; and In the spacer region, all nucleoside moieties are free of sugar modifications and all nucleoside moieties are optionally free of base modifications; Wherein, the sugar modification is selected from: 2'-O-methylated sugar modification, 2'-O-methoxyethylated sugar modification, or a combination thereof; the base modification is methyl modification of the 5-position of cytosine (C) base; as well as, (c) The internucleoside bonds of the oligonucleotides are all phosphorothioate internucleoside bonds.

4. The compound according to any one of claims 1 to 3, characterized in that The oligonucleotide has the sequence shown in SEQ ID NO.

2.

5. The compound according to claim 1, characterized in that (a) The oligonucleotide has the sequence shown in SEQ ID NO.2; (b) The double-Gap modification means that each nucleoside portion in the oligonucleotide is modified in the manner shown in Formula I; P1-S1-P2-S2-P3 (I) In Formula I, P1 represents the modified region consisting of 3 to 4 consecutive nucleotides located at the 5′ end; P2 represents a modified region consisting of 3 or 4 consecutive nucleotides; P3 represents a modified region consisting of 3 or 4 consecutive nucleotides; S1 represents a spacer region consisting of 5 or 6 consecutive nucleotides; S2 represents a spacer region consisting of 5 or 6 consecutive nucleotides; and, In the modified region, all nucleoside moieties have sugar modifications, and all nucleoside moieties optionally have base modifications; and In the spacer region, all nucleoside moieties are free of sugar modifications and all nucleoside moieties are optionally free of base modifications; Wherein, the sugar modification is 2'-O-methoxyethyl sugar modification; the base modification is methyl modification of the 5-position of cytosine (C) base; as well as, (c) The internucleoside bonds of the oligonucleotides are all phosphorothioate internucleoside bonds.

6. The compound according to claim 1, characterized in that The compound is selected from Table A and Table B Table A In Table A, A / T / G / C represent conventional unmodified deoxyribonucleotide residues; A / T / G / C represent 2'-O-methyl modified nucleoside bases; and the internucleoside bonds of each oligonucleotide are phosphorothioate internucleoside bonds; Table B In Table B, A / T / G represent conventional unmodified deoxyribonucleotide residues and C represent 5-methyl modified deoxyribonucleotide residues; A / T / G / C represent 2'-O-methoxyethyl modified nucleotide bases; and the internucleoside bonds of each oligonucleotide are phosphorothioate internucleoside bonds.

7. The compound according to claim 6, characterized in that The compound is PA0093 or PA0094.

8. A pharmaceutical composition comprising the compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable carrier.

9. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 8 in the preparation of a drug for treating and / or preventing diseases associated with hepatitis B virus or hepatitis D virus.

10. The use according to claim 9, characterized in that The disease includes hepatitis B virus, hepatitis D virus, liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC), or a combination thereof.

11. A method for regulating the expression of HBV DNA / RNA, HBsAg and / or HBeAg, the method comprising the step of contacting a subject with a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, hydrate or solvate thereof, thereby regulating the expression of HBV DNA / RNA, HBsAg and / or HBeAg.

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