Adenine-rich phosphorothioate oligonucleotides and their application in anti-hepatitis virus

Abstract

The present invention discloses an adenine-rich phosphorothioate oligonucleotide and its application in anti-hepatitis virus. Specifically, the present invention discloses a m AAC n A repetitive sequence-rich adenine phosphorothioate oligonucleotide (ARON) is used for inhibiting hepatitis B surface antigen (HBsAg) and for treating hepatitis B virus infection or hepatitis B virus / hepatitis D virus co-infection. The adenine phosphorothioate oligonucleotide (ARON) is 15-40 nucleotides in length and contains adenine nucleotides and cytosine nucleotides, wherein the content of adenine nucleotides exceeds 50%, has high antiviral activity and a sufficiently large safety window.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and in particular to an adenine-rich phosphorothioate oligonucleotide and an application thereof in resisting hepatitis virus. Background Art

[0002] Data released by the World Health Organization show that more than 200 million people were chronically infected with HBV and 887,000 people died from complications caused by HBV infection in 2015. Although some antiviral drugs have been approved for the treatment of HBV infection, the existing drug therapies or combination therapies are unable to induce effective immune responses or HBsAg seroconversion that can provide lasting control of infection or functional cure, except in a small number of patients (<3%).

[0003] 90% of adults infected with HBV can recover on their own, but 90% of infants and young children infected with HBV will develop chronic hepatitis. Chronic HBV infection may lead to liver fibrosis, further developing into cirrhosis and hepatocellular carcinoma (HCC). In addition, studies have shown that hepatitis B increases the risk of pancreatic cancer. Cure or functional cure of chronic HBV infection is a huge unmet clinical need.

[0004] After HBV infects human liver cells, two different particles are mainly produced. One is Dane particles, which are the complete HBV virus itself, including the viral nucleocapsid assembled by hepatitis B core antigen (HBcAg) and viral nucleic acid (RcDNA), and has a viral envelope composed of hepatitis B surface antigen (HBsAg); the other is subviral particles (SVP), which are non-infectious particles composed of lipids, cholesterol, cholesterol esters and hepatitis B surface antigen (HBsAg). The hepatitis B surface antigen contained in SVP accounts for the vast majority of hepatitis B surface antigen in the patient's blood (>99.9%). HBV-infected liver cells 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 the intervention of drugs on the virus.

[0005] Hepatitis D virus (HDV) is a satellite virus of HBV. It relies on hepatitis B surface antigen (HBsAg) to form its complete infectious HDV virus particles. HDV infection can only occur in patients with concomitant HBV infection. HDV / HBV co-infection complications significantly increase the progression rate of liver fibrosis to cirrhosis. For patients with chronic HDV infection, there is currently only one intervention method, interferon treatment, and there are no marketed drugs that directly target the HDV virus. Existing treatments are ineffective and have significant side effects.

[0006] 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, so they are the most widely used. Interferon needs to be injected, individual reactions vary greatly, adverse reactions are obvious, and the efficacy is poor. Nucleoside drugs only act on the replication process of the virus from pgRNA to rcDNA, and have no inhibitory effect on other links in the life cycle of hepatitis B virus. After long-term treatment, the negative conversion rate of hepatitis B e antigen (HBeAg) is still low, and very few patients can turn negative for hepatitis B surface antigen (HBsAg). After 48 weeks of treatment with entecavir (354 cases) and tenofovir (176 cases), the negative conversion rates of hepatitis B surface antigen (HBsAg) in patients with positive hepatitis B e antigen (HBeAg) were 2% and 3.2%, respectively, and the negative conversion rates of hepatitis B surface antigen (HBsAg) in patients with negative hepatitis B e antigen (HBeAg) were 0.3% and 0%, respectively. Since the existing treatment options cannot cure hepatitis B, patients need to take medication for a long time, which may expose patients to major side effects. For example, long-term use of adefovir dipivoxil and tenofovir disoproxil fumarate can cause nephrotoxicity and bone toxicity.

[0007] A large amount of hepatitis B surface antigen (HBsAg) in the form of subviral particles (SVP) in the blood of patients with chronic HBV infection can neutralize specific hepatitis B surface antibodies (HBsAb) secreted by B lymphocytes, thereby leading to immune tolerance, while only a small number of HBV virus particles can escape immune detection, which may be one of the important reasons why HBV maintains chronic infection. Although studies have shown that the three viral antigens hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and hepatitis B e antigen (HBeAg) all have immunosuppressive properties, hepatitis B surface antigen (HBsAg) accounts for the vast majority of all viral antigens in the blood of HBV-infected subjects and may be the most important inducer of host immunity suppression. Hepatitis B surface antigen (HBsAg) seroconversion (HBsAg clearance from the blood and the appearance of free HBsAb) is a recognized prognostic indicator for functional control of viral infection during treatment. Another key reason why HBV maintains the characteristics of chronic infection is that it synthesizes a stable circular DNA repository, namely HBV covalently closed circular DNA (cccDNA), in the nucleus of infected hepatocytes with the help of host DNA repair enzymes. cccDNA can exist stably in hepatocytes for a long time and can be continuously replenished. It can produce HBV viral nucleic acid RcDNA and mRNA required to encode all viral antigens through transcription and reverse transcription. Transcriptional inhibition or elimination of cccDNA is crucial for curing or functionally curing HBV infection. Long-term treatment with nucleoside (acid) analogs cannot completely eliminate cccDNA or inhibit its transcription, so the expression level of hepatitis B surface antigen (HBsAg) is almost unaffected by nucleoside (acid) drugs. Immunomodulation can mediate humoral and cellular immunity, thereby inhibiting cccDNA transcription or eliminating infected cells, but a large antigen load will greatly inhibit the immune process. Therefore, a significant reduction in antigen, especially hepatitis B surface antigen (HBsAg), combined with immunomodulation is an effective means to help patients achieve lasting immune control.

[0008] Currently, there is a lack of phosphorothioate oligonucleotides with high activity and a sufficiently large safety window for the treatment of HBV and HBV / HDV co-infection. Summary of the invention

[0009] The object of the present invention is to provide a phosphorothioate oligonucleotide with high activity and a sufficiently large safety window for use in treating HBV and HBV / HDV co-infection.

[0010] In a first aspect of the present invention, a compound is provided, or an optical isomer, a pharmaceutically acceptable salt, a hydrate, or a solvate thereof, wherein the compound is an adenine-rich phosphorothioate oligonucleotide (ARON), wherein the adenine-rich phosphorothioate oligonucleotide comprises (ARON m AACn Repeating sequences;

[0011] wherein m is independently 0 or 1; n is a positive number ≥ 4; and the length of the adenine-rich phosphorothioate oligonucleotide is 15-40 nucleotides;

[0012] 80-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

[0013] In another preferred embodiment, 95-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

[0014] In another preferred embodiment, all phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

[0015] In another preferred embodiment, the sequence of the adenine-rich phosphorothioate oligonucleotide is (A m AAC n1 (AAAC) (A m AAC n2 , wherein n1 and n2 are each independently an integer of 0-10 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), n3 is 0, 1, 2, 3 or 4, and

[0016] m and n are defined as above.

[0017] In another preferred embodiment, the sequence of the adenine-rich phosphorothioate oligonucleotide is (R)(A m AAC n'2 ,(A m AAC n'1 (R), (R)(CA m AA) n'2 or (CA m AA) n'1 (R), wherein each R is independently selected from: A or C, n'1 and n'2 are each independently an integer of 3-10 (i.e., 3, 4, 5, 6, 7, 8, 9, 10),

[0018] The definition of m is as described above.

[0019] In another preferred embodiment, each A m AAC (or CA m AA) fragments are independently selected from the group consisting of AAAC, AAC, CAAA, CAA.

[0020] In another preferred embodiment, when n is a non-integer, the non-integer part of A mThe AAC fragment is selected from: A, AA, AC, AAA, AAC, CA, CAA.

[0021] In another preferred embodiment, (AAAC) The fragment is selected from the following group: none, C, A, AA, AC, AAA, AAC, AAAC, CA, CAA, CAAA.

[0022] In another preferred embodiment, when n is an integer, (A m AAC) fragment is selected from the group consisting of AAAC, AAC, CAAA, CAA.

[0023] In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide comprises (A m AAC n Repeating sequence, n is a positive number ≥ 4; m is independently 0 or 1, and at least one m is 1; preferably, all m are 1.

[0024] In another preferred embodiment, the length of the adenine-rich phosphorothioate oligonucleotide is 16-36 nucleotides.

[0025] In another preferred embodiment, the length of the adenine-rich phosphorothioate oligonucleotide is 17-33 nucleotides.

[0026] In another preferred embodiment, n is 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9 or 10.

[0027] In another preferred embodiment, the sequences of the adenine-rich phosphorothioate oligonucleotides are as shown in Table A (ie, as shown in SEQ ID NOs: 10-16 and SEQ ID NOs: 7-8).

[0028] In another preferred embodiment, the sequence of the adenine-rich phosphorothioate oligonucleotide (ARON) is the oligonucleotide shown in SEQ ID NO: 10-16.

[0029] In another preferred embodiment, the sequence of the adenine-rich phosphorothioate oligonucleotide (ARON) is the oligonucleotide shown in SEQ ID NO:11.

[0030] In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown in SEQ ID NO: 12.

[0031] In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide represented by SEQ ID NO: 13.

[0032] In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide represented by SEQ ID NO: 14.

[0033] In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide represented by SEQ ID NO: 15.

[0034] In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide represented by SEQ ID NO: 16.

[0035] In another preferred embodiment, at least one 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is modified.

[0036] In another preferred embodiment, the 2' sugar groups in the adenine-rich phosphorothioate oligonucleotides are all modified.

[0037] In another preferred embodiment, the modification of the 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is selected from the following group: 2'-O-alkyl modification, 2'-hydroxyl modification, 2'-amino modification, 2' halogen modification or 2'-O-methoxyethyl (2'MOE) modification, preferably 2'O-methyl modification.

[0038] In another preferred embodiment, in the adenine-rich phosphorothioate oligonucleotide, the sugar group is a deoxyribose group.

[0039] In another preferred embodiment, one or more cytosines in the adenine-rich phosphorothioate oligonucleotide are 5-methylcytosine.

[0040] In another preferred embodiment, the cytosine in the adenine-rich phosphorothioate oligonucleotides are all 5-methylcytosine.

[0041] The second aspect of the present invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of the compound as described in the first aspect, or its optical isomers, pharmaceutically acceptable salts, hydrates or solvates; and a pharmaceutically acceptable adjuvant, diluent or carrier.

[0042] The third aspect of the present invention provides a use of the compound described in the first aspect, or its optical isomers, pharmaceutically acceptable salts, hydrates or solvates, or the use of the pharmaceutical composition described in the second aspect, for preparing a pharmaceutical composition for treating and / or preventing diseases related to viral infection.

[0043] In another preferred embodiment, the diseases include, for example, HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HPV (human papillomavirus) and HIV (human immunodeficiency virus).

[0044] In another preferred embodiment, the infectious disease is a disease related to HBV infection and HBV / HDV co-infection.

[0045] In another preferred embodiment, the disease is selected from: hepatitis B virus infection (HBV), hepatitis C virus infection (HCV), hepatitis D virus infection (HDV).

[0046] In another preferred embodiment, the disease is selected from: HPV, HIV.

[0047] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The antiviral effects of phosphorothioate oligonucleotides of different lengths in the AAV-HBV mouse model were shown;

[0049] Figure 1 A, 1B show the antiviral activity of phosphorothioate oligonucleotides of different lengths administered intraperitoneally once a week to C57 mice infected with AAV-HBV, evaluated by qPCR detection of serum HBV-DNA at the end of treatment;

[0050] Figure 1 C, 1D show the antiviral activity of phosphorothioate oligonucleotides of different lengths administered intraperitoneally once a week to C57 mice infected with AAV-HBV, evaluated by measuring serum HBsAg by ELISA at the end of treatment;

[0051] Figure 1 E, 1F show the antiviral activity of phosphorothioate oligonucleotides of different lengths administered intraperitoneally twice weekly to C57 mice infected with AAV-HBV, evaluated by qPCR detection of serum HBV-DNA at the end of treatment;

[0052] Figure 1 G, 1H show the antiviral activity of phosphorothioated oligonucleotides of different lengths administered intraperitoneally twice weekly to C57 mice infected with AAV-HBV, evaluated by measuring serum HBsAg by ELISA at the end of treatment.

[0053] Figure 2 The changes of serum biochemical indexes albumin (ALB) and creatinine (CRE) in C57 mice infected with AAV-HBV after injection of phosphorothioate oligonucleotides of different lengths.

[0054] Figure 3 The antiviral effects of phosphorothioate oligonucleotides with different base compositions in the AAV-HBV mouse model were shown.

[0055] Figure 3 A, 3B show the antiviral activity of phosphorothioate oligonucleotides with different base compositions administered intraperitoneally once a week to C57 mice infected with AAV-HBV, evaluated by qPCR detection of serum HBV-DNA at the end of treatment;

[0056] Figure 3 C, 3D show the antiviral activity of phosphorothioate oligonucleotides with different base compositions administered intraperitoneally once a week to C57 mice infected with AAV-HBV, and evaluated by measuring serum HBsAg by ELISA at the end of treatment.

[0057] Figure 4 The antiviral effects of different doses of adenine phosphorothioate-rich oligonucleotides in the AAV-HBV mouse model, including

[0058] Figure 4 A, 4B show the antiviral activity of different doses of PA0028 administered intraperitoneally once a week to C57 mice infected with AAV-HBV, evaluated by qPCR detection of serum HBV-DNA at the end of treatment;

[0059] Figure 4 C, 4D show the antiviral activity of different doses of PA0028 administered intraperitoneally once a week to C57 mice infected with AAV-HBV, and evaluated by measuring serum HBsAg by ELISA at the end of treatment.

[0060] Figure 5 The antiviral effects of adenine phosphorothioate-rich oligonucleotides of different lengths in the AAV-HBV mouse model, among which,

[0061] Figure 5 A, 5B show the antiviral activity of adenine phosphorothioate-rich oligonucleotides (ARONs) of different lengths administered intraperitoneally once a week to C57 mice infected with AAV-HBV, evaluated by qPCR detection of serum HBV-DNA at the end of treatment;

[0062] Figure 5C, 5D show the antiviral activity of ARONs of different lengths administered intraperitoneally once a week to C57 mice infected with AAV-HBV, and evaluated by measuring serum HBsAg by ELISA at the end of treatment. DETAILED DESCRIPTION

[0063] After long-term and in-depth research, the inventors have verified the antiviral activity of phosphorothioate oligonucleotide compounds in rodent HBV persistent infection animal models for the first time through exploration and optimization of experimental conditions; and found that there is no significant difference in the anti-HBsAg activity of oligonucleotides with a length of 20-40nt; for the first time, it was found that phosphorothioate oligonucleotides with different base compositions have different anti-HBsAg activities, and adenine-rich phosphorothioate oligonucleotides (ARON) with an adenine content greater than 50% (such as 75%) have the best activity in inhibiting HBV replication and anti-HBsAg; it was also found that the shortest length of this type of adenine-rich phosphorothioate oligonucleotide with anti-HBV activity is 16nt. Based on the above findings, the inventors have completed the present invention.

[0064] In the present invention, “A m AAC fragments" when written from left to right, when connected to other fragments in the molecule, also include "CA m AA", m is defined as above.

[0065] In the present invention, "2' sugar modification" includes "2' ribose modification" and "2' deoxyribose modification", wherein the modification is selected from the following group: 2'-O-alkyl (such as 2'-O-methyl), 2'-hydroxyl, 2'-amino, 2'-halogen or 2'-O-methoxyethyl (2'-MOE).

[0066] In the present invention, the terms "comprising", "including" or "comprising" indicate that various components can be applied 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".

[0067] In the present invention, the term "pharmaceutically acceptable" ingredient 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 reaction), that is, has a reasonable benefit / risk ratio.

[0068] In the present invention, the term "effective amount" refers to the amount of a therapeutic agent to treat, alleviate or prevent a target disease or condition, or to an amount that exhibits a detectable therapeutic or preventive effect. The exact effective amount for a subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is useless to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation, which a clinician is able to judge.

[0069] 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.

[0070] The term "oligonucleotide" refers to an oligomer of ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA). The term includes oligonucleotides consisting of modified nucleobases, sugars, and phosphodiester bonds between nucleosides, and oligonucleotides with similar functions of non-naturally occurring parts. Such modified or substituted oligonucleotides can 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 for nuclease-mediated degradation. Oligonucleotides can be single-stranded or double-stranded, including single-stranded molecules such as antisense oligonucleotides (ASOs), and aptamers and miRNA, and double-stranded molecules such as small interfering RNA (siRNA) or small hairpin RNA (shRNA). The optionally modified oligonucleotides of the present invention can include various modifications, such as stable modifications, and therefore can include at least one modification in phosphodiester bonds and / or on sugars and / or bases. For example, oligonucleotides can include, but are not limited to, one or more modifications, or can be completely modified so as to contain all bonds or sugars or bases with the modifications described. Modified bonds may include phosphorothioate bonds and phosphorodithioate bonds. Additional useful modifications 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 (2'MOE)), 2'amino modifications, 2'halogen modifications (such as 2'-fluoro substitutions); acyclic nucleotide analogs. Other 2' modifications are also well known in the art and may be used, such as locked nucleic acids. Specifically, the oligonucleotide has modified bonds throughout or has each bond modified, for example, phosphorothioate; has a 3'-cap and / or a 5'-cap: including a terminal 3'-5' bond. Base modifications may include 5' methylation of cytosine bases (5' methylcytosine) and / or 4' thiolation of uracil bases (4' thiouracil). When the synthesis conditions are chemically compatible, then different chemically compatible modified bonds can be combined, such as 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 used to further fully modify the oligonucleotide.

[0071] The term "phosphorothioate nucleotide" refers to a nucleotide with an altered phosphate backbone in which the sugar moieties are linked by phosphorothioate bonds. The phosphorothioate bonds contain sulfur atoms as replacements for non-bridging oxygen atoms in the phosphate backbone of the oligonucleotide sequence.

[0072] The phosphorothioate esterification refers to the replacement of the phosphoroximate internucleoside bonds of the oligonucleotide with phosphorothioate internucleoside bonds.

[0073] An exemplary phosphorothioate bond of the present invention is shown below:

[0074] 3′,5′-phosphorothioate diester bond

[0075]

[0076] As used herein, the term "adenine-rich phosphorothioate oligonucleotide" or its English abbreviation "ARON" (Adenosine Rich Oligo Nucleotides) refers to a phosphorothioate oligonucleotide containing adenine in a ratio of more than 50% (such as 75%, 80%, 85%, 90%) of all bases, and its antiviral activity does not depend on Toll-like receptor recognition, hybridization with target RNA or DNA, or aptamer interaction that depends on the sequence to form secondary or tertiary structure.

[0077] Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can be optionally in R configuration or S configuration, or a mixture of R configuration and S configuration.

[0078] 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 include compounds containing variable amounts of water formed when prepared by low-pressure sublimation drying.

[0079] Active ingredients

[0080] The active ingredient of the present invention is adenine-rich phosphorothioate oligonucleotide (ARON), or its optical isomers, pharmaceutically acceptable salts, hydrates, or solvates,

[0081] The adenine-rich phosphorothioate oligonucleotide comprises (A m AAC n Repeating sequences;

[0082] wherein m is independently 0 or 1; n is a positive number ≥ 4; and the length of the adenine-rich phosphorothioate oligonucleotide is 15-40 nucleotides;

[0083] 80-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

[0084] Preferably, all phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

[0085] Preferably, n is an integer; or when n is a non-integer, the length of the adenine-rich phosphorothioate oligonucleotide sequence is an integer.

[0086] Preferably, the sequence of the adenine phosphorothioate-rich oligonucleotide is (A m AAC n1 (AAAC) (A m AAC n2 , wherein n1 and n2 are each independently an integer of 0-10, n3 is 0, 1, 2, 3 or 4, and

[0087] m and n are defined as above.

[0088] Preferably, in the above sequences, the integer part A m The AAC fragment is selected from the group consisting of AAAC, AAC, CAAA, CAA.

[0089] Preferably, in the above sequences, when n is a non-integer, the non-integer part of A m AAC segments (such as (AAAC) n3 is 1, 2 or 3) is selected from one or several oligonucleotides in the AAAC fragment (such as A, C, AA, AC, AAA, AAC, CAA).

[0090] Preferably, in the above sequences, the non-integer part of A m AAC segments (such as (AAAC) n3 is 1, 2 or 3) depends on the non-integer part of n (e.g. ), for example, when the non-integer part is 0.25 (i.e., n3 is 1), the number of oligonucleotides in the non-integer part is 1, i.e., A or C; when the non-integer part is 0.5 (i.e., n3 is 2), the number of oligonucleotides in the non-integer part is 2, i.e., AA or AC, and so on.

[0091] Preferably, (AAAC) The fragment is selected from the group consisting of: none, A, AA, AC, AAA, AAC, AAAC, CAA, CAAA.

[0092] Preferably, in the above sequences, n is 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9 or 10.

[0093] Preferably, in each of the above sequences, the length of the adenine-rich phosphorothioate oligonucleotide is 16-36 nucleotides.

[0094] Preferably, in each of the above sequences, the length of the adenine-rich phosphorothioate oligonucleotide is 17-33 nucleotides.

[0095] Preferably, the adenine-rich phosphorothioate oligonucleotide is an AAAC repeat sequence, and its length is 16-40 nucleotides, more preferably, 16-36 nucleotides.

[0096] Preferably, the adenine-rich phosphorothioate oligonucleotide is an AAC repeat sequence, and its length is 15-39 nucleotides.

[0097] Preferably, the adenine-rich phosphorothioate oligonucleotide comprises an AAAC sequence and an AAC sequence, and has a length of 15-39 nucleotides.

[0098] Preferably, in each of the above sequences, at least one 2' sugar group of the adenine phosphorothioate-rich oligonucleotide (ARON) is modified, and more preferably, all 2' sugar groups are modified; wherein the modification is selected from the following group: 2'-O-alkyl modification, 2'-hydroxyl modification, 2'-amino modification, 2'-halogen modification, and 2'-O-methoxyethyl modification.

[0099] Preferably, in the above sequences, the sugar group of the adenine-rich phosphorothioate oligonucleotide is a deoxyribose group.

[0100] Preferably, in each of the above sequences, one or more cytosines in the adenine-rich phosphorothioate oligonucleotides are 5-methylcytosines, and more preferably, all cytosines are 5-methylcytosines.

[0101] Preferably, the sequence of the adenine-rich phosphorothioate oligonucleotide is as shown in Table A.

[0102]

[0103] Preferably, the sequence of the adenine-rich phosphorothioate oligonucleotide is as shown in SEQ ID NO: 10-16.

[0104] Preferably, the adenine-rich phosphorothioate oligonucleotide is the oligonucleotide represented by SEQ ID NO:11.

[0105] Preferably, at least one 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is modified, and more preferably, all 2' sugar groups of the adenine-rich phosphorothioate oligonucleotide are modified.

[0106] Preferably, the modification of the 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is selected from the group consisting of 2'-O-alkyl modification, 2' amino modification, and 2' halogen modification. Preferably, the modification of the 2' sugar group is 2'O-methyl modification or 2'O-methoxyethyl (2'MOE).

[0107] Preferably, in the above-mentioned adenine-rich phosphorothioate oligonucleotide, the phosphodiester bond is a phosphorothioate diester bond.

[0108] It should be understood that the compounds of the present invention may exist in various thermodynamically stable isomers after preparation, such as tautomers, conformational isomers, meso compounds and optical isomers with enantiomeric or diastereomeric relationships, etc. The above-mentioned changes will be obvious to those skilled in the art after reading the disclosure of the present invention.

[0109] Preparation of phosphorothioate oligonucleotides

[0110] The adenine-rich phosphorothioate oligonucleotide of the present invention can be prepared and synthesized by conventional synthesis methods in the oligonucleic acid industry. For example, phosphoramidite solid phase synthesis can be used under the control of a device such as GE OP100, and the oxidant iodine can be replaced by the sulfur transfer reagent phenylacetyl disulfide (PADS) to convert the phosphate bond into a phosphorothioate bond, thereby preparing a phosphorothioate oligonucleotide.

[0111] Pharmaceutical compositions and methods of administration

[0112] The phosphorothioate oligonucleotide (ARON) can be used in combination with other drugs known to treat or improve similar symptoms. During co-administration, the original drug administration and dosage can remain unchanged, and the phosphorothioate oligonucleotide is administered simultaneously or subsequently. When the phosphorothioate oligonucleotide is administered simultaneously with one or more other drugs, a pharmaceutical composition containing one or more known drugs and the phosphorothioate oligonucleotide can be preferably used. Drug combination also includes administering the phosphorothioate oligonucleotide and one or more other known drugs in overlapping time periods. When the phosphorothioate oligonucleotide is used in combination with one or more other drugs, the dosage of the phosphorothioate oligonucleotide or the known drug may be lower than the dosage of their individual medications.

[0113] The indicative effective dosing regimen for administering the adenine-rich phosphorothioate oligonucleotide of the present invention to humans is once a week (QW) as described in Example III, with a single dose of 3 mg / kg (based on body surface area conversion); following the dosing regimen commonly used for other phosphorothioate oligonucleotides (e.g., antisense oligonucleotides ASO); the conventional use of 100-500 mg of the compound administered parenterally per week is well established in the art.

[0114] According to the disclosure presented herein, it is useful to treat a subject having HBV infection or HBV / HDV co-infection using a pharmaceutically acceptable adenine-rich phosphorothioate oligonucleotide formulation.

[0115] The compositions described herein can be administered by any suitable means, for example, by oral ingestion; by oral inhalation; by subcutaneous, intravenous injection or infusion; in a dosage unit formulation containing a non-toxic pharmaceutically acceptable carrier or diluent. For example, the present composition can be administered in a formulation suitable for immediate release or sustained release.

[0116] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0117] Main advantages of the invention:

[0118] 1. For the first time, the antiviral activity of phosphorothioate oligonucleotide compounds was verified in a rodent HBV persistent infection animal model; and it was found that there was no significant difference in the anti-HBsAg activity of oligonucleotides with a length of 20-40nt;

[0119] 2. It was discovered for the first time that phosphorothioate oligonucleotides with different base compositions have different activities against hepatitis B surface antigen. Adenine-rich phosphorothioate oligonucleotides (ARONs) with an adenine content of more than 50% (e.g., 75%) have better activities in inhibiting hepatitis B virus replication and against hepatitis B surface antigen.

[0120] 3. The 16nt adenine-rich phosphorothioate oligonucleotide is the shortest oligonucleotide with significant anti-HBV activity.

[0121] 4. The adenine phosphorothioate-rich oligonucleotide of the present invention has high antiviral activity and a sufficiently large safety window.

[0122] 5. Because HBV virus has commonalities with HIV and HPV, the present invention can also be applied to treat infections caused by HIV and HPV.

[0123] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0124] Example 1 Antiviral effects of phosphorothioate oligonucleotides of different lengths in the AAV-HBV mouse model

[0125] Phosphorothioate oligonucleotides of different lengths were tested in c57 mice infected with adeno-associated virus carrying HBV 1.3 ploidy and continuously replicating HBV-DNA and expressing HBV antigens to establish their antiviral activity. These phosphorothioate oligonucleotides are PA0001 (SEQ ID NO: 1), PA0005 (SEQ ID NO: 5), PA0008 (SEQ ID NO: 6) and PA0028 (SEQ ID NO: 11). Table 1 provides the chemical description of these oligonucleotides.

[0126] Table 1

[0127]

[0128]

[0129] dA = deoxyriboadenylation

[0130] dC = deoxyribocytidine

[0131] Use 5X10 10 rAAV8-1.3HBV (Eupatorium avium and) were injected into male C57BL / 6 mice through the tail vein to prepare a mouse model of persistent hepatitis B infection. After confirming that the HBV virus was stably replicated, the mice were randomly divided into 10 groups (4 mice / group) according to body weight. Groups 1, 3, 5, 7, and 9 were intraperitoneally injected with solvent (10 ml / kg) (i.e., blank control group) and 90 mg / kg of PA0001, PA0005, PA0008, and PA0028 once a week (QW), and the vehicle group was used as the control group. Groups 2, 4, 6, 8, and 10 were intraperitoneally injected with solvent (10 ml / kg) and 90 mg / kg of PA0001, PA0005, PA0008, and PA0028 twice a week (BIW), and the vehicle group was used as the control group. After 6 weeks of administration, blood was collected twice a week, and the hepatitis B virus titer in the serum was analyzed by qPCR, and the surface antigen concentration in the serum was analyzed by ELISA, and a curve graph was drawn.

[0132] result

[0133] Serum HBV titer (see Figure 1 A, 1B, 1E, 1F), serum surface antigen concentration (see Figure 1C, 1D, 1G, 1H), during the experiment, 2 animals died in group 5 (PA0005 QW), 1 animal died in group 4 (PA0001 BIW), 1 animal died in group 6 (PA0005 BIW), and no animal died in other groups. Significant splenomegaly was observed in animals in groups 3, 5, 4, and 6 (PA 0001 QW, PA0005 QW, PA0001 BIW, PA0005 BIW), while no significant splenomegaly was observed in other groups. Splenomegaly may be related to immune activation caused by excessive oligonucleotides.

[0134] Figure 2 A and Figure 2 B shows the changes in serum biochemical indicators albumin (ALB) and creatinine (CRE) in C57 mice infected with AAV-HBV after injection of phosphorothioate oligonucleotides of different lengths. At the end of the experiment, compared with the control group, the serum albumin (ALB) of animals in the groups treated with phosphorothioate oligonucleotides PA0001, PA0005, PA0008 and PA0028 decreased, and the decrease in twice-weekly dosing (BIW) was greater than that in once-weekly dosing (QW). Among the animals treated twice a week, the decrease in serum albumin in the PA0001 group was the largest. At the end of the experiment, compared with the control group, the serum creatinine concentration of animals in each group did not increase, but decreased slightly.

[0135] The summary results are shown in Table 2:

[0136] Table 2

[0137]

[0138]

[0139] #NO 1-4 Administer once a week 5-8 Administer twice a week

[0140] PA0005, NO 6 mice died on August 2, NO 1, 4 mice died on August 20

[0141] PA0001, NO 6 mice died on August 16

[0142] The above results show that:

[0143] a. All oligonucleotides can lead to the reduction of serum HBsAg and HBV-DNA.

[0144] b. Oligonucleotides with the same repeating sequence and a length of 20 to 40 nt (PA0001, PA0005, PA0008) have considerable antiviral effects, and the toxicity of the oligonucleotide is positively correlated with the length. The longer the length, the greater the toxicity. The severity of splenomegaly caused is also positively correlated with the length of the oligonucleotide. The longer the length, the more significant the splenomegaly.

[0145] c. Antiviral comparison of oligonucleotides of the same length (20 nt) and different sequences (PA0008 and PA0028): PA0008 with the sequence ACAC repeats had no HBV titer <4log 10 IU / ml (0%), while PA0028 with the sequence AAAC repeats had HBV titers <4log in all 4 animals 10 IU / ml (100%); Comparison of reducing blood surface antigen: BIW administration, PA0008 and PA0028 have the same HBsAg>1log, while QW administration, PA0008 HBsAg>1log is 25% (1 / 4), PA0028 HBsAg>1log is 75% (3 / 4). Therefore, the sequence with AAAC repeat sequence has better efficacy and higher safety.

[0146] Example II Antiviral effects of phosphorothioate oligonucleotides with different base compositions in AAV-HBV mouse model

[0147] Phosphorothioate oligonucleotides of the same length but different base composition were tested in c57 mice infected with adeno-associated virus carrying HBV 1.3 ploidy and continuously replicating HBV-DNA and expressing HBV antigens to evaluate their antiviral activity. These phosphorothioate oligonucleotides are PA0008 (SEQ ID NO: 6), PA0027 (SEQ ID NO: 17), PA0028 (SEQ ID NO: 11), PA0029 (SEQ ID NO: 2), PA0030 (SEQ ID NO: 3) and PA0031 (SEQ ID NO: 4), and the chemical descriptions of these oligonucleotides are provided in Table 3.

[0148] Table 3

[0149]

[0150] dA = deoxyriboadenylation

[0151] dC = deoxyribocytidine

[0152] dT = deoxyribothymidine

[0153] Use 5X10 10rAAV8-1.3HBV (Eupatorium aviculare) was injected into male C57BL / 6 mice through the tail vein to prepare a mouse model of persistent hepatitis B infection. After confirming the stable replication of HBV virus, the mice were randomly divided into 7 groups (5 / group) according to body weight, and the solvent (10ml / kg) and 90mg / kg of PA0008, PA00027, PA0028, PA0029, PA0030, and PA0031 were intraperitoneally injected once a week (QW), and the solvent group served as the control group. After 12 weeks of administration, blood was collected twice a week, and the HBV titer in the serum was analyzed by qPCR, and the surface antigen content in the serum was analyzed by ELISA, and a curve graph was drawn.

[0154] result

[0155] Serum HBV titer (see Figure 3 A, 3B), serum surface antigen concentration (see Figure 3 C, 3D), the summary results are as follows Table 4:

[0156] Table 4

[0157]

[0158]

[0159] In groups PA0008, PA0028, and PA0031, 2, 2, and 1 animal had a decrease in serum HBsAg >1 log, respectively. 10 These animals also had a concomitant decrease in serum HBV-DNA, especially PA0028, where 100% of the animals had DNA > 2log 10 The antiviral activity of PA0027, PA0029 and PA0030 was weak or no, which may be related to the formation of secondary structure. In addition, the weight gain of animals in the PA0008 group during administration was significantly lower than that in the control group, while there was no significant difference in the weight gain of animals in the PA0028 group compared with the control group.

[0160] The above results show that:

[0161] a. The antiviral effect of oligonucleotides of the same length is related to the base composition.

[0162] b. Both PA0008, in which adenine accounts for 50%, and PA0028, in which adenine accounts for 75%, showed significant antiviral activity, and the PA0028 group had the most animals with decreased HBV-DNA, with a greater decrease, and was more advantageous.

[0163] Example III Antiviral effects of different doses of adenine-rich phosphorothioate oligonucleotides in the AAV-HBV mouse model

[0164] Different doses of adenine-rich phosphorothioate oligonucleotide PA0028 were tested in c57 mice infected with adeno-associated virus carrying HBV 1.3 ploidy and continuously replicating HBV-DNA and expressing HBV antigens to evaluate the dose-dependency of its antiviral activity. Table 5 provides the chemical description of PA0028.

[0165] Table 5

[0166]

[0167] dA = deoxyriboadenylation

[0168] dC = deoxyribocytidine

[0169] Use 5X10 10 rAAV8-1.3HBV (Eupatorium aviculare) was injected into male C57BL / 6 mice through the tail vein to prepare a mouse model of persistent hepatitis B infection. After confirming the stable replication of HBV virus, the mice were randomly divided into 4 groups (5 mice in each group) according to body weight, and PA0028 was injected intraperitoneally once a week (QW) at doses of 0 (vehicle), 10 mg / kg, 30 mg / kg, and 90 mg / kg, respectively. The vehicle group served as the control group (blank). After 12 weeks of administration, blood was collected twice a week, and the HBV titer in the serum was analyzed by qPCR, and the surface antigen content in the serum was analyzed by ELISA, and a curve graph was drawn.

[0170] result

[0171] Serum HBV titer (see Figure 4 A, 4B), serum surface antigen concentration (see Figure 4 C, 4D), the summary results are shown in Table 6:

[0172] Table 6

[0173]

[0174] The HBsAg and HBV-DNA in the animal serum of the vehicle control group (Vehicle) showed a stable fluctuation, and the HBV-DNA in the animal serum of the three different dose groups of PA0028 showed >1log 10 The HBV-DNA of some animals decreased to the lower limit of quantification (LLOQ), and 60% of the animals (3 / 5) had DNA>2log 10 Therefore, 10 mg / kg is considered to be the effective dose that causes a significant decrease in HBV-DNA.

[0175] The results show that:

[0176] In both the 30mg / kg and 90mg / kg dose groups, 80% of the animals (4 / 5) had serum HBsAg >1.5log 10 There was no significant difference in the antiviral effect between 30mg / kg and 90mg / kg, so 30mg / kg can be used as the effective dose to cause HBsAg reduction.

[0177] Example IV Antiviral Effects of Adenine-Rich Phosphorothioate Oligonucleotides of Different Lengths in AAV-HBV Mouse Model

[0178] Different lengths of adenine-rich phosphorothioate oligonucleotides were tested in c57 mice infected with adeno-associated virus carrying HBV 1.3 ploidy and continuously replicating HBV-DNA and expressing HBV antigens to establish their antiviral activity. These phosphorothioate oligonucleotides are PA00017 (SEQ ID NO: 9), PA0018 (SEQ ID NO: 10) and PA0028 (SEQ ID NO: 11), and the chemical descriptions of these oligonucleotides are provided in Table 7.

[0179] Table 7

[0180]

[0181]

[0182] dA = deoxyriboadenylation

[0183] dC = deoxyribocytidine

[0184] Use 5X10 10 rAAV8-1.3HBV (Eupatorium aviculare) was injected into male C57BL / 6 mice through the tail vein to prepare a mouse model of persistent hepatitis B infection. After confirming the stable replication of HBV virus, the mice were randomly divided into 4 groups according to body weight, and the solvent (10ml / kg) and 90mg / kg of PA00017, PA00018, and PA0028 were intraperitoneally injected once a week (QW), and the solvent group served as the control group. After 12 weeks of administration, blood was collected twice a week, and the HBV titer in the serum was analyzed by qPCR, and the surface antigen content in the serum was analyzed by ELISA, and a curve graph was drawn.

[0185] result

[0186] Serum HBV titer (see Figure 5 A, 5B), serum surface antigen concentration (see Figure 5 C, 5D), the summary results are as follows Table 8:

[0187] Table 8

[0188] Compound Name PA0017 PA0018 PA0028 Sequence length 12nt 16nt 20nt <![CDATA[HBsAg>1.5log 10 Number of decreases]]> 0 / 4 2 / 4 3 / 4 <![CDATA[DNA maximum > 1 log 10 descending number]]> 0 / 4 4 / 4 4 / 4

[0189] There was no significant decrease in HBsAg and HBV-DNA in the serum of animals in the Vehicle group and the PA0017 group, but the HBV-DNA in the serum of animals in the PA0018 and PA0028 groups was >1log 10 The HBV-DNA of some animals decreased to the lower limit of quantification (LLOQ). Serum HBsAg of 50% (2 / 4) and 75% (3 / 4) of the animals in these two groups, respectively, was >1.5log 10 of reduction.

[0190] The results show that:

[0191] Among oligonucleotides with the same repeating sequence and a length of 12 to 20 nt (PA0017, PA0018, and PA0028), the activity is positively correlated with the length, and the longer the length, the better the activity.

[0192] discuss

[0193] In the above embodiments, the 90 mg / kg phosphorothioate oligonucleotides with a length of >20 nt in Example I can cause toxicity leading to animal death, and Example III shows that the dose of ≥30 mg / kg fails to significantly increase the anti-HBV activity, and the dose of 90 mg / kg of each compound administered in Example IV is a higher dose. Therefore, it can be seen that 16 nt is the shortest length that has significant anti-HBV activity as an adenine-rich phosphorothioate oligonucleotide.

[0194] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Zhejiang Plato Pharmaceutical Technology Co., Ltd. <120> Adenine-rich phosphorothioate oligonucleotides and their application in anti-hepatitis virus <130> P2020-0687 <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence() <400> 1 acacacac acacacac acacacac ac 32 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence() <400> 2 acccacccac ccacccaccc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence() <400> 3 cccccccccc cccccccccc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence() <400> 4 tctctctctc tctctctctc 20 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence() <400> 5 acacacac acacacacac acacacacac acacacacac 40 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence() <400> 6 acacacacac acacacacac 20 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence() <400> 7 aacaacaaca acaacaacaa c 21 <210> 8 <211> twenty one <212> DNA <213> Artificial Sequence() <400> 8 aacaaacaac aaacaacaaa c 21 <210> 9 <211> 12 <212> DNA <213> Artificial Sequence() <400> 9 aaacaaacaa ac 12 <210> 10 <211> 16 <212> DNA <213> Artificial Sequence() <400> 10 aaacaaacaa acaaac 16 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence() <400> 11 aaacaaacaa acaaacaaac 20 <210> 12 <211> twenty four <212> DNA <213> Artificial Sequence() <400> 12 aaacaaacaa acaaacaaac aaac 24 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence() <400> 13 aaacaaacaa acaaacaaac aaacaaac 28 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence() <400> 14 aaacaaacaa acaaacaaac aaacaaacaa ac 32 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence() <400> 15 aaacaaacaa acaaacaaac aaacaaacaa acaaac 36 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence() <400> 16 aaacaaacaa acaaacaaac aaacaaacaa acaaacaaac 40 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence() <400> 17 aaaaaaaaaaaa aaaaaaaaa 20

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: The compound is an adenine-rich phosphorothioate oligonucleotide, wherein the adenine-rich phosphorothioate oligonucleotide is an oligonucleotide as shown in SEQ ID NO: 10 or an oligonucleotide as shown in SEQ ID NO: 11; 80-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown in SEQ ID NO:

10.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown in SEQ ID NO:

11.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: At least one 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is modified; wherein the modification is selected from the following group: 2'-O-alkyl modification, 2'-hydroxyl modification, 2'-amino modification, 2'-halogen modification, 2'-O-methoxyethyl modification.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The sugar group of the adenine-rich phosphorothioate oligonucleotide is a deoxyribose group.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: One or more cytosines in the adenine-rich phosphorothioate oligonucleotide are 5-methylcytosine.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: 95-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The phosphates in the adenine-rich phosphorothioate oligonucleotide are all phosphorothioates.

9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The cytosine in the adenine-rich phosphorothioate oligonucleotide is 5-methylcytosine.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

11. Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 10, characterized in that: For preparing a pharmaceutical composition for treating and / or preventing diseases associated with viral infection; The viral infection-related disease is selected from the group consisting of HBV infection and HBV / HDV co-infection.

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