CN114057816B

CN114057816B - Adenine-rich phosphorothioate oligonucleotides and their application in anti-hepatitis virus

Info

Publication number: CN114057816B
Application number: CN202010753174.0A
Authority: CN
Inventors: 席志坚; 伍中山; 陆春平
Original assignee: Zhejiang Palo Alto Pharmaceuticals Inc
Current assignee: Zhejiang Palo Alto Pharmaceuticals Inc
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2025-02-11
Anticipated expiration: 2040-07-30
Also published as: WO2022022158A1; CN114057816A

Abstract

The invention discloses adenine-rich phosphorothioate oligonucleotide and application thereof in resisting hepatitis virus. Specifically, the invention discloses an adenine-rich phosphorothioate oligonucleotide (ARON) containing (A _mAAC)_n) repetitive sequence for inhibiting hepatitis B surface antigen (HBsAg) and application thereof for treating hepatitis B virus infection or hepatitis B virus/hepatitis delta virus co-infection, wherein the length of the adenine-rich phosphorothioate oligonucleotide (ARON) is 15-40 nucleotides, and the adenine-rich phosphorothioate oligonucleotide contains adenine nucleotides and cytosine nucleotides, wherein the content of the adenine nucleotides exceeds 50%, and the adenine-rich oligonucleotide has higher antiviral activity and a safety window is large enough.

Description

Adenine-enriched phosphorothioate oligonucleotide and application thereof in resisting hepatitis virus

Technical Field

The invention relates to the field of medical biology, in particular to adenine-rich phosphorothioate oligonucleotide and application thereof in resisting hepatitis virus.

Background

The published data of the world health organization show that more than 2 million people chronically infect HBV worldwide in 2015, 88.7 tens of thousands of people die due to complications caused by HBV infection. Although some antiviral drugs have been approved globally for the treatment of HBV infection, therapies or combination therapies of existing drugs, except in a small fraction of patients (< 3%), fail to elicit an effective immune response or serological shift of HBsAg that can provide persistent control or functional cure of the infection.

90% Of adults infected with HBV are self-healing, but 90% develop chronic hepatitis after infants are infected with HBV. Chronic HBV infection may lead to liver fibrosis, further developing cirrhosis and hepatocellular carcinoma (HCC). In addition, there are studies showing that hepatitis B increases the risk of pancreatic cancer. Cure or functional cure with HBV chronic infection is a tremendous unmet clinical need.

Two different particles are mainly produced after HBV infection of human hepatocytes, one is Dane particle, namely the whole HBV virus itself, comprising a viral nucleocapsid assembled from hepatitis B core antigen (HBcAg) and viral nucleic acid (RcDNA) and having a viral envelope composed of hepatitis B surface antigen (HBsAg), and the other is subviral particle (SVP), which is a non-infectious particle composed of lipid, cholesterol ester and hepatitis B surface antigen (HBsAg). The hepatitis B surface antigen contained in SVP accounts for the vast majority (> 99.9%) of hepatitis B surface antigen in patient 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 the interference of drugs to viruses.

Hepatitis Delta Virus (HDV) is a satellite virus of HBV, relying on hepatitis b surface antigen (HBsAg) to form its complete infectious HDV viral particles, HDV infection can only occur in patients with HBV infection. HDV/HBV co-infection complications significantly increase the rate of progression of liver fibrosis to cirrhosis. For patients suffering from HDV chronic infection, only one intervention means for interferon treatment exists at present, no marketed medicine for directly targeting HDV virus exists, and the existing treatment method has poor curative effect and remarkable side effect.

The medicine for treating hepatitis B clinically includes mainly interferon medicine and nucleotide medicine. The interferon drugs include common interferon and polyethylene glycol modified long-acting interferon, the latter including perhexiline (PEG-IFN alpha-2 a) and pelargonic (PEG-IFN alpha-2 b). The nucleoside (acid) drugs comprise lamivudine, telbivudine, adefovir dipivoxil, tenofovir Disoproxil Fumarate (TDF), tenofovir Alafenamide Fumarate (TAF), entecavir, etc. The nucleoside drugs can effectively control the replication of viruses and improve liver functions, so that the application is most widely used. The interferon needs injection administration, has large individual response difference, obvious adverse reaction and poor curative effect. The nucleoside medicine only acts on the replication process of the virus from pgRNA to rcDNA, and has no inhibition effect on other links in the life cycle of the hepatitis B virus. For long-term treatment, the conversion rate of hepatitis B e antigen (HBeAg) is still low, and few patients can convert hepatitis B surface antigen (HBsAg) into negative. Entecavir (354 cases) and tenofovir (176 cases) were treated for 48 weeks, with negative conversion rates of hepatitis B surface antigen (HBsAg) of 2% and 3.2% in patients positive for hepatitis B e antigen (HBeAg), and negative conversion rates of 0.3% and 0% in patients negative for hepatitis B e antigen (HBeAg), respectively. Because the existing treatment schemes cannot cure hepatitis B, patients are required to take medicines for a long time, and the patients may face serious side effects, for example, long-time administration of adefovir dipivoxil and tenofovir disoproxil fumarate can cause nephrotoxicity and bone toxicity.

A large amount of hepatitis B surface antigen (HBsAg) in the form of subviral particles (SVP) in the blood of HBV chronically infected patients can neutralize the specific hepatitis B surface antibody (HBsAg) secreted by B lymphocytes, thereby leading to immune tolerance, while only a small number of HBV viral particles can escape from immune examination, which may be one of the important causes of HBV maintenance chronic infection. Although studies have shown that all three viral antigens, hepatitis b surface antigen (HBsAg), hepatitis b core antigen (HBcAg), and hepatitis b e antigen (HBeAg), 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 is likely to be the most predominant inducer that inhibits host immunity. Serological conversion of hepatitis B surface antigen (HBsAg) (clearance of HBsAg from blood, appearance of free HBsAb) is a well-established prognostic indicator for functional control of viral infection in treatment. Another key reason for HBV to maintain the chronically infected character is that it synthesizes a stable circular DNA repository, HBV covalently closed circular DNA (cccDNA), in the nucleus of the infected hepatocytes by means of host DNA repair enzymes. cccDNA can exist stably in hepatocytes for a long period of time and can be continuously supplemented, which can produce nucleic acid RcDNA of HBV virus and mRNA required for encoding all viral antigens by transcription and reverse transcription. Transcriptional inhibition or clearance of cccDNA is critical for the cure or functional cure of HBV infection. Long-term treatment with nucleoside (nucleotide) analogs does not completely clear cccDNA nor inhibit its transcription, and thus hepatitis b surface antigen (HBsAg) expression levels are hardly affected by nucleoside (nucleotide) drugs. Immunomodulation can mediate fluid and cellular immunity, thereby inhibiting cccDNA transcription or clearing infected cells, but large antigen loads can greatly inhibit the immune process, thus greatly reducing antigens, particularly hepatitis b surface antigen (HBsAg), in combination with immunomodulation is an effective means of helping patients achieve durable immune control.

Currently, phosphorothioate oligonucleotides with high activity and a sufficiently large safety window are lacking for the treatment of HBV and HBV/HDV coinfection.

Disclosure of Invention

The object of the present invention is to provide phosphorothioated oligonucleotides which are highly active and have a sufficiently large safety window for use in the treatment of HBV and HBV/HDV coinfection.

In a first aspect of the invention, there is provided a compound, or an optical isomer, a pharmaceutically acceptable salt, hydrate, or solvate thereof, said compound being an adenine-rich phosphorothioate oligonucleotide (ARON), wherein said adenine-rich phosphorothioate oligonucleotide comprises (a _mAAC)_n repeat;

Wherein each m is independently 0 or 1, n is a positive number greater than or equal to 4, and the adenine-rich phosphorothioate oligonucleotide is 15-40 nucleotides in length;

80-100% of the phosphate in the adenine-rich phosphorothioate oligonucleotide is phosphorothioate.

In another preferred embodiment, 95-100% of the phosphate in the adenine-rich phosphorothioate oligonucleotide is phosphorothioate.

In another preferred embodiment, the phosphorothioate oligonucleotides are phosphorothioate throughout the adenine-rich phosphorothioate oligonucleotide.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide has the sequence (A _mAAC)_n1 (AAAC)(A _mAAC)_n2) wherein n1 and n2 are each independently an integer from 0 to 10 (i.e., 0,1, 2,3, 4, 5, 6, 7, 8, 9, 10), n3 is 0,1, 2,3, or 4, and

M and n are as defined above.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide has the sequence (R) (A _mAAC)_n'2、(A_mAAC)_n'1(R)、(R)(CA_mAA)_n'2 or (CA _mAA)_n'1 (R), wherein each R is independently selected from A or C, n '1 and n'2 are each independently an integer from 3 to 10 (i.e., 3, 4, 5, 6, 7, 8, 9, 10),

M is defined as above.

In another preferred embodiment, each A _m AAC (or CA _m AA) fragment is independently selected from the group consisting of AAAC, AAC, CAAA, CAA.

In another preferred embodiment, when n is a non-integer, the A _m AAC fragment of the non-integer portion is selected from A, AA, AC, AAA, AAC, CA, CAA.

In another preferred embodiment, (AAAC)The fragment is selected from the group consisting of none and C, A, AA, AC, AAA, AAC, AAAC, CA, CAA, CAAA.

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

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide comprises (A _mAAC)_n repeats, n is a positive number of ≡4; m is each independently 0 or 1 and at least one m is 1, preferably m is 1.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is 16-36 nucleotides in length.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is 17-33 nucleotides in length.

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

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is provided in Table A (i.e., as shown in SEQ ID NOS: 10-16 and SEQ ID NOS: 7-8).

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide (ARON) has the sequence shown in SEQ ID NO. 10-16.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide (ARON) has the sequence shown in SEQ ID NO. 11.

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

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide shown in SEQ ID NO. 13.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide shown in SEQ ID NO. 14.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide shown in SEQ ID NO. 15.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide shown by SEQ ID NO. 16.

In another preferred embodiment, the at least one 2' glycosyl of the adenine-rich phosphorothioated oligonucleotide is modified.

In another preferred embodiment, the 2' sugar groups of the adenine-rich phosphorothioate oligonucleotide are modified.

In another preferred embodiment, the modification of the 2 'glycosyl group of the adenine phosphorothioate oligonucleotide is selected from the group consisting of a 2' -O-alkyl modification, a2 '-hydroxy modification, a 2' -amino modification, a2 'halogen modification, and a 2' -O-methoxyethyl (2 'MOE) modification, preferably a 2' O-methyl modification.

In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is one in which the sugar moiety is a deoxyribose moiety.

In another preferred embodiment, one or more of the cytosines in the adenine-rich phosphorothioated oligonucleotide is 5-methylcytosine.

In another preferred embodiment, the cytosine in the adenine-rich phosphorothioate oligonucleotide is 5-methylcytosine.

In a second aspect of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the first aspect, or an optical isomer, pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable adjuvant, diluent or carrier.

In a third aspect, the present invention provides a compound according to the first aspect, or an optical isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, or a pharmaceutical composition according to the second aspect, for use in the preparation of a pharmaceutical composition for the treatment and/or prevention of a disease associated with a viral infection.

In another preferred example, the diseases include, for example, HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HPV (human papilloma virus), HIV (human immunodeficiency virus) and the like.

In another preferred embodiment, the infectious disease is a disease associated with HBV infection and HBV/HDV co-infection.

In another preferred embodiment, the disease is selected from the group consisting of viral hepatitis B infection (HBV), viral hepatitis C infection (HCV), and viral hepatitis D infection (HDV).

In another preferred embodiment, the disease is selected from HPV, HIV.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows antiviral effects of phosphorothioate oligonucleotides of different lengths in AAV-HBV mouse model, wherein

FIGS. 1A,1B show antiviral activity of phosphorothioate oligonucleotides of different lengths administered by intraperitoneal injection weekly to AAV-HBV-infected C57 mice, assessed by qPCR detection of serum HBV-DNA at the end of treatment;

FIGS. 1C,1D show antiviral activity of phosphorothioate oligonucleotides of varying lengths administered by intraperitoneal injection weekly to AAV-HBV-infected C57 mice, assessed by ELISA detection of serum HBsAg at the end of treatment;

FIGS. 1E,1F show antiviral activity of phosphorothioate oligonucleotides of different lengths given twice weekly intraperitoneal injections to C57 mice infected with AAV-HBV, assessed by qPCR detection of serum HBV-DNA at the end of treatment;

FIGS. 1G,1H show antiviral activity of phosphorothioate oligonucleotides of different lengths given twice weekly intraperitoneal injections to C57 mice infected with AAV-HBV, assessed by ELISA detection of serum HBsAg at the end of treatment.

FIG. 2 shows the changes in serum biochemical indicators Albumin (ALB), creatinine (CRE) of phosphorothioate oligonucleotides of different lengths given to AAV-HBV infected C57 mice.

FIG. 3 shows the antiviral effect of phosphorothioate oligonucleotides of different base composition in AAV-HBV mouse model, wherein

FIGS. 3A,3B show antiviral activity of phosphorothioate oligonucleotides of different base composition administered by intraperitoneal injection weekly to AAV-HBV-infected C57 mice, assessed by qPCR detection of serum HBV-DNA at the end of treatment;

FIG. 3C,3D shows antiviral activity of phosphorothioated oligonucleotides of different base composition administered by intraperitoneal injection weekly to AAV-HBV-infected C57 mice, assessed by ELISA detection of serum HBsAg at the end of treatment.

FIG. 4 antiviral effects of different doses of adenine-rich phosphorothioate oligonucleotides in AAV-HBV mouse model, wherein

FIGS. 4A,4B show antiviral activity of various doses of PA0028 given by intraperitoneal injection weekly to C57 mice infected with AAV-HBV, assessed by qPCR detection of serum HBV-DNA at the end of treatment;

FIGS. 4C,4D show antiviral activity of PA0028 given weekly intraperitoneal injections to C57 mice infected with AAV-HBV, assessed by ELISA detection of serum HBsAg at the end of treatment.

FIG. 5 antiviral effects of different lengths of adenine-rich phosphorothioate oligonucleotides in AAV-HBV mouse models, wherein,

FIGS. 5A,5B show antiviral activity of various lengths of adenine-rich phosphorothioate oligonucleotide (ARON) given by intraperitoneal injection weekly to C57 mice infected with AAV-HBV, assessed by qPCR detection of serum HBV-DNA at the end of treatment;

FIGS. 5C,5D show antiviral activity of various lengths of adenine-rich phosphorothioate oligonucleotide (ARON) given by intraperitoneal injection weekly to C57 mice infected with AAV-HBV, assessed by ELISA detection of serum HBsAg at the end of treatment.

Detailed Description

Through long-term and intensive researches, the inventor verifies the antiviral activity of phosphorothioate oligonucleotide compounds in rodent HBV continuous infection animal models for the first time through exploring and optimizing experimental conditions, discovers that the activity of the oligonucleotide with the length of 20-40nt against hepatitis B surface antigens is not obviously different, discovers that the phosphorothioate oligonucleotide with different base compositions has different activity against hepatitis B surface antigens for the first time, and discovers that adenine-rich phosphorothioate oligonucleotide (ARON) with the adenine content of more than 50 percent (such as 75 percent) has the best activity of inhibiting replication of hepatitis B virus and resisting hepatitis B surface antigens, and discovers that the adenine-rich phosphorothioate oligonucleotide has the shortest length of resisting hepatitis B virus activity of 16nt. Based on the above findings, the inventors have completed the present invention.

In the present invention, "a _m AAC fragment" when it is linked to other fragments in the molecule by writing from left to right, it also includes the right to left writing form "CA _m AA", m being as defined above.

In the present invention, "2 'glycosyl modification" includes "2' ribose modification" and "2 'deoxyribose modification" wherein the modification is selected from the group consisting of 2' -O-alkyl (e.g., 2 '-O-methyl), 2' -hydroxy, 2 '-amino, 2' -halogen, or 2 '-O-methoxyethyl (2' -MOE).

In the present invention, the terms "comprising," "including," or "containing" mean that the various ingredients may be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

In the present invention, the term "pharmaceutically acceptable" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Thus, it is not useful to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.

As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention with bases that are suitable for use as medicaments.

The term "oligonucleotide" refers to an oligomer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA). The term includes oligonucleotides composed of modified nucleobases, sugars and internucleoside phosphodiester linkages, as well as functionally similar oligonucleotides having non-naturally occurring portions. Such 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. Oligonucleotides may be single-stranded or double-stranded, including single-stranded molecules such as antisense oligonucleotides (ASOs), and aptamers and mirnas, etc., as well as double-stranded molecules such as small interfering RNAs (sirnas) or small hairpin RNAs (shrnas). The optionally modified oligonucleotides described herein may include various modifications, such as stable modifications, and thus may include at least one modification in the phosphodiester linkage and/or on the sugar and/or base. For example, an oligonucleotide may include, but is not limited to, one or more modifications, or may be modified entirely so as to contain all bonds or sugars or bases having the modifications. Modified linkages may include phosphorothioate linkages and phosphorodithioate linkages. Additional useful modifications include, but are not limited to, modifications at the 2 'position of the sugar, including 2' -O-alkyl modifications (e.g., 2 'O-methyl modifications, 2' O-methoxyethyl (2 'MOE)), 2' amino modifications, 2 'halogen modifications (e.g., 2' -fluoro substitutions), acyclic nucleotide analogs. Other 2' modifications are also well known in the art and may be used, such as locking nucleic acids. In particular, the oligonucleotides have modified bonds throughout or have each bond modified, e.g., phosphorothioate, have 3 '-caps and/or 5' -caps, including terminal 3'-5' bonds. The base modification may include 5 'methylation of cytosine bases (5' methylcytosine) and/or 4 'sulfation of uracil bases (4' thiouracil). When the synthesis conditions are chemically compatible, then different chemically compatible modified linkages can be combined, for example, oligonucleotides having a phosphorothioate linkage, a 2' ribose modification (e.g., 2' O-methylation), and a modified base (e.g., 5' methylcytosine). All of these various modifications (e.g., each phosphorothioate linkage, each 2' modified ribose, and each modified base) can be used to further fully modify the oligonucleotide.

The term "phosphorothioated nucleotide" refers to a nucleotide having an altered phosphate backbone in which the sugar moiety is linked by a phosphorothioate linkage. In the phosphate backbone of the oligonucleotide sequence, the phosphorothioate linkage contains a sulfur atom as a substitute for the non-bridging oxygen atom.

Phosphorothioate refers to the replacement of phosphorothioate internucleoside linkages of an oligonucleotide by phosphorothioate internucleoside linkages.

Exemplary phosphorothioate linkages in the present invention are shown below:

3',5' -phosphorothioate diester bonds

As used herein, the term "adenine-rich phosphorothioate oligonucleotide" or its english abbreviation "ARON" (Adenosine Rich Oligo Nucleotides) refers to phosphorothioate oligonucleotides containing adenine in a proportion of more than 50% (e.g., 75%, 80%, 85%, 90%) of total bases, whose antiviral activity is not dependent on Toll-like receptor recognition, hybridization with target RNA or DNA, or sequence-dependent aptamer interactions to form secondary or tertiary structures.

Unless otherwise indicated, all compounds present 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). Among all the compounds of the invention, each chiral carbon atom may optionally be in the R configuration or in the S configuration, or in a mixture of R and S configurations.

Some of the compounds of the present invention may be crystallized or recrystallized from water or various organic solvents, in which case various solvates may be formed. Solvates of the present invention include stoichiometric solvates such as hydrates and the like, as well as compounds containing variable amounts of water formed when prepared by the low pressure sublimation drying process.

Active ingredient

The active ingredient of the invention is adenine-rich phosphorothioate oligonucleotide (ARON), or optical isomer, pharmaceutically acceptable salt, hydrate or solvate thereof,

The adenine-rich phosphorothioate oligonucleotide comprises (a _mAAC)_n repeat;

Preferably, all of the phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.

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

Preferably, the adenine-rich phosphorothioate oligonucleotide has the sequence (A _mAAC)_n1 (AAAC)(A _mAAC)_n2 wherein n1 and n2 are each independently an integer of 0 to 10, n3 is 0, 1,2, 3 or 4, and

M and n are as defined above.

Preferably, in each of the above sequences, the A _m AAC fragment of the integer portion is selected from the group consisting of AAAC, AAC, CAAA, CAA.

Preferably, in each of the above sequences, when n is a non-integer, the A _m AAC fragment of the non-integer portion (e.g., AAAC)N3 is 1,2 or 3) is selected from one or several oligonucleotides (e.g. A, C, AA, AC, AAA, AAC, CAA) in the AAAC fragment.

Preferably, in each of the above sequences, the non-integer portion of the A _m AAC fragment (e.g., AAAC)N 3is 1,2 or 3) depending on the non-integer portion 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.

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

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

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

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

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

Preferably, the adenine-rich phosphorothioate oligonucleotide is an AAC repeat and is 15-39 nucleotides in length.

Preferably, the adenine-rich phosphorothioate oligonucleotide comprises an AAAC sequence and an AAC sequence and is 15-39 nucleotides in length.

Preferably, in each of the above sequences, at least one 2' glycosyl group of the adenine-rich phosphorothioated oligonucleotide (ARON) is modified, more preferably, the 2' glycosyl group is modified, wherein the modifications are selected from the group consisting of 2' -O-alkyl modifications, 2' -hydroxy modifications, 2' -amino modifications, 2' -halogen modifications, 2' -O-methoxyethyl modifications.

Preferably, in each of the above sequences, the glycosyl group of the adenine-rich phosphorothioate oligonucleotide is a deoxyribosyl group.

Preferably, in each of the above sequences, one or more of the cytosines in the adenine-rich phosphorothioated oligonucleotide is 5-methylcytosine, preferably, the cytosines are each 5-methylcytosine.

Preferably, the adenine-rich phosphorothioate oligonucleotide has the sequence shown in Table A

Preferably, the adenine-rich phosphorothioate oligonucleotide has the sequence shown in SEQ ID NO. 10-16.

Preferably, the adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown by SEQ ID NO. 11.

Preferably, at least one 2 'glycosyl of the adenine-rich phosphorothioate oligonucleotide is modified, more preferably, both 2' glycosyl of the adenine-rich phosphorothioate oligonucleotide are modified.

Preferably, the modification of the 2 'glycosyl of the adenine-rich phosphorothioated oligonucleotide is selected from the group consisting of a 2' -O-alkyl modification, a 2 'amino modification, a 2' halogen modification, preferably the modification of the 2 'glycosyl is a 2' O-methyl modification or a 2 'O-methoxyethyl (2' MOE).

Preferably, in the adenine-rich phosphorothioate oligonucleotide described above, the phosphodiester linkage is a phosphorothioate linkage.

It is to be understood that various thermodynamically stable isomers, such as tautomers, conformational isomers, meso compounds, and optical isomers having an enantiomeric or diastereomeric relationship, etc., may exist after preparation of the compounds of the present invention, and that such modifications will be apparent to those skilled in the art upon review of the present disclosure.

Phosphorothioate oligos preparation of nucleotides

The adenine-rich phosphorothioate oligonucleotides of the present invention may be prepared and synthesized by conventional synthetic methods in the oligonucleotide industry. For example, phosphorothioate oligonucleotides can be prepared by solid phase synthesis of phosphoramidites under the control of equipment such as GE OP100, using the sulfur transfer reagent diphenylacetyl disulfide (PADS) in place of the oxidant iodine (iodine), to convert the phosphoester linkage to a phosphorothioate linkage.

Pharmaceutical compositions and methods of administration

The phosphorothioated oligonucleotides (ARONs) may be used in combination with other drugs known to treat or ameliorate similar conditions. When administered in combination, the mode and dosage of administration of the original drug may be maintained unchanged while the phosphorothioated oligonucleotide is administered simultaneously or subsequently. When the phosphorothioate oligonucleotide is administered simultaneously with the other drug or drugs, it may be preferable to use a pharmaceutical composition containing both the known drug or drugs and the phosphorothioate oligonucleotide. Drug combination also includes administration of phosphorothioated oligonucleotides with other known drug(s) over overlapping time periods. When phosphorothioate oligonucleotides are used in combination with one or more other drugs, the dosage of phosphorothioate oligonucleotides or known drugs may be lower than the dosage of their individual drugs.

The prompter effective dosing regimen for administration of adenine-rich phosphorothioate oligonucleotides of the present invention to humans, once a week (QW) as described in example III, a single dose of 3mg/kg (based on body surface area conversion), following dosing regimens commonly used for other phosphorothioate oligonucleotides (e.g., antisense oligonucleotides ASOs), is well established in the art for parenteral administration of 100-500mg of compound per week.

In accordance with the disclosure presented herein, it is useful to treat subjects with HBV infection or HBV/HDV co-infection with a pharmaceutically acceptable adenine-rich phosphorothioate oligonucleotide formulation.

The compositions described herein may be administered by any suitable means, e.g., oral ingestion, oral inhalation, by subcutaneous, intravenous injection or infusion, in dosage unit formulations containing non-toxic pharmaceutically acceptable carriers or diluents. For example, the present compositions may be adapted for administration in immediate release or sustained release formulations.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective administration dose, and the daily administration dose is usually 1 to 2000mg, preferably 50 to 1000mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The invention has the main advantages that:

1. The antiviral activity of phosphorothioate oligonucleotide compounds is verified in a rodent HBV continuous infection animal model for the first time, and no obvious difference in the activity of the oligonucleotide with the length of 20-40nt against hepatitis B surface antigen is found;

2. The phosphorothioate oligonucleotide (ARON) which is rich in adenine and has the adenine content of more than 50 percent (such as 75 percent) has better activity of inhibiting replication of hepatitis B virus and resisting hepatitis B surface antigen;

3. an adenine-rich phosphorothioate oligonucleotide of length 16nt is the shortest oligonucleotide with significant anti-hepatitis B virus activity.

4. The adenine-rich phosphorothioate oligonucleotide of the invention has higher antiviral activity and a sufficiently large safety window.

5. Because HBV viruses and HIV have commonalities with HPV, the present invention may be equally applicable to the treatment of infection by HIV and HPV.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example I antiviral Effect of phosphorothioated oligonucleotides of different Length in AAV-HBV mouse model

Phosphorothioate oligonucleotides of different lengths were tested in c57 mice infected with adeno-associated virus carrying HBV1.3 ploidy and continuously replicating HBV-DNA and expressing HBV antigens to establish their antiviral activity. These phosphorothioated 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 a chemical description of these oligonucleotides.

TABLE 1

DA = deoxyriboadenosine

Dc=deoxyribocytidine

A model of persistent hepatitis B infection mice was prepared by tail vein injection of male C57BL/6 mice with 5X10 ¹⁰ rAAV8-1.3HBV (Acanthopanax senticosus). After stable replication of HBV virus was determined to be randomly divided into 10 groups (4 groups) by body weight, groups 1, 3, 5, 7, 9 were respectively intraperitoneally injected with vehicle (10 ml/kg) (i.e., blank control group) and 90mg/kg of PA0001, PA0005, PA0008, PA0028, vehicle group as control group. Groups 2, 4, 6, 8, 10 were given twice weekly (BIW) intraperitoneal injections of vehicle (10 ml/kg) and 90mg/kg of PA0001, PA0005, PA0008, PA0028, respectively, as control groups. After 6 weeks of administration, blood was taken twice a week, the serum was analyzed for hepatitis B virus titer by qPCR method, the serum was analyzed for surface antigen concentration by ELISA method, and a graph was drawn.

Results

Hepatitis B virus titer in serum (see FIGS. 1A, 1B, 1E, 1F), surface antigen concentration in serum (see FIGS. 1C, 1D, 1G, 1H), 2 animals died in group 5 (PA 0005 QW), 1 animal died in group 4 (PA 0001 BIW), 1 animal died in group 6 (PA 0005 BIW), no animal died in other groups, significant splenomegaly was seen in animals in group 3,5,4,6 (PA 0001 QW,PA0005 QW,PA0001 BIW,PA0005 BIW), no significant spleen changes were seen in other groups, and splenomegaly was likely to be associated with immune activation by excess oligonucleotides.

FIGS. 2A and 2B show the changes in serum biochemical indicators Albumin (ALB) and Creatinine (CRE), respectively, of phosphorothioated oligonucleotides of different lengths administered to AAV-HBV infected C57 mice. At the end of the experiment, the phosphorothioated oligonucleotides PA0001, PA0005, PA0008 and PA0028 were all reduced in animal serum Albumin (ALB) in the group administered with respect to the control group, and the reduction in BIW was greater than once weekly (QW). The greatest decrease in serum albumin was observed in the PA0001 group in animals given twice weekly. At the end of the experiment, there was no increase in serum creatinine concentration and a slight decrease in serum creatinine concentration for each group relative to the control group.

The results are summarized in Table 2 below:

TABLE 2

The administration of # NO 1-4 once a week and twice a week of 5-8

PA0005, 8 month 2 day death of NO 6 mice NO 1,4 mice 8 month 20 day death

PA0001, NO 6 mice die 8 months and 16 days

The above results indicate that:

a. all oligonucleotides can result in a decrease in serum HBsAg and HBV-DNA.

B. The oligonucleotides (PA 0001, PA0005 and PA 0008) with the same repeated sequence and the length of 20-40 nt have equivalent antiviral effects, and the toxicity of the oligonucleotides is positively correlated with the length, the longer the length is, the greater the toxicity is, the severity of spleen enlargement is positively correlated with the length of the oligonucleotides, and the longer the length is, the more obvious the spleen enlargement is.

C. Antiviral comparison of oligonucleotides of equal length (20 nt), different sequences (PA 0008 and PA 0028) BIW administration of PA0008 with sequence ACAC repeat <4log ₁₀ IU/ml (0%) and PA0028 with sequence AAAC repeat <4log ₁₀ IU/ml (100%) all 4 animals hepatitis B virus titres, comparison in reducing surface antigens in blood BIW administration of PA0008 and PA0028 with the same HBsAg >1log and QW administration, HBsAg >1log of PA0008 25% (1/4) and HBsAg >1log of PA0028 75% (3/4). Therefore, the sequence with the AAAC repeated sequence has better drug effect and higher safety.

EXAMPLE II antiviral Effect of phosphorothioate oligonucleotides of different base compositions in AAV-HBV mouse model

Phosphorothioate oligonucleotides of the same length but different base composition were tested in c57 mice infected with adeno-associated virus carrying HBV1.3 ploidy and continuously replicating HBV-DNA and expressing HBV antigen to evaluate their antiviral activity. These phosphorothioated 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 Table 3 provides a chemical description of these oligonucleotides.

TABLE 3 Table 3

DA = deoxyriboadenosine

Dc=deoxyribocytidine

DT = deoxyribothymidine

A model of persistent hepatitis B infection mice was prepared by tail vein injection of male C57BL/6 mice with 5X10 ¹⁰ rAAV8-1.3HBV (Acanthopanax senticosus). After stable replication of HBV virus was confirmed, it was randomly divided into 7 groups (5 groups) by body weight, and vehicle (10 ml/kg) and 90mg/kg of PA0008, PA00027, PA0028, PA0029, PA0030, and PA0031 were respectively injected intraperitoneally once a week (QW), and vehicle groups were used as control groups. After 12 weeks of administration, blood was taken twice a week, the serum was analyzed for hepatitis B virus titer by qPCR method and for surface antigen content by ELISA method, and a graph was drawn.

Results

Hepatitis B virus titers in serum (see FIGS. 3A, 3B), surface antigen concentrations in serum (see FIGS. 3C, 3D), and the summary results are given in Table 4 below:

TABLE 4 Table 4

The serum HBsAg reduction of 2, 2 and 1 animals in the PA0008, PA0028, PA0031 groups, respectively, was >1log ₁₀, which animals were also accompanied by a reduction in serum HBV-DNA, in particular a reduction in PA0028,100% of animal DNA >2log ₁₀, possibly associated with the formation of secondary structures, with weak or no antiviral activity of PA0027, PA0029 and PA0030, and in addition, the weight gain of animals during the administration of the PA0008 group was significantly lower than that of the control group, whereas the weight gain of animals in the PA0028 group was not significantly different from that of the control group.

The above results indicate that:

a. the antiviral effect of oligonucleotides of the same length is related to the base composition.

B. PA0008 with 50% adenine and PA0028 with 75% adenine both showed significant antiviral activity, and most animals with HBV-DNA decrease in PA0028 group, the decrease amplitude was larger, with advantage.

Example III antiviral Effect of different doses of adenine-rich phosphorothioate oligonucleotides in AAV-HBV mouse models

Different doses of adenine-rich phosphorothioated oligonucleotide PA0028 were tested in c57 mice infected with adeno-associated virus carrying HBV1.3 ploidy (AAV-HBV, acanthopanax) and continuously replicating HBV-DNA and expressing HBV antigens to evaluate the dose-dependent relationship of their antiviral activity, table 5 provides a chemical description of PA 0028.

TABLE 5

DA = deoxyriboadenosine

Dc=deoxyribocytidine

A model of persistent hepatitis B infection mice was prepared by tail vein injection of male C57BL/6 mice with 5X10 ¹⁰ rAAV8-1.3HBV (Acanthopanax senticosus). After stable replication of HBV virus, it was determined that HBV virus was randomly divided into 4 groups (5 groups) by body weight, and the intraperitoneal injection dose (QW) was 0 (vehicle), 10mg/kg, 30mg/kg, 90mg/kg, respectively, and the vehicle group was used as a control group (blank). After 12 weeks of administration, blood was taken twice a week, the serum was analyzed for hepatitis B virus titer by qPCR method, the serum was analyzed for surface antigen content by ELISA method, and a graph was drawn.

Results

Hepatitis B virus titers in serum (see FIGS. 4A, 4B), surface antigen concentrations in serum (see FIGS. 4C, 4D), and the summary results are shown in Table 6 below:

TABLE 6

The serum of animals in the Vehicle control group (Vehicle) showed smooth fluctuations of HBsAg and HBV-DNA, and the serum HBV-DNA of animals in three different dose groups of PA0028 showed a trend of >1log ₁₀ fluctuation decrease, and part of the animals HBV-DNA was reduced to the lower limit of quantification (LLOQ) with 60% of animals (3/5) DNA >2log ₁₀ decrease, so 10mg/kg was considered as the effective dose causing significant HBV-DNA reduction.

The results show that:

80% of animals (4/5) showed a >1.5log ₁₀ reduction in serum HBsAg in both 30mg/kg and 90mg/kg dose groups, and there was no significant difference in antiviral effect between the 30mg/kg and 90mg/kg doses, so 30mg/kg could be taken as the effective dose to cause the HBsAg reduction.

EXAMPLE IV antiviral Effect of adenine-rich phosphorothioate oligonucleotides of different lengths in AAV-HBV mouse model

The antiviral activity was established by testing various lengths of adenine-rich phosphorothioated oligonucleotides in c57 mice infected with adeno-associated virus carrying HBV 1.3-fold (AAV-HBV, acanthopanax) and continuously replicating HBV-DNA and expressing HBV antigens. These phosphorothioated oligonucleotides are PA00017 (SEQ ID NO: 9), PA0018 (SEQ ID NO: 10) and PA0028 (SEQ ID NO: 11), and Table 7 provides a chemical description of these oligonucleotides.

TABLE 7

DA = deoxyriboadenosine

Dc=deoxyribocytidine

A model of persistent hepatitis B infection mice was prepared by tail vein injection of male C57BL/6 mice with 5X10 ¹⁰ rAAV8-1.3HBV (Acanthopanax senticosus). After stable replication of HBV virus was confirmed, it was randomly divided into 4 groups by body weight, once weekly (QW) and 90mg/kg of PA00017, PA00018 and PA0028 were respectively injected intraperitoneally with vehicle (10 ml/kg), and vehicle groups were used as control groups. After 12 weeks of administration, blood was taken twice a week, the serum was analyzed for hepatitis B virus titer by qPCR method, the serum was analyzed for surface antigen content by ELISA method, and a graph was drawn.

Results

Hepatitis B virus titers in serum (see FIGS. 5A, 5B), surface antigen concentrations in serum (see FIGS. 5C, 5D), and the summary results are shown in Table 8 below:

TABLE 8

Names of Compounds	PA0017	PA0018	PA0028
				Sequence length	12nt	16nt	20nt
HBsAg >1.5log ₁₀ decrease number	0/4	2/4	3/4
				DNA max >1log ₁₀ decrease	0/4	4/4	4/4

There was no significant decrease in HBsAg and HBV-DNA in the animal serum of Vehicle and PA0017, the animal serum HBV-DNA of PA0018 and PA0028 showed a trend of >1log ₁₀ fluctuation decrease, and part of the animal HBV-DNA was reduced to the lower limit of quantification (LLOQ), which showed >1.5log ₁₀ decrease in serum HBsAg of 50% (2/4) and 75% (3/4), respectively.

The results show that:

the same repeated sequence, the activity of the oligonucleotides (PA 0017, PA0018 and PA 0028) with the length of 12-20 nt is positively correlated with the length, and the longer the length is, the better the activity is.

Discussion of the invention

In each of the above examples, phosphorothioate oligonucleotides of 90mg/kg length >20nt in example I could cause toxicity leading to death of animals, and example III indicated that a dose of > 30mg/kg failed to significantly increase anti-HBV activity, and the dose of 90mg/kg of each compound administered in example IV was the higher dose, thus, it can be seen that 16nt is the shortest length with significant anti-HBV activity as adenine-rich phosphorothioate oligonucleotides.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

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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 or pharmaceutically acceptable salt thereof according to claim 1, wherein the adenine-rich phosphorothioate oligonucleotide is an oligonucleotide as shown in SEQ ID NO: 10.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the adenine-rich phosphorothioate oligonucleotide is an oligonucleotide as shown in SEQ ID NO: 11.

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

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

6. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein one or more cytosines in the adenine-rich phosphorothioate oligonucleotide are 5-methylcytosine.

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

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

9. The compound or pharmaceutically acceptable salt thereof according to claim 1, 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 it is used to prepare a pharmaceutical composition for treating and/or preventing diseases related to viral infection;

The viral infection-related disease is selected from the group consisting of HBV infection and HBV/HDV co-infection.