WO2022022158A1 - 富含腺嘌呤硫代磷酸酯化寡核苷酸及其抗肝炎病毒的应用 - Google Patents

富含腺嘌呤硫代磷酸酯化寡核苷酸及其抗肝炎病毒的应用 Download PDF

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WO2022022158A1
WO2022022158A1 PCT/CN2021/101064 CN2021101064W WO2022022158A1 WO 2022022158 A1 WO2022022158 A1 WO 2022022158A1 CN 2021101064 W CN2021101064 W CN 2021101064W WO 2022022158 A1 WO2022022158 A1 WO 2022022158A1
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adenine
rich
hbv
hepatitis
oligonucleotides
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French (fr)
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席志坚
伍中山
陆春平
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浙江柏拉阿图医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the invention relates to the field of medicine and biology, in particular to an adenine-rich phosphorothioate oligonucleotide and its anti-hepatitis virus application.
  • HBV infection may lead to liver fibrosis, which further develops into cirrhosis and hepatocellular carcinoma (HCC).
  • HCC cirrhosis and hepatocellular carcinoma
  • HBV infection of human hepatocytes mainly produces two different particles, one is Dane particles, that is, the complete HBV virus itself, including the viral nucleocapsid assembled from 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 a subviral particle (SVP), which is a non-infectious particle composed of lipids, cholesterol, cholesterol esters, and hepatitis B surface antigen (HBsAg) .
  • SVP subviral particle
  • the HBsAg contained in SVP accounts for the vast majority (>99.9%) of HBsAg in the blood of patients.
  • HBV-infected hepatocytes also secrete an e-antigen (HBeAg) into the blood.
  • Hepatitis B surface antigen HBsAg
  • HBsAb hepatitis B surface antibody
  • HBcAb hepatitis B core antibody
  • HBeAg hepatitis B e antigen
  • HBeAb hepatitis B e antibody
  • Hepatitis delta virus is a satellite virus of HBV and relies on hepatitis B surface antigen (HBsAg) to form its complete infectious HDV virion.
  • HDV infection can only occur in patients with concomitant HBV infection.
  • Complications of HDV/HBV co-infection significantly increased the rate of progression from fibrosis to cirrhosis.
  • interferon therapy is an intervention method, and there is no marketed drug directly targeting HDV virus.
  • the existing treatment methods have poor efficacy and significant side effects.
  • the drugs used in clinical treatment of hepatitis B mainly include interferon and nucleoside (acid) drugs.
  • Interferon drugs include common interferon and polyethylene glycol-modified long-acting interferon, the latter including Pegasin (PEG-IFN ⁇ -2a) and PegIntron (PEG-IFN ⁇ -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 virus replication and improve liver function, so they are the most widely used.
  • hepatitis B e antigen HBeAg
  • HBsAg hepatitis B surface antigen
  • hepatitis B surface antigen (HBsAg) negative rate in hepatitis B e antigen (HBeAg) positive patients was 2% and 3.2%, respectively.
  • Hepatitis B surface antigen (HBsAg) negative conversion rates were 0.3% and 0% in HBeAg-negative patients, respectively. Since the existing treatment options cannot cure hepatitis B, patients are required to take long-term medication, which may expose patients to significant side effects. For example, long-term use of adefovir dipivoxil and tenofovir disoproxil fumarate can lead to nephrotoxicity and bone toxicity. .
  • hepatitis B surface antigen (HBsAg) in the form of subviral particles (SVP) in the blood of patients with chronic HBV infection can neutralize the specific hepatitis B surface antibody (HBsAb) secreted by B lymphocytes, thereby leading to immune tolerance.
  • HBsAb hepatitis B surface antibody
  • a small number of HBV virus particles can escape immune inspection, which may be one of the important reasons why HBV remains chronically infected.
  • hepatitis B surface antigen (HBsAg)
  • HBcAg hepatitis B core antigen
  • HBeAg hepatitis B e antigen
  • HBV covalently closed circular DNA (cccDNA)
  • cccDNA HBV covalently closed circular DNA
  • cccDNA can exist stably in hepatocytes for a long time and can be continuously replenished, and it can generate 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 curative or functional cure of HBV infection.
  • hepatitis B surface antigen hepatitis B surface antigen (HBsAg)
  • Immunomodulation can mediate humoral and cellular immunity, thereby inhibiting the transcription of cccDNA or eliminating infected cells, but a large antigen load will greatly inhibit the immune process, thus greatly reducing antigens, especially hepatitis B surface antigen (HBsAg), binding Immunomodulation is an effective means of helping patients achieve durable immune control.
  • the purpose of the present invention is to provide a phosphorothioate oligonucleotide with high activity and a sufficiently large safety window for the treatment of HBV and HBV/HDV co-infection.
  • the first aspect of the present invention provides a compound, or an optical isomer, pharmaceutically acceptable salt, hydrate, or solvate thereof, the compound is adenine-rich phosphorothioate oligonucleoside acid (ARON), wherein the adenine-rich phosphorothioate oligonucleotides comprise ( Am AAC) n repeats ;
  • ARON adenine-rich phosphorothioate oligonucleoside acid
  • 80-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotides are phosphorothioates.
  • 95-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotides are phosphorothioates.
  • all the phosphates in the adenine-rich phosphorothioate oligonucleotides are phosphorothioates.
  • sequence of the adenine-rich phosphorothioate oligonucleotide is wherein, n1 and n2 are each independently an integer from 0 to 10 (ie, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and n3 is 0, 1, 2, 3, or 4 ,and
  • 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, and n'1 and n'2 are each independently 3- an integer of 10 (i.e. 3, 4, 5, 6, 7, 8, 9, 10),
  • n is defined as above.
  • each Am AAC (or CA m AA ) fragment is independently selected from the group consisting of AAAC, AAC, CAAA, CAA.
  • the Am AAC fragment of the non-integer part is selected from: A, AA, AC, AAA, AAC, CA, CAA.
  • (AAAC) Fragments are selected from the group consisting of none, C, A, AA, AC, AAA, AAC, AAAC, CA, CAA, CAAA.
  • the (A m AAC) fragment is selected from the following group: AAAC, AAC, CAAA, CAA.
  • 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, m is all 1.
  • the length of the adenine-rich phosphorothioate oligonucleotide is 16-36 nucleotides.
  • the length of the adenine-rich phosphorothioate oligonucleotide is 17-33 nucleotides.
  • n is 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9 or 10.
  • sequences of the adenine-rich phosphorothioate oligonucleotides are shown in Table A (that is, as shown in SEQ ID NOs: 10-16 and SEQ ID NOs: 7-8) Show.
  • sequence of the adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown in SEQ ID NO: 10-16.
  • sequence of the adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown in SEQ ID NO: 11.
  • the adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown in SEQ ID NO: 12.
  • the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide represented by SEQ ID NO: 13.
  • the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide represented by SEQ ID NO: 14.
  • the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide shown in SEQ ID NO: 15.
  • the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide represented by SEQ ID NO: 16.
  • At least one 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is modified.
  • the 2' sugar groups in the adenine-rich phosphorothioate oligonucleotides are all modified.
  • 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.
  • the sugar group is a deoxyribose group.
  • one or more cytosines in the adenine-rich phosphorothioate oligonucleotide are 5-methylcytosine.
  • the cytosines in the adenine-rich phosphorothioate oligonucleotides are all 5-methylcytosines.
  • the second aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound described in the first aspect, or an optical isomer, a pharmaceutically acceptable salt, a hydrate or an optical isomer thereof. solvates; and pharmaceutically acceptable adjuvants, diluents or carriers.
  • the third aspect of the present invention provides the use of the compound described in the first aspect, or an optical isomer, pharmaceutically acceptable salt, hydrate or solvate thereof, or the use of the pharmaceutical composition described in the second aspect. Use, for preparing a pharmaceutical composition for treating and/or preventing viral infection-related diseases.
  • the diseases include, for example, HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HPV (human papilloma virus) and HIV (human immune system). Defective virus) etc.
  • the infectious disease is a disease related to HBV infection and HBV/HDV co-infection.
  • the disease is selected from the group consisting of: hepatitis B virus infection (HBV), hepatitis C virus infection (HCV), and hepatitis D virus infection (HDV).
  • HBV hepatitis B virus infection
  • HCV hepatitis C virus infection
  • HDV hepatitis D virus infection
  • the disease is selected from: HPV, HIV.
  • Figure 1 shows the antiviral effects of phosphorothioate oligonucleotides of different lengths in the AAV-HBV mouse model
  • Figures 1A, 1B show the antiviral activity of phosphorothioate oligonucleotides of different lengths administered weekly to AAV-HBV-infected C57 mice by intraperitoneal injection, as assessed by qPCR detection of serum HBV-DNA at the end of treatment ;
  • Figures 1C, 1D show the antiviral activity of phosphorothioate oligonucleotides of different lengths administered weekly to C57 mice infected with AAV-HBV by intraperitoneal injection, as assessed by ELISA detection of serum HBsAg at the end of treatment;
  • Figures 1E, 1F show the antiviral activity of phosphorothioate oligonucleotides of different lengths administered twice weekly by intraperitoneal injection to AAV-HBV-infected C57 mice, detected by qPCR at the end of treatment for serum HBV-DNA. Evaluation;
  • Figures 1G, 1H show the antiviral activity of phosphorothioated oligonucleotides of different lengths administered twice weekly by intraperitoneal injection to C57 mice infected with AAV-HBV, as assessed by ELISA detection of serum HBsAg at the end of treatment.
  • Figure 2 shows the changes of serum biochemical indexes albumin (ALB) and creatinine (CRE) of C57 mice infected with AAV-HBV by injection of phosphorothioate oligonucleotides of different lengths.
  • Figure 3 shows the antiviral effects of phosphorothioate oligonucleotides with different base compositions in the AAV-HBV mouse model, wherein
  • Figures 3A, 3B show the antiviral activity of phosphorothioate oligonucleotides with different base constitutions administered weekly by intraperitoneal injection to C57 mice infected with AAV-HBV, and serum HBV-DNA was detected by qPCR at the end of treatment to evaluate;
  • Figures 3C, 3D show the antiviral activity of phosphorothioate oligonucleotides with different base compositions administered weekly by intraperitoneal injection to AAV-HBV-infected C57 mice, as assessed by ELISA detection of serum HBsAg at the end of treatment .
  • Figures 4A, 4B show the antiviral activity of different doses of PA0028 administered by intraperitoneal injection once a week to AAV-HBV-infected C57 mice, as assessed by qPCR detection of serum HBV-DNA at the end of treatment;
  • Figures 4C, 4D show the antiviral activity of different doses of PA0028 administered by intraperitoneal injection once a week to C57 mice infected with AAV-HBV, as assessed by ELISA detection of serum HBsAg at the end of treatment.
  • Figures 5A, 5B show the antiviral activity of adenine-rich phosphorothioate oligonucleotides (ARON) of various lengths administered weekly intraperitoneally to C57 mice infected with AAV-HBV by qPCR at the end of treatment Detect serum HBV-DNA to evaluate;
  • ARON adenine-rich phosphorothioate oligonucleotides
  • Figures 5C, 5D show the antiviral activity of adenine-rich phosphorothioate oligonucleotides (ARON) of various lengths administered weekly by intraperitoneal injection to AAV-HBV-infected C57 mice by ELISA at the end of treatment Serum HBsAg was detected for evaluation.
  • ARON adenine-rich phosphorothioate oligonucleotides
  • Am AAC fragment when written from left to right, when it is connected to other fragments in the molecule, also includes “CA m AA” written from right to left, and m is defined as above.
  • “2' glycosyl 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'-hydroxy, 2'-amino, 2'-halogen or 2'-O-methoxyethyl (2'-MOE).
  • the terms “comprising”, “including” or “comprising” mean that the various ingredients can be used together in the mixture or composition of the present invention.
  • the terms “consisting essentially of” and “consisting of” are encompassed by the term “comprising”.
  • the term "pharmaceutically acceptable” ingredients refers to substances that are suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergy), ie, have a reasonable benefit/risk ratio.
  • the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • the precise effective amount for a subject depends 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. Therefore, it is useless to prespecify the exact effective amount. However, for a given situation, routine experimentation can be used to determine the effective amount, as is the judgment of the clinician.
  • the term "pharmaceutically acceptable salt” refers to a salt of a compound of the present invention with a base that is suitable for use as a medicament.
  • oligonucleotide refers to an oligomer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of modified nucleobases, sugars, and internucleoside phosphodiester linkages, as well as functionally similar oligonucleotides having non-naturally occurring moieties.
  • modified or substituted oligonucleotides may be superior to the native form due to desirable properties such as, for example, reduced immunoreactivity, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and/or increased resistance to nuclease-mediated Degradation stability is improved.
  • Oligonucleotides can be single-stranded or double-stranded, including single-stranded molecules, such as antisense oligonucleotides (ASOs), and aptamers and miRNAs, etc., and double-stranded molecules, such as small interfering RNAs (siRNAs) or Small hairpin RNA (shRNA).
  • ASOs antisense oligonucleotides
  • shRNA Small hairpin RNA
  • the optionally modified oligonucleotides described herein may include various modifications, such as stabilization modifications, and thus may include at least one modification in the phosphodiester linkage and/or on the sugar and/or base.
  • an oligonucleotide may include, but is not limited to, one or more modifications, or may be completely modified to contain all linkages or sugars or bases with the modifications described.
  • Modified linkages can 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 (eg, 2'O-methyl modifications, 2'O-methoxyethyl (2'MOE )), 2'amino modification, 2'halogen modification (eg, 2'-fluoro substitution); acyclic nucleotide analogs.
  • Other 2' modifications are also well known in the art and can be used, such as locked nucleic acids.
  • oligonucleotides have modified linkages throughout or have each linkage modified, e.g., phosphorothioate; have 3'-caps and/or 5'-caps: include terminal 3'- 5' key.
  • Base modifications may include 5' methylation of cytosine bases (5' methylcytosine) and/or 4' thiolation of uracil bases (4' thiouracil).
  • different chemically compatible modified linkages can be combined, such as having linkages with phosphorothioate linkages, 2' ribose modifications (eg 2'O-methylation) and modified linkages oligonucleotides of the base (eg 5'methylcytosine).
  • the oligonucleotides can be further fully modified with all of these different modifications (eg, each phosphorothioate linkage, each 2' modified ribose sugar, and each modified base).
  • phosphorothioated nucleotide refers to a nucleotide having an altered phosphate backbone in which the sugar moieties are linked by phosphorothioate linkages.
  • the phosphorothioate linkage contains a sulfur atom as a replacement for a non-bridging oxygen atom.
  • the phosphorothioate esterification refers to the substitution of phosphorothioate internucleoside bonds of oligonucleotides by phosphorothioate internucleoside bonds.
  • adenine-rich phosphorothioate oligonucleotide or its English abbreviation "ARON” (Adenosine Rich Oligo Nucleotides) refers to those containing adenine in a proportion of more than 50% of all bases (such as 75 %, 80%, 85%, 90%) of phosphorothioated oligonucleotides whose antiviral activity is not dependent on Toll-like receptor recognition, hybridization to target RNA or DNA, or sequence-dependent formation of secondary or tertiary Hierarchical aptamer interactions.
  • each chiral carbon atom can optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.
  • 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 low pressure sublimation drying.
  • the active ingredient of the present invention is adenine-rich phosphorothioate oligonucleotide (ARON), or an optical isomer, pharmaceutically acceptable salt, hydrate, or solvate thereof,
  • ARON adenine-rich phosphorothioate oligonucleotide
  • the adenine-rich phosphorothioate oligonucleotides comprise ( Am AAC) n repeats ;
  • 80-100% of the phosphates in the adenine-rich phosphorothioate oligonucleotides are phosphorothioates.
  • all the phosphates in the adenine-rich phosphorothioate oligonucleotides are phosphorothioates.
  • n is an integer; or when n is a non-integer, the length of the adenine-rich phosphorothioate oligonucleotide sequence is an integer.
  • the sequence of the adenine-rich phosphorothioate oligonucleotide is where n1 and n2 are each independently an integer from 0 to 10, n3 is 0, 1, 2, 3, or 4, and
  • the Am AAC fragment of the integer part is selected from the group consisting of AAAC , AAC, CAAA, CAA.
  • the non-integer part of Am AAC fragment (such as (AAAC) n3 is 1, 2 or 3) selected from one or several oligonucleotides in the AAAC fragment (eg A, C, AA, AC, AAA, AAC, CAA).
  • the non-integer part of Am AAC fragment (such as (AAAC)
  • the number of nucleotides where n3 is 1, 2, or 3) depends on the non-integer part of n (eg, ) value to determine, for example, when the non-integer part is 0.25 (that is, n3 is 1), the number of oligonucleotides in the non-integer part is 1, that is, A or C; for example, when the non-integer part is 0.5 (that is, n3 When it is 2), the number of oligonucleotides in the non-integer part is 2, namely AA or AC, and so on.
  • (AAAC) Fragments are selected from the group consisting of none, A, AA, AC, AAA, AAC, AAAC, CAA, CAAA.
  • n is 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9 or 10.
  • the length of the adenine-rich phosphorothioate oligonucleotide is 16-36 nucleotides.
  • the length of the adenine-rich phosphorothioate oligonucleotide is 17-33 nucleotides.
  • the adenine-rich phosphorothioate oligonucleotide is an AAAC repeat, and its length is 16-40 nucleotides, more preferably, 16-36.
  • the adenine-rich phosphorothioate oligonucleotide is an AAC repeat sequence, and its length is 15-39 nucleotides.
  • the adenine-rich phosphorothioate oligonucleotide comprises AAAC sequence and AAC sequence, and its length is 15-39 nucleotides.
  • At least one 2' sugar group of the adenine-rich phosphorothioate oligonucleotide (ARON) is modified, more preferably, all 2' sugar groups are modified wherein, the modification is selected from the group consisting of: 2'-O-alkyl modification, 2'-hydroxyl modification, 2'-amino modification, 2'-halogen modification, 2'-O-methoxyethyl modification .
  • the sugar group of the adenine-rich phosphorothioate oligonucleotide is deoxyribose.
  • one or more cytosines in the adenine-rich phosphorothioate oligonucleotides are 5-methylcytosines, preferably, all cytosines are 5-methylcytosines base cytosine.
  • the sequence of the adenine-rich phosphorothioate oligonucleotide is as shown in Table A
  • sequences of the adenine-rich phosphorothioate oligonucleotides are shown in SEQ ID NOs: 10-16.
  • the adenine-rich phosphorothioate oligonucleotide is the oligonucleotide shown in SEQ ID NO: 11.
  • At least one 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is modified, more preferably, the adenine-rich phosphorothioate oligonucleotide
  • the 2' sugar groups in the acid are all modified.
  • the modification of the 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is selected from the following group: 2'-O-alkyl modification, 2' amino modification, 2' halogen modification,
  • the modification of the 2' glycosyl group is a 2'O-methyl modification or 2'O-methoxyethyl (2'MOE).
  • the phosphodiester bond is a phosphorothioate bond.
  • thermodynamically stable isomers such as tautomers, conformers, meso compounds and optical isomers with enantiomeric or diastereomeric relationships, may exist after the compounds of the present invention are prepared etc., the above modifications will be apparent to those skilled in the art after reading the disclosure of the present invention.
  • the adenine-rich phosphorothioate oligonucleotides of the present invention can be prepared and synthesized by conventional synthesis methods in the oligonucleotide industry.
  • the phosphoramidite solid-phase synthesis method can be used, and the sulfur transfer reagent diphenylacetyl disulfide (PADS) is used to replace the oxidant iodine (iodine), which can convert the phosphate bond into phosphorothioate ester bonds to produce thiooligonucleotides.
  • PADS sulfur transfer reagent diphenylacetyl disulfide
  • compositions and methods of administration are provided.
  • the phosphorothioated oligonucleotides can be used in combination with other drugs known to treat or ameliorate similar conditions. When co-administered, the mode and dose of the original drug can be kept unchanged while the phosphorothioated oligonucleotide is administered concurrently or subsequently. When the phosphorothioated oligonucleotide is administered simultaneously with one or more other drugs, it may be preferable to use a pharmaceutical composition containing both one or more known drugs and the phosphorothioated oligonucleotide . Drug combinations also include the administration of phosphorothioated oligonucleotides with one or more other known drugs for overlapping time periods. When phosphorothioated oligonucleotides are administered in combination with one or more other drugs, the doses of phosphorothioated oligonucleotides or known drugs may be lower than they would be administered alone.
  • a suggestive and effective dosing regimen for the administration of the adenine phosphorothioated oligonucleotide-rich agents of the present invention to humans, as described in Example III, is once a week (QW), a single dose 3 mg/kg (based on body surface area); follow dosing regimen typically used for other phosphorothioated oligonucleotides (eg, antisense oligonucleotide ASO); weekly parenterals are well established in the art Routine use of 100-500 mg of compound is administered.
  • compositions described herein may be administered by any suitable means, eg, oral ingestion; oral inhalation; by subcutaneous, intravenous injection or infusion; may be in the presence of a non-toxic pharmaceutically acceptable carrier or diluent in dosage unit formulations.
  • the compositions may be administered in formulations suitable for immediate or sustained release.
  • a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) in need of treatment, and the dose is the effective dose considered pharmaceutically, for a 60kg body weight, the daily dose is
  • the administration dose is usually 1 to 2000 mg, preferably 50 to 1000 mg.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • phosphorothioate oligonucleotides with different bases have different anti-hepatitis B surface antigen activities.
  • the adenine-rich phosphorothioate oligonucleotide with a length of 16 nt is the shortest oligonucleotide with significant anti-HBV activity.
  • the adenine-rich phosphorothioate oligonucleotide of the present invention has high antiviral activity and a sufficiently large safety window.
  • the present invention can also be applied to the treatment of infection caused by HIV and HPV.
  • Example 1 Antiviral effect of phosphorothioate oligonucleotides of different lengths in AAV-HBV mouse model
  • Phosphorothioate oligonucleotides of different lengths were tested to establish their antiviral activity in c57 mice infected with adeno-associated virus carrying HBV 1.3 ploidy and persistently replicating HBV-DNA and expressing HBV antigens.
  • 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 chemical descriptions of these oligonucleotides.
  • a mouse model of persistent hepatitis B infection was established by injecting male C57BL/6 mice with 5 ⁇ 10 10 rAAV8-1.3HBV (Wujia and) through the tail vein. After confirming the stable replication of HBV virus, they were randomly divided into 10 groups according to their body weight (4 animals/group). group) and 90 mg/kg of PA0001, PA0005, PA0008, and PA0028, and the vehicle group served as the control group. Groups 2, 4, 6, 8, and 10 were injected with vehicle (10ml/kg) and 90mg/kg of PA0001, PA0005, PA0008, and PA0028 by intraperitoneal injection twice a week (BIW), respectively. The vehicle group served as the control group. After 6 weeks of administration, blood was drawn twice a week, the titer of hepatitis B virus in serum was analyzed by qPCR, the concentration of surface antigen in serum was analyzed by ELISA, and the curve was drawn.
  • the hepatitis B virus titer in serum (see Figure 1A, 1B, 1E, 1F), the concentration of surface antigen in serum (see Figure 1C, 1D, 1G, 1H), in the experiment group 5 (PA0005QW) 2 animals died, the first 1 animal died in group 4 (PA0001BIW), 1 animal died in group 6 (PA0005BIW), no animal died in other groups, animals in groups 3, 5, 4, 6 (PA 0001QW, PA0005QW, PA0001BIW, PA0005BIW) were seen Significant spleen enlargement, no significant spleen changes were seen in other groups, spleen enlargement may be related to immune activation caused by excess oligonucleotides.
  • FIG. 2A and Figure 2B respectively show the changes of serum biochemical indexes albumin (ALB) and creatinine (CRE) in C57 mice infected with AAV-HBV by injection of phosphorothioate oligonucleotides of different lengths.
  • ARB serum biochemical indexes albumin
  • CRE creatinine
  • All oligonucleotides can lead to the reduction of serum HBsAg and HBV-DNA.
  • Oligonucleotides (PA0001, PA0005, PA0008) with the same repeat sequence and length of 20-40 nt have equivalent antiviral effects, and the toxicity of oligonucleotides is positively correlated with the length. The longer the length, the greater the toxicity. The severity of causing splenomegaly was also positively correlated with the length of the oligonucleotide, the longer the length, the more pronounced the splenomegaly.
  • Phosphorothioate oligonucleotides with 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 antigen to evaluate their resistance to viral 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:11) ID NO: 3) and PA0031 (SEQ ID NO: 4), Table 3 provides chemical descriptions of these oligonucleotides.
  • a mouse model of persistent hepatitis B infection was established by injecting male C57BL/6 mice with 5 ⁇ 10 10 rAAV8-1.3HBV (Wujia and) through the tail vein. After confirming the stable replication of HBV virus, they were randomly divided into 7 groups according to body weight (5 animals/group), and were injected with vehicle (10ml/kg) and 90mg/kg of PA0008, PA00027, PA0028, PA0029, PA0030, PA0031, the vehicle group served as the control group. After 12 weeks of administration, blood was collected twice a week, the titer of hepatitis B virus in serum was analyzed by qPCR method, the surface antigen content in serum was analyzed by ELISA method, and the curve was drawn.
  • oligonucleotides of the same length is related to the base composition.
  • adenine-rich phosphorothioate oligos were tested in c57 mice infected with adeno-associated virus (AAV-HBV, Wujia sum) carrying HBV 1.3 ploidy and persistently replicating HBV-DNA and expressing HBV antigens
  • AAV-HBV, Wujia sum adeno-associated virus carrying HBV 1.3 ploidy and persistently replicating HBV-DNA and expressing HBV antigens
  • the nucleotide PA0028 was used to evaluate the dose-dependent relationship of its antiviral activity, and Table 5 provides a chemical description of PA0028.
  • a mouse model of persistent hepatitis B infection was established by injecting male C57BL/6 mice with 5 ⁇ 10 10 rAAV8-1.3HBV (Wujia and) through the tail vein. After confirming the stable replication of HBV virus, they were randomly divided into 4 groups according to their body weight (5 in each group), and once a week (QW) were injected intraperitoneally with PA0028 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, the titer of hepatitis B virus in serum was analyzed by qPCR, the content of surface antigen in serum was analyzed by ELISA, and the curve was drawn.
  • the HBsAg and HBV-DNA in the animal serum of the vehicle control group showed a steady fluctuation, and the animal serum HBV-DNA of the three different dose groups of PA0028 all showed a downward trend of fluctuation > 1log 10 , and the HBV-DNA of some animals decreased Up to the lower limit of quantification (LLOQ), 60% of animals (3/5) had a >2 log 10 reduction in DNA, so 10 mg/kg was considered to be the effective dose that caused a significant reduction in HBV-DNA.
  • Adenine-rich phosphorothioate oligos of different lengths were tested in c57 mice infected with adeno-associated virus (AAV-HBV, Wujia sum) carrying HBV 1.3 ploidy and persistently replicating HBV-DNA and expressing HBV antigens nucleotides to establish its antiviral activity.
  • AAV-HBV adeno-associated virus
  • These phosphorothioated oligonucleotides are PA00017 (SEQ ID NO:9), PA0018 (SEQ ID NO:10) and PA0028 (SEQ ID NO:11) and Table 7 provides chemical descriptions of these oligonucleotides.
  • a mouse model of persistent hepatitis B infection was established by injecting male C57BL/6 mice with 5 ⁇ 10 10 rAAV8-1.3HBV (Wujia and) through the tail vein. After confirming the stable replication of HBV virus, they were randomly divided into 4 groups according to their body weight, and the vehicle (10ml/kg) and 90mg/kg PA00017, PA00018 and PA0028 were injected intraperitoneally once a week (QW), respectively. The vehicle group served as the control group. After 12 weeks of administration, blood was collected twice a week, the titer of hepatitis B virus in serum was analyzed by qPCR, the content of surface antigen in serum was analyzed by ELISA, and the curve was drawn.
  • the activity is positively correlated with the length, and the longer the length, the better the activity.
  • phosphorothioate oligonucleotides with a length > 20 nt at a dose of 90 mg/kg in Example I can cause toxicity leading to animal death
  • Example III shows that a dose of ⁇ 30 mg/kg fails to Significantly increased anti-HBV activity
  • the administered dose of each compound in Example IV was 90 mg/kg as a higher dose, therefore, it can be seen that 16nt has significant anti-HBV activity as adenine-rich phosphorothioate oligonucleotides the shortest length.

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Abstract

涉及一种富含腺嘌呤硫代磷酸酯化寡核苷酸及其抗肝炎病毒的应用。具体地，涉及一种包含(AmAAC)n重复序列的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)用于抑制乙肝表面抗原(HBsAg)及其用于治疗乙型肝炎病毒感染或乙型肝炎病毒/丁型肝炎病毒共感染的用途，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)长度为15-40个核苷酸，包含腺嘌呤核苷酸和胞嘧啶核苷酸，其中，腺嘌呤核苷酸的含量超过50％，具有较高的抗病毒活性且安全窗足够大。

Description

富含腺嘌呤硫代磷酸酯化寡核苷酸及其抗肝炎病毒的应用 技术领域
本发明涉及医药生物领域，具体涉及一种富含腺嘌呤硫代磷酸酯化寡核苷酸及其抗肝炎病毒的应用。
背景技术
世界卫生组织公布的数据显示，2015年全球有超过2亿人慢性感染HBV，有88.7万人因HBV感染引起的并发症而死亡。虽然全球已经批准了一些抗病毒药物用于治疗HBV感染，但是现有药物的疗法或组合疗法除了在小部分患者(<3％)外，无法引起能够提供感染的持久控制或者功能性治愈的有效免疫反应或HBsAg的血清学转换。
感染HBV的成年人90％能够自愈，但是婴幼儿感染HBV后，90％会发展成为慢性肝炎。慢性HBV感染可能会导致肝纤维化，进一步发展为肝硬化和肝细胞癌(HCC)。另外，有研究表明乙肝会增加胰腺癌风险。治愈或功能性治愈以HBV慢性感染是巨大的未满足的临床需求。
HBV感染人肝细胞后主要产生两种不同的颗粒，一种是Dane颗粒，也就是完整的HBV病毒本身，包括由乙肝核心抗原(HBcAg)和病毒核酸(RcDNA)组装而成的病毒核衣壳，且具有由乙肝表面抗原(HBsAg)组成的病毒包膜；另一种是亚病毒颗粒(SVP)，其是由脂质、胆固醇、胆固醇酯和乙肝表面抗原(HBsAg)组成的非感染性颗粒。SVP所包含的乙肝表面抗原占患者血液中乙肝表面抗原的绝大多数(>99.9％)。HBV感染的肝细胞还分泌一种e-抗原(HBeAg)到血液中。乙肝表面抗原(HBsAg)、乙肝表面抗体(HBsAb)、乙肝核心抗体(HBcAb)、乙肝e抗原(HBeAg)以及乙肝e抗体(HBeAb)是评价药物对病毒干预情况的重要分子标记物。
丁型肝炎病毒(HDV)是HBV的卫星病毒，依赖乙肝表面抗原(HBsAg)形成其完整的具有感染性的HDV病毒颗粒，HDV感染仅可在伴随HBV感染的患者中发生。HDV/HBV共感染并发症显著增加了肝纤维化至肝硬化的进展速率。对于HDV慢性感染的患者，目前仅有干扰素治疗一种干预手段，无直接靶向HDV病毒的上市药物，现有治疗方法疗效不佳，副作用显著。
临床上用于治疗乙型肝炎的药物主要有干扰素类和核苷(酸)类药物。干扰素类药物有普通干扰素和聚乙二醇修饰的长效干扰素，后者包括派罗欣(PEG-IFNα-2a)和佩乐能(PEG-IFNα-2b)。核苷(酸)类药物包括拉米夫定、替比夫定、阿德福韦酯、富马酸替诺福韦二吡呋酯(TDF)、富马酸替诺福韦艾拉酚胺(TAF)、恩替卡韦等。这些核苷类药物能有效的控制病毒的复制，改善肝功能，因而应用最为广泛。干扰素需要注射给药，个体反应差异大，不良反应明显，且疗效不佳。核苷类药物仅作用于病毒从pgRNA到rcDNA的复制过程，对乙肝病毒生命周期中其他的环节没有抑制作用。长期治疗，乙 肝e抗原(HBeAg)转阴率仍然较低，极少数患者乙肝表面抗原(HBsAg)能够转阴。恩替卡韦(354例)和替诺福韦(176例)治疗48周，在乙肝e抗原(HBeAg)阳性的患者中乙肝表面抗原(HBsAg)转阴率分别为2％和3.2％，在乙肝e抗原(HBeAg)阴性的患者中乙肝表面抗原(HBsAg)转阴率分别为0.3％和0％。由于现有的治疗方案不能治愈乙肝，需要患者长期服药，可能使患者面临重大副作用，例如长期服用阿德福韦酯和富马酸替诺福韦二吡呋酯均可导致肾毒性和骨毒性。
HBV慢性感染患者血液中大量的以亚病毒颗粒(SVP)形式存在的乙肝表面抗原(HBsAg)可以中和B淋巴细胞分泌的特异性乙肝表面抗体(HBsAb)，进而导致免疫耐受，而仅占少数的HBV病毒颗粒则能够逃逸免疫检查，这可能是HBV保持慢性感染的重要原因之一。尽管研究表明，三种病毒抗原乙肝表面抗原(HBsAg)、乙肝核心抗原(HBcAg)、乙肝e抗原(HBeAg)都具有免疫抑制性质，但乙肝表面抗原(HBsAg)在HBV感染对象的血液中占所有病毒抗原的绝大多数，并有可能是抑制宿主免疫的最主要的诱导物。乙肝表面抗原(HBsAg)的血清学转换(HBsAg从血液中清除，游离的HBsAb的出现)是治疗时，病毒感染获得功能性控制的公认的预后指标。HBV保持慢性感染特征的另一关键原因是其在感染的肝细胞的细胞核中借助宿主DNA修复酶合成了稳定的环状DNA存储库，即HBV共价闭合环状DNA(cccDNA)。cccDNA可在肝细胞中长期稳定存在，并能够持续获得补充，其可通过转录和逆转录产生HBV病毒的核酸RcDNA及编码全部病毒抗原所需的mRNA。cccDNA的转录抑制或清除对于治愈或功能性治愈HBV感染是至关重要的。核苷(酸)类似物长期治疗并不能彻底清除cccDNA，也不能抑制其转录，因此乙肝表面抗原(HBsAg)表达水平几乎不受核苷(酸)类药物的影响。免疫调节可介导体液和细胞免疫，进而抑制cccDNA转录或者清除被感染的细胞，但大的抗原负荷会极大的抑制该免疫过程，因此大幅降低抗原，尤其是乙肝表面抗原(HBsAg)，结合免疫调节是帮助患者获得持久免疫控制的有效手段。
目前，尚缺乏活性高且安全窗足够大的硫代磷酸酯化寡核苷酸，以用于治疗HBV和HBV/HDV共感染。
发明内容
本发明目的是提供一种活性高且安全窗足够大的硫代磷酸酯化寡核苷酸，以用于治疗HBV和HBV/HDV共感染。
本发明的第一方面，提供一种化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，所述的化合物为富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)，其中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸包含(A mAAC) n重复序列；
其中，m各自独立地为0或1；n为≥4的正数；且所述富含腺嘌呤硫代磷酸酯化寡核苷酸的长度为15-40个核苷酸；
所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中80-100％的磷酸酯为硫代磷酸酯。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中95-100％的磷酸酯为硫代磷酸酯。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中的磷酸酯全部为硫代磷酸酯。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的序列为
Figure PCTCN2021101064-appb-000001
Figure PCTCN2021101064-appb-000002
其中，n1和n2各自独立地为0-10(即0、1、2、3、4、5、6、7、8、9、10)的整数，n3为0、1、2、3或4，且
Figure PCTCN2021101064-appb-000003
m和n的定义如上所述。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的序列为(R)(A mAAC) n'2、(A mAAC) n'1(R)、(R)(CA mAA) n'2或(CA mAA) n'1(R)，其中，各R独立的选自：A或C，n'1和n'2各自独立地为3-10(即3、4、5、6、7、8、9、10)的整数，
m的定义如上所述。
在另一优选例中，各A mAAC(或CA mAA)片段独立地选自下组：AAAC、AAC、CAAA、CAA。
在另一优选例中，当n为非整数时，非整数部分的A mAAC片段选自：A、AA、AC、AAA、AAC、CA、CAA。
在另一优选例中，(AAAC)
Figure PCTCN2021101064-appb-000004
片段选自下组：无、C、A、AA、AC、AAA、AAC、AAAC、CA、CAA、CAAA。
在另一优选例中，当n为整数时，(A mAAC)片段选自下组：AAAC、AAC、CAAA、CAA。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸包含(A mAAC) n重复序列，n为≥4的正数；m各自独立地为0或1，且至少有一个m为1；优选地m均为1。
在另一优选例中，所述富含腺嘌呤硫代磷酸酯化寡核苷酸的长度为16-36个核苷酸。
在另一优选例中，所述富含腺嘌呤硫代磷酸酯化寡核苷酸的长度为17-33个核苷酸。
在另一优选例中，n为4、4.5、5、5.5、6、6.5、7、8、9或10。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的序列如表A(即如SEQ ID NO：10-16和SEQ ID NO：7-8所示)所示。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)的序列如SEQ ID NO：10-16所示的寡核苷酸。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)的序列为如SEQ ID NO：11所示的寡核苷酸。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为SEQ ID NO：12所示的寡核苷酸。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为任选修饰的由SEQ ID NO：13所示的寡核苷酸。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为任选修饰的由SEQ ID NO：14所示的寡核苷酸。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为任选修饰的由SEQ ID NO：15所示的寡核苷酸。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为任选修饰的由SEQ ID NO：16所示的寡核苷酸。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的至少一个2'糖基是修饰的。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中的2'糖基均是修饰的。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的2'糖基的修饰选自下组：2'-O-烷基修饰、2'-羟基修饰、2'-氨基修饰、2'卤素修饰或2'-O-甲氧基乙基(2'MOE)修饰，优选地为2'O-甲基修饰。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中，糖基为脱氧核糖基。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中一个或多个胞嘧啶为5-甲基胞嘧啶。
在另一优选例中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中的胞嘧啶均为5-甲基胞嘧啶。
本发明第二方面，提供一种药物组合物，所述的药物组合物包括治疗有效量的如第一方面所述的化合物，或其光学异构体、药学上可接受的盐、水合物或溶剂化物；和药学上可接受的辅助剂、稀释剂或载体。
本发明第三方面，提供一种第一方面所述的化合物，或其光学异构体、药学上可接受的盐、水合物或溶剂化物的用途，或第二方面所述的药物组合物的用途，用于制备治疗和/或预防病毒感染相关的疾病的药物组合物。
在另一优选例中，所述的疾病包括，例如HBV(乙型肝炎病毒)、HCV(丙型肝炎病毒)、HDV(丁型肝炎病毒)、HPV(人乳头瘤病毒)和HIV(人类免疫缺陷病毒)等。
在另一优选例中，所述的感染疾病为HBV感染和HBV/HDV共感染相关的疾病。
在另一优选例中，所述的疾病选自：乙型病毒性肝炎感染(HBV)、丙型病毒性肝炎感染(HCV)、丁型病毒性肝炎感染(HDV)。
在另一优选例中，所述的疾病选自：HPV、HIV。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了不同长度的硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果；其中
图1A,1B显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同长度的硫代磷酸酯化寡核苷酸的抗病毒活性，在治疗结束时通过qPCR检测血清HBV-DNA来评价；
图1C,1D显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同长度的硫代磷酸酯化寡核苷酸的抗病毒活性，在治疗结束时通过ELISA检测血清HBsAg来评价；
图1E,1F显示每周两次腹腔注射给予被AAV-HBV感染的C57小鼠不同长度的硫代磷酸酯化寡核苷酸的抗病毒活性，在治疗结束时通过qPCR检测血清HBV-DNA来评价；
图1G,1H显示每周两次腹腔注射给予被AAV-HBV感染的C57小鼠不同长度的硫代磷酸酯化寡核苷酸的抗病毒活性，在治疗结束时通过ELISA检测血清HBsAg来评价。
图2显示注射给予被AAV-HBV感染的C57小鼠不同长度的硫代磷酸酯化寡核苷酸的血清生化指标白蛋白(ALB)、肌酐(CRE)的变化。
图3显示不同碱基构成的硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果，其中
图3A,3B显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同碱基构成的硫代磷酸酯化寡核苷酸的抗病毒活性，在治疗结束时通过qPCR检测血清HBV-DNA来评价；
图3C,3D显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同碱基构成的硫代磷酸酯化寡核苷酸的抗病毒活性，在治疗结束时通过ELISA检测血清HBsAg来评价。
图4不同剂量的富含腺嘌呤硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果，其中
图4A,4B显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同剂量的PA0028的抗病毒活性，在治疗结束时通过qPCR检测血清HBV-DNA来评价；
图4C,4D显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同剂量的PA0028的抗病毒活性，在治疗结束时通过ELISA检测血清HBsAg来评价。
图5不同长度的富含腺嘌呤硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果，其中，
图5A,5B显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同长度的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)的抗病毒活性，在治疗结束时通过qPCR检测血清HBV-DNA来评价；
图5C,5D显示每周一次腹腔注射给予被AAV-HBV感染的C57小鼠不同长度的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)的抗病毒活性，在治疗结束时通过ELISA检测血清HBsAg来评价。
具体实施方式
本发明人经过长期而深入的研究，通过对实验条件的探索和优化，首次在啮齿类HBV持续感染动物模型中验证了硫代磷酸酯化寡核苷酸类化合物的抗病毒活性；并发现长度为20-40nt的寡核苷酸抗乙肝表面抗原的活性无显著差异；首次发现不同碱基构成的硫代磷酸酯化寡核苷酸抗乙肝表面抗原的活性不同，腺嘌呤含量大于50％(如75％)的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)，抑制乙肝病毒复制和抗乙肝表面抗原的活性最好；还发现这类富含腺嘌呤硫代磷酸酯化寡核苷酸具有抗乙肝病毒活性的最短长度为16nt。基于上述发现，发明人完成了本发明。
本发明中，“A mAAC片段”当通过从左向右书写时，其与分子中的其他片段连接时，其也同样包括从右向左书写方式“CA mAA”，m的定义如上。
本发明中，“2'糖基修饰”包括“2'核糖修饰”和“2'脱氧核糖修饰”，其中，所述的修饰选自下组：2'-O-烷基(如2'-O-甲基)、2'-羟基、2'-氨基、2'-卤素或2'-O-甲氧基乙基(2'-MOE)。
本发明中，术语“含有”、“包括”或“包含”表示各种成分可以一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
本发明中，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比的物质。
本发明中，术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。
如本文所用，术语“药学上可接受的盐”指本发明化合物与碱所形成的适合用作药物的盐。
术语“寡核苷酸”是指核糖核酸(RNA)和/或脱氧核糖核酸(DNA)的低聚物。该术语包括由修饰的核碱基、糖和核苷间磷酸二酯键构成的寡核苷酸，以及具有非天然存在的部分的功能类似的寡核苷酸。这种修饰或取代的寡核苷酸可以由于诸如以下的期望的性质而优于自然形式：例如，免疫反应性降低、细胞摄取增强、对核酸靶标的亲和力增强和/或对核酸酶介导的降解的稳定性提高。寡核苷酸可以是单链或双链的，包括单链分子，诸如反义寡核苷酸(ASO)、和适配体和miRNA等，以及双链分子，诸如小干扰RNA(siRNA)或小发夹RNA(shRNA)。本发明所述的任选修饰的寡核苷酸可以包括各种修饰，例如稳定修饰，并且因此可以在磷酸二酯键中和/或在糖和/或碱基上包括至少一种修饰。例如，寡核苷酸可包括但不限于，一种或多种修饰，或可完全经修饰以便含有具有所述的修饰的所有键或糖或碱基。经过修饰的键可以包括硫代磷酸酯键和二硫代磷酸酯键。额外有用的 修饰包括但不限于，在糖的2'位置的修饰，包括2'-O-烷基修饰(如2'O-甲基修饰、2'O-甲氧基乙基(2'MOE))、2'氨基修饰、2'卤素修饰(如2'-氟取代)；无环核苷酸类似物。其他2'修饰也是本领域中熟知的并且可加以使用，诸如锁核酸。具体地，寡核苷酸具有遍及各处的经修饰的键或具有经修饰的每个键，例如，硫代磷酸酯；具有3'-帽和/或5'-帽:包括末端3'-5'键。碱基修饰可包括胞嘧啶碱基的5'甲基化(5'甲基胞嘧啶)和/或尿嘧啶碱基的4'硫基化(4'硫尿嘧啶)。当合成条件是化学相容的时候，则可组合不同的化学相容的修饰的键，例如具有带有硫代磷酸酯键、2'核糖修饰(例如2'O-甲基化)和经修饰的碱基(例如5'甲基胞嘧啶)的寡核苷酸。可利用所有这些不同的修饰(例如每个硫代磷酸酯化键、每个2'修饰的核糖和每个经修饰的碱基)进一步完全修饰寡核苷酸。
术语“硫代磷酸酯化的核苷酸”指具有改变的磷酸酯主链的核苷酸，其中，糖部分通过硫代磷酸酯键连接。在寡核苷酸序列的磷酸酯主链中，硫代磷酸酯键含有硫原子作为非桥连氧原子的替代物。
所述的硫代磷酸酯化是指寡核苷酸的氧代磷酸酯核苷间键被硫代磷酸酯核苷间键取代。
本发明中示意性的硫代磷酸酯键如下所示：
Figure PCTCN2021101064-appb-000005
如本文所用，术语“富含腺嘌呤硫代磷酸酯化寡核苷酸”或其英文缩写“ARON”(Adenosine Rich Oligo Nucleotides)是指含有腺嘌呤占全部碱基比例为50％以上(如75％、80％、85％、90％)的硫代磷酸酯化寡核苷酸，其抗病毒活性并非取决于Toll样受体识别、与靶标RNA或DNA的杂交或依赖序列形成二级或三级结构的适配体相互作用。
除非特别说明，本发明中，所有出现的化合物均意在包括所有可能的光学异构体，如单一手性的化合物，或各种不同手性化合物的混合物(即外消旋体)。本发明的所有化合物之中，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。
本发明中的一些化合物可能用水或各种有机溶剂结晶或重结晶，在这种情况下，可能形成各种溶剂化物。本发明的溶剂合物包括化学计量的溶剂化物如水合物等，也包括在用低压升华干燥法制备时形成的包含可变量水的化合物。
活性成分
本发明的活性成分是富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，
所述的富含腺嘌呤硫代磷酸酯化寡核苷酸包含(A mAAC) n重复序列；
其中，m各自独立地为0或1；n为≥4的正数；且所述富含腺嘌呤硫代磷酸酯化寡核苷酸的长度为15-40个核苷酸；
所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中80-100％的磷酸酯为硫代磷酸酯。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中的磷酸酯全部为硫代磷酸酯。
优选地，n为整数；或当n为非整数时，满足富含腺嘌呤硫代磷酸酯化寡核苷酸序列的长度为整数。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的序列为
Figure PCTCN2021101064-appb-000006
Figure PCTCN2021101064-appb-000007
其中，n1和n2各自独立地为0-10的整数，n3为0、1、2、3或4，且
Figure PCTCN2021101064-appb-000008
m和n的定义如上所述。
优选地，上述各序列中，整数部分的A mAAC片段选自下组：AAAC、AAC、CAAA、CAA。
优选地，上述各序列中，当n为非整数时，非整数部分的A mAAC片段(如(AAAC)
Figure PCTCN2021101064-appb-000009
n3为1、2或3)选自AAAC片段中某一个或某几个寡核苷酸(如A、C、AA、AC、AAA、AAC、CAA)。
优选地，上述各序列中，非整数部分的A mAAC片段(如(AAAC)
Figure PCTCN2021101064-appb-000010
n3为1、2或3)的核苷酸个数依据n的非整数部分(如，
Figure PCTCN2021101064-appb-000011
)的值来确定，如当非整数部分为0.25(即n3为1)时，非整数部分的寡核苷酸个数为1个，即A或C；如当非整数部分为0.5(即n3为2)时，非整数部分的寡核苷酸个数为2个，即AA或AC，依次类推。
优选地，(AAAC)
Figure PCTCN2021101064-appb-000012
片段选自下组：无、A、AA、AC、AAA、AAC、AAAC、CAA、CAAA。
优选地，上述各序列中，n为4、4.5、5、5.5、6、6.5、7、8、9或10。
优选地，上述各序列中，所述富含腺嘌呤硫代磷酸酯化寡核苷酸的长度为16-36个核苷酸。
优选地，上述各序列中，所述富含腺嘌呤硫代磷酸酯化寡核苷酸的长度为17-33个核苷酸。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为AAAC重复序列，且其长度为16-40个核苷酸，更佳地，16-36个。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为AAC重复序列，且其长度为15-39个核苷酸。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸包含AAAC序列和AAC序列，且其长度为15-39个核苷酸。
优选地，上述各序列中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)的至少一个2'糖基是修饰的，更佳地，2'糖基均是修饰的；其中，所述的修饰选自下组：2'-O-烷基修饰、2'-羟基修饰、2'-氨基修饰、2'-卤素修饰、2'-O-甲氧基乙基修饰。
优选地，上述各序列中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的糖基为脱氧核糖基。
优选地，上述各序列中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中一个或多个胞嘧啶为5-甲基胞嘧啶，较佳地，胞嘧啶均为5-甲基胞嘧啶。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的序列如表A所示
表A
Figure PCTCN2021101064-appb-000013
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的序列如SEQ ID NO：10-16所示。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为由SEQ ID NO：11所示的寡核苷酸。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的至少一个2'糖基是经过修饰的，更优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中的2'糖基均是经过修饰的。
优选地，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的2'糖基的修饰选自下组：2'-O-烷基修饰、2'氨基修饰、2'卤素修饰，优选地，2'糖基的修饰为2'O-甲基修饰或2'O-甲氧基乙基(2'MOE)。
优选地，上述的富含腺嘌呤硫代磷酸酯化寡核苷酸中，磷酸二酯键为硫代磷酸二酯键。
应理解，本发明的化合物制备后可能存在各种热力学稳定的异构体,如互变异构体、构象异构体、内消旋化合物和具有对映或非对映关系的光学异构体等，上述改变形式在阅读了本发明的公开之后，对于本领域技术人员而言是显而易见的。
硫代磷酸酯化寡核苷酸的制备
本发明中的富含腺嘌呤硫代磷酸酯化寡核苷酸可以用寡核酸工业的常规合成方法进行制备合成。例如可以在GE OP100这种设备的控制下，使用亚磷酰胺固相合成法，并用硫转移试剂二硫化二苯乙酰(PADS)替代氧化剂碘(iodine)，可将磷酸酯键变成硫代磷酸酯键，从而制备出硫代寡聚核苷酸。
药物组合物和施用方法
所述硫代磷酸酯化寡核苷酸(ARON)可以与已知的治疗或改进相似病状的其他药物联用。联合给药时，原来药物的给药方式和剂量可以保持不变，而同时或随后施用硫代磷酸酯化寡核苷酸。当硫代磷酸酯化寡核苷酸与其它一种或几种药物同时施用时，可以优选使用同时含有一种或几种已知药物和硫代磷酸酯化寡核苷酸的药用组合物。药物联用也包括在重叠的时间段施用硫代磷酸酯化寡核苷酸与其它一种或几种已知药物。当硫代磷酸酯化寡核苷酸与其它一种或几种药物进行药物联用时，硫代磷酸酯化寡核苷酸或已知药物的剂量可能比它们单独用药的剂量低。
针对本发明的富含腺嘌呤硫代磷酸酯化寡核苷酸的药剂施用于人体的提示性有效的给药方案，如实施例III所述的每周给药一次(QW)，单次剂量为3mg/kg(基于体表面积换算)；遵循通常用于其他硫代磷酸酯化寡核苷酸(例如反义寡核苷酸ASO)的给药方案；本领域中充分确立每周胃肠外施用100-500mg化合物的常规使用。
根据本文中所呈现的公开内容，利用药学上可接受的富含腺嘌呤硫代磷酸酯化寡核苷酸制剂治疗具有HBV感染或HBV/HDV共感染的对象是有用的。
本文所述的组合物可通过任何合适的手段来施用，例如，经口摄入；经口吸入；通过皮下、静脉注射或输注；可以在含有无毒性的药学上可接受的载体或稀释剂的剂量单位制剂中。例如，可以适用于立即释放或缓释的制剂施用本组合物。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂 量通常为1～2000mg，优选50～1000mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
发明主要优点：
1、首次在啮齿类HBV持续感染动物模型中验证了硫代磷酸酯化寡核苷酸类化合物的抗病毒活性；并发现长度为20-40nt的寡核苷酸抗乙肝表面抗原的活性无显著差异；
2、首次发现不同碱基构成的硫代磷酸酯化寡核苷酸抗乙肝表面抗原的活性不同，腺嘌呤含量超过50％(如75％)的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)，具有较好的抑制乙肝病毒复制和抗乙肝表面抗原的活性；
3、长度为16nt的富含腺嘌呤硫代磷酸酯化寡核苷酸为最短的具有显著的抗乙肝病毒活性的寡核苷酸。
4、本发明的富含腺嘌呤硫代磷酸酯化寡核苷酸具有较高的抗病毒活性且安全窗足够大。
5、因为HBV病毒和HIV与HPV有共性，所以本发明可以同样适用于治疗HIV与HPV所引起的感染。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例I不同长度的硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果
在感染携带HBV1.3倍体的腺相关病毒并持续复制HBV-DNA及表达HBV抗原的c57小鼠中测试不同长度的硫代磷酸酯化寡核苷酸，来建立其抗病毒活性。这些硫代磷酸酯化寡核苷酸是PA0001(SEQ ID NO:1)、PA0005(SEQ ID NO:5)、PA0008(SEQ ID NO:6)和PA0028(SEQ ID NO:11)。表1提供这些寡核苷酸的化学描述。
表1
Figure PCTCN2021101064-appb-000014
Figure PCTCN2021101064-appb-000015
dA＝脱氧核糖腺苷
dC＝脱氧核糖胞苷
用5X10 10个rAAV8-1.3HBV(五加和)通过尾静脉注射雄性C57BL/6小鼠，制备持续性乙肝感染小鼠模型。确定HBV病毒稳定复制后按体重随机分为10组(4只/组)，第1、3、5、7、9组每周一次(QW)分别腹腔注射溶媒(10ml/kg)(即空白对照组)和90mg/kg的PA0001、PA0005、PA0008、PA0028，溶媒组作为对照组。第2、4、6、8、10组每周两次(BIW)分别腹腔注射溶媒(10ml/kg)和90mg/kg的PA0001、PA0005、PA0008、PA0028，溶媒组作为对照组。经过6周给药，每周取血两次，qPCR方法分析血清中乙肝病毒滴度，ELISA方法分析血清中表面抗原浓度，绘制曲线图。
结果
血清中乙肝病毒滴度(见图1A、1B、1E、1F)，血清中表面抗原浓度(见图1C、1D、1G、1H)，实验中第5组(PA0005QW)有2只动物死亡，第4组(PA0001BIW)有1只动物死亡，第6组(PA0005BIW)有1只动物死亡，其他组无动物死亡，第3,5,4,6组(PA 0001QW，PA0005QW，PA0001BIW，PA0005BIW)动物可见显著的脾脏肿大，其他组未见显著的脾脏变化，脾脏肿大可能与过量寡核苷酸引起的免疫激活有关。
图2A和图2B分别显示注射给予被AAV-HBV感染的C57小鼠不同长度的硫代磷酸酯化寡核苷酸的血清生化指标白蛋白(ALB)、肌酐(CRE)的变化。实验终点，相对于对照组，硫代磷酸酯化寡核苷酸PA0001、PA0005、PA0008和PA0028给药组的动物血清白蛋白(ALB)均有降低，且每周两次给药(BIW)的降低幅度大于每周给药一次(QW)。每周两次给的动物中PA0001组血清白蛋白降低幅度最大。实验终点，相对于对照组，各组动物的血清肌酐浓度未见升高，有轻微下降。
汇总结果如下表2：
表2
Figure PCTCN2021101064-appb-000016
Figure PCTCN2021101064-appb-000017
#NO 1-4一周给药一次5-8一周给药两次
PA0005，NO 6小鼠8月2日死亡NO 1,4小鼠8月20日死亡
PA0001，NO 6小鼠8月16日死亡
上述结果表明：
a.所有寡核苷酸皆可导致血清HBsAg和HBV-DNA的降低。
b.相同重复序列，长度为20～40nt的寡核苷酸(PA0001、PA0005、PA0008)具有相当的抗病毒效应，并且寡核苷酸的毒性与长度呈正相关，长度越长，毒性越大，引起脾脏肿大的严重程度也与寡核苷酸的长度呈正相关，长度越长，脾脏肿大越显著。
c.同等长度(20nt)，不同序列(PA0008和PA0028)寡核苷酸的抗病毒比较：BIW给药具有序列ACAC重复序列的PA0008无乙肝病毒滴度<4log 10IU/ml(0％)，而具有序列AAAC重复序列的PA0028，全部4只动物乙肝病毒滴度<4log 10IU/ml(100％)；降低血中表面抗原方面比较：BIW给药，PA0008和PA0028具有相同的HBsAg>1log，而QW给药，PA0008的HBsAg>1log为25％(1/4)，PA0028的HBsAg>1log为75％(3/4)。因此，具有AAAC重复序列的序列，具有更好的药效和更高的安全性。
实施例II不同碱基构成的硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果
在感染携带HBV1.3倍体的腺相关病毒并持续复制HBV-DNA及表达HBV抗原的c57小鼠中测试长度相同但碱基构成不同的硫代磷酸酯化寡核苷酸，来评价其抗病毒活性。这些硫代磷酸酯化寡核苷酸是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)和PA0031(SEQ ID NO:4)，表3提供这些寡核苷酸的化学描述。
表3
Figure PCTCN2021101064-appb-000018
Figure PCTCN2021101064-appb-000019
dA＝脱氧核糖腺苷
dC＝脱氧核糖胞苷
dT＝脱氧核糖胸苷
用5X10 10个rAAV8-1.3HBV(五加和)通过尾静脉注射雄性C57BL/6小鼠，制备持续性乙肝感染小鼠模型。确定HBV病毒稳定复制后按体重随机分为7组(5只/组)，每周一次(QW)分别腹腔注射溶媒(10ml/kg)和90mg/kg的PA0008、PA00027、PA0028、PA0029、PA0030、PA0031，溶媒组作为对照组。经过12周给药，每周取血两次，qPCR方法分析血清中乙肝病毒滴度、ELISA方法分析血清中表面抗原含量，绘制曲线图。
结果
血清中乙肝病毒滴度(见图3A、3B)，血清中表面抗原浓度(见图3C、3D)，汇总结果如下表4：
表4
Figure PCTCN2021101064-appb-000020
Figure PCTCN2021101064-appb-000021
PA0008、PA0028、PA0031组分别有2只、2只和1只动物血清HBsAg下降幅度>1log 10这些动物也同时伴随着血清HBV-DNA的降低，特别是PA0028，100％的动物DNA>2log 10的下降；可能与其形成二级结构有关，PA0027、PA0029和PA0030抗病毒活性弱，或无抗病毒活性；此外，PA0008组给药期间动物体重增长显著低于对照组，而PA0028组动物的体重增长情况与对照组无显著性差异。
上述结果表明：
a.相同长度的寡核苷酸的抗病毒效应与碱基构成有关。
b.腺嘌呤占比为50％的PA0008和腺嘌呤占比为75％的PA0028均表现出显著的抗病毒活性，且PA0028组的HBV-DNA下降的动物最多，下降幅度更大，更具有优势。
实施例III不同剂量的富含腺嘌呤硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果
在感染携带HBV1.3倍体的腺相关病毒(AAV-HBV，五加和)并持续复制HBV-DNA及表达HBV抗原的c57小鼠中测试不同剂量的富含腺嘌呤硫代磷酸酯化寡核苷酸PA0028，来评价其抗病毒活性的剂量依赖关系，表5提供PA0028的化学描述。
表5
Figure PCTCN2021101064-appb-000022
dA＝脱氧核糖腺苷
dC＝脱氧核糖胞苷
用5X10 10个rAAV8-1.3HBV(五加和)通过尾静脉注射雄性C57BL/6小鼠，制备持续性乙肝感染小鼠模型。确定HBV病毒稳定复制后按体重随机分为4组(每组5只)，每周一次(QW)分别腹腔注射剂量为0(溶媒)、10mg/kg、30mg/kg、90mg/kg的PA0028，溶媒组作为对照组(空白)。经过12周给药，每周取血两次，qPCR方法分析血清中乙肝病毒滴度，ELISA方法分析血清中表面抗原含量，绘制曲线图。
结果
血清中乙肝病毒滴度(见图4A、4B)，血清中表面抗原浓度(见图4C、4D)，汇总结果如下表6：
表6
Figure PCTCN2021101064-appb-000023
溶媒对照组(Vehicle)的动物血清中HBsAg和HBV-DNA呈现平稳的波动，PA0028的三个不同剂量组的动物血清HBV-DNA均呈现>1log 10的波动下降的趋势，部分动物HBV-DNA降低至定量下限(LLOQ)，都有60％的动物(3/5)DNA>2log 10下降，所以认为10mg/kg是引起HBV-DNA显著降低的起效剂量。
结果表明：
30mg/kg和90mg/kg两个剂量组都有80％的动物(4/5)的血清HBsAg出现>1.5log 10的降低，且30mg/kg与90mg/kg剂量的抗病毒作用无显著性差异，因此可以将30mg/kg作为引起HBsAg降低的起效剂量。
实施例IV不同长度的富含腺嘌呤硫代磷酸酯化寡核苷酸在AAV-HBV小鼠模型中的抗病毒效果
在感染携带HBV1.3倍体的腺相关病毒(AAV-HBV，五加和)并持续复制HBV-DNA及表达HBV抗原的c57小鼠中测试不同长度的富含腺嘌呤硫代磷酸酯化寡核苷酸，来建立其抗病毒活性。这些硫代磷酸酯化寡核苷酸是PA00017(SEQ ID NO:9)，PA0018(SEQ ID NO:10)和PA0028(SEQ ID NO:11)，表7提供这些寡核苷酸的化学描述。
表7
Figure PCTCN2021101064-appb-000024
dA＝脱氧核糖腺苷
dC＝脱氧核糖胞苷
用5X10 10个rAAV8-1.3HBV(五加和)通过尾静脉注射雄性C57BL/6小鼠，制备持续性乙肝感染小鼠模型。确定HBV病毒稳定复制后按体重随机分为4组，每周一次(QW)分别腹腔注射溶媒(10ml/kg)和90mg/kg的PA00017、PA00018、PA0028，溶媒组作为对照组。经过12周给药，每周取血两次，qPCR方法分析血清中乙肝病毒滴度，ELISA方法分析血清中表面抗原含量，绘制曲线图。
结果
血清中乙肝病毒滴度(见图5A、5B)，血清中表面抗原浓度(见图5C、5D)，汇总结果如下表8：
表8
化合物名称 PA0017 PA0018 PA0028
序列长度 12nt 16nt 20nt
HBsAg>1.5log 10的下降数 0/4 2/4 3/4
DNA最大>1log 10下降数 0/4 4/4 4/4
Vehicle组和PA0017组的动物血清中HBsAg和HBV-DNA未见显著的降低，PA0018和PA0028组的动物血清HBV-DNA呈现>1log 10的波动下降的趋势，部分动物HBV-DNA降低至定量下限(LLOQ)，这两组分别有50％(2/4)和75％(3/4)的动物的血清HBsAg出现>1.5log 10的降低。
结果表明：
相同重复序列，长度为12～20nt的寡核苷酸(PA0017、PA0018、PA0028)中，其活性与长度呈正相关，长度越长，活性越好。
讨论
上述各实施例中，实施例I中剂量为90mg/kg的长度>20nt的硫代磷酸酯化寡核苷酸可引起导致动物死亡的毒性，且实施例III表明≥30mg/kg的剂量未能显著增加抗HBV活性，实施例IV中各化合物的施用的剂量90mg/kg为较高剂量，因此，可以看出16nt是作为富含腺嘌呤硫代磷酸酯化寡核苷酸具有显著抗HBV活性的最短长度。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，所述的化合物为富含腺嘌呤硫代磷酸酯化寡核苷酸，其中，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸包含(A mAAC) n重复序列；
    其中，m各自独立地为0或1；n为≥4的正数；且所述富含腺嘌呤硫代磷酸酯化寡核苷酸的长度为15-40个核苷酸；
    所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中80-100％的磷酸酯为硫代磷酸酯。
  2. 如权利要求1所述的化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的序列为
    Figure PCTCN2021101064-appb-100001
    Figure PCTCN2021101064-appb-100002
    其中，n1和n2各自独立地为0-10的整数，n3为0、1、2、3或4，且
    Figure PCTCN2021101064-appb-100003
    m和n的定义如权利要求1所述。
  3. 如权利要求1或2所述的化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，各A mAAC片段独立地选自下组：AAAC、AAC、CAAA、CAA。
  4. 如权利要求1所述的化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸为如SEQ ID NO：10-16和SEQ ID NO：7-8所示的寡核苷酸。
  5. 如权利要求1所述的化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸(ARON)为SEQ ID NO：11所示的寡核苷酸。
  6. 如权利要求1所述的化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的至少一个2'糖基是修饰的；其中，所述的修饰选自下组：2'-O-烷基修饰、2'-羟基修饰、2'-氨基修饰、2'-卤素修饰、2'-O-甲氧基乙基修饰。
  7. 如权利要求1所述的化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸的糖基为脱氧核糖基。
  8. 如权利要求1所述的化合物，或其光学异构体、药学上可接受的盐、水合物、或溶剂化物，其特征在于，所述的富含腺嘌呤硫代磷酸酯化寡核苷酸中一个或多个胞嘧啶为5-甲基胞嘧啶。
  9. 一种药物组合物，其特征在于，所述的药物组合物包括治疗有效量的如权利要求1-8中任一项所述的化合物，或其光学异构体、药学上可接受的盐、水合物或溶剂化物；和药学上可接受的辅助剂、稀释剂或载体。
  10. 如权利要求1-8任一项所述的化合物，或其光学异构体、药学上可接受的盐、水合物或溶剂化物的用途，或如权利要求9所述的药物组合物的用途，其特征在于，用于制备治疗和/或预防病毒感染相关的疾病的药物组合物。
PCT/CN2021/101064 2020-07-30 2021-06-18 富含腺嘌呤硫代磷酸酯化寡核苷酸及其抗肝炎病毒的应用 WO2022022158A1 (zh)

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