CN114057816A - Adenine-rich phosphorothioate oligonucleotide and application thereof in resisting hepatitis virus - Google Patents
Adenine-rich phosphorothioate oligonucleotide and application thereof in resisting hepatitis virus Download PDFInfo
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- CN114057816A CN114057816A CN202010753174.0A CN202010753174A CN114057816A CN 114057816 A CN114057816 A CN 114057816A CN 202010753174 A CN202010753174 A CN 202010753174A CN 114057816 A CN114057816 A CN 114057816A
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- adenine
- rich
- phosphorothioate
- oligonucleotide
- pharmaceutically acceptable
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Abstract
The invention discloses an adenine-rich phosphorothioate oligonucleotide and application thereof in resisting hepatitis viruses. Specifically, the invention discloses a composition comprising (A)mAAC)nRepeated sequence adenine-rich phosphorothioate oligonucleotide (ARON) for inhibiting hepatitis B surface antigen (HBsAg) and its use for treating hepatitis B virus infection or hepatitis B virus/hepatitis D virus co-infection, the length of the adenine-rich phosphorothioate oligonucleotide (ARON) is 15-40 nucleotides, and comprises adenine nucleotide and cytosine nucleotide, whereinThe content of adenine nucleotide is more than 50%, and the product has high antiviral activity and a large safety window.
Description
Technical Field
The invention relates to the field of medical biology, in particular to an adenine-rich phosphorothioate oligonucleotide and application thereof in resisting hepatitis viruses.
Background
Data published by the world health organization show that over 2 million people are chronically infected with HBV 2015 worldwide, and 88.7 million people die due to complications caused by HBV infection. Although some antiviral drugs have been approved globally for the treatment of HBV infection, current drug therapy or combination therapy, except in a small fraction of patients (< 3%), does not elicit an effective immune response or serological switch of HBsAg that can provide a long-lasting control or functional cure of the infection.
90% of adults infected with HBV are able to heal themselves, but 90% of infants develop chronic hepatitis after being infected with HBV. Chronic HBV infection may lead to liver fibrosis, further progression to cirrhosis and hepatocellular carcinoma (HCC). In addition, there are studies that indicate that hepatitis b increases the risk of pancreatic cancer. Curative or functional cure of chronic infection with HBV is a great unmet clinical need.
After HBV infects human hepatocytes, two different particles are mainly produced, one is Dane particles, i.e., complete HBV itself, including viral nucleocapsid assembled from hepatitis B core antigen (HBcAg) and viral nucleic acid (RcDNA), and having 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 esters, and hepatitis b surface antigen (HBsAg). SVP comprises hepatitis B surface antigen that makes up the vast majority (> 99.9%) of hepatitis B surface antigens in the blood of patients. 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 intervention condition of drugs on viruses.
Hepatitis D Virus (HDV) is a satellite virus of HBV that relies on the hepatitis b surface antigen (HBsAg) to form its complete, infectious HDV viral particles, and HDV infection can only occur in patients who are accompanied by HBV infection. HDV/HBV co-infection complications significantly increase the rate of progression from liver fibrosis to cirrhosis. For patients with HDV chronic infection, only one intervention means is available for interferon treatment at present, no medicine for directly targeting HDV virus is available on the market, and the existing treatment method has poor curative effect and obvious side effect.
The clinical medicines for treating hepatitis B mainly include interferons and nucleoside (acid) medicines. The interferon drugs comprise common interferon and long-acting interferon modified by polyethylene glycol, wherein the latter comprises peroxin (PEG-IFN alpha-2 a) and pellucinol (PEG-IFN alpha-2 b). The nucleoside (acid) drugs include lamivudine, telbivudine, adefovir dipivoxil, Tenofovir Disoproxil Fumarate (TDF), Tenofovir Alafenamide Fumarate (TAF), entecavir and the like. These nucleoside drugs are most widely used because they can effectively control viral replication and improve liver function. The interferon needs to be injected for administration, has large individual reaction difference, obvious adverse reaction and poor curative effect. Nucleoside drugs only act on the replication process of the virus from pgRNA to rcDNA, and have no inhibiting effect on other links in the life cycle of hepatitis B virus. The hepatitis B e antigen (HBeAg) turns negative for long-term treatment, and hepatitis B surface antigen (HBsAg) can turn negative for few patients. 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 hepatitis b e antigen (HBeAg) -positive patients, and 0.3% and 0% in hepatitis b surface antigen (HBsAg) -negative patients, respectively. Because the existing treatment scheme can not cure hepatitis B, the patient needs to take the medicine for a long time, and the patient may be confronted with serious side effects, for example, the long-term taking of adefovir dipivoxil and tenofovir disoproxil fumarate can cause renal toxicity and bone toxicity.
The large amount of hepatitis B surface antigen (HBsAg) in the form of subviral particles (SVP) in the blood of patients with chronic HBV infection can neutralize the specific hepatitis B surface antibody (HBsAb) secreted by B lymphocytes, thereby causing immune tolerance, while only a few HBV viral particles can escape from the immune examination, which may be one of the important reasons for maintaining chronic HBV infection. Although studies have shown that the three viral antigens hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg) all have immunosuppressive properties, hepatitis B surface antigen (HBsAg) accounts for the vast majority of all viral antigens in the blood of HBV infected subjects and is likely to be the most prominent inducer of host immunity. Serological switch of hepatitis b surface antigen (HBsAg) (clearance of HBsAg from the blood, appearance of free HBsAb) is a well-established prognostic indicator of functional control of viral infection at the time of treatment. Another key reason that HBV maintains the chronic infection profile is that it synthesizes a stable circular DNA store, i.e. HBV covalently closed circular DNA (cccdna), in the nucleus of infected hepatocytes by means of host DNA repair enzymes. cccDNA, which is stably present in hepatocytes for a long period of time and can be continuously supplemented, can produce nucleic acid RcDNA of HBV virus and mRNA encoding all viral antigens by transcription and reverse transcription. Transcriptional inhibition or clearance of cccDNA is crucial for curative or functional cure of HBV infection. Long-term treatment with nucleoside (acid) analogues cannot completely eliminate cccDNA and cannot inhibit its transcription, so that the expression level of hepatitis b surface antigen (HBsAg) is hardly affected by nucleoside (acid) drugs. Immunomodulation mediates humoral and cellular immunity, which in turn inhibits cccDNA transcription or eliminates infected cells, but a large antigen load greatly inhibits this immune process, thus greatly reducing antigens, especially hepatitis b surface antigen (HBsAg), combined with immunomodulation is an effective means to help patients achieve durable immune control.
At present, there is a lack of phosphorothioate oligonucleotides with high activity and a safety window large enough for the treatment of HBV and HBV/HDV co-infection.
Disclosure of Invention
The object 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.
In a first aspect of the present invention, there is provided a compound, or an optical isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, wherein the compound is an adenine-rich phosphorothioate oligonucleotide (ARON), wherein the adenine-rich phosphorothioate oligonucleotide comprises (a)mAAC)nA repeat sequence;
wherein each m is independently 0 or 1; n is a positive number not less than 4; and the length of the adenine-rich phosphorothioate oligonucleotide is 15-40 nucleotides;
80-100% of the phosphate ester in the adenine-rich phosphorothioate oligonucleotide is phosphorothioate.
In another preferred embodiment, 95-100% of the phosphate in said adenine-rich phosphorothioate oligonucleotide is phosphorothioate.
In another preferred embodiment, all the phosphates in the adenine-rich phosphorothioate oligonucleotide are phosphorothioates.
In another preferred embodiment, the sequence of the adenine-rich phosphorothioate oligonucleotide is (A)mAAC)n1(AAAC)
(AmAAC)n2Wherein n1 and n2 are each independently an integer of 0-10 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), n3 is 0, 1, 2, 3, or 4, and
m and n are as defined above.
In another preferred embodiment, the adenine phosphorothioate-rich oligonucleotide has the sequence (R) (A)mAAC)n'2、(AmAAC)n'1(R)、(R)(CAmAA)n'2Or (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 as defined above.
In another preferred embodiment, each AmAAC (or CA)mAA) fragments are independently selected from the group consisting of: AAAC, AAC, CAAA, CAA.
In another preferred embodiment, when n is a non-integer, A of the non-integer partmThe AAC fragment is selected from: A. AA, AC, AAA, AAC, CA, CAA.
In another preferred embodiment, (AAAC)
The fragments are selected from the group consisting of: none, C, A, AA, AC, AAA, AAC, AAAC, CA, CAA, CAAA.
In another preferred embodiment, when n is an integer, (A)mAAC) fragments are selected from the group consisting of: AAAC, AAC, CAAA, CAA.
In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide comprises (A)mAAC)nRepeating the sequence, wherein n is a positive number more than or equal to 4; each m is independently 0 or 1, and at least one m is 1; preferably m are both 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 oligonucleotides are represented by the sequence shown 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 phosphorothioate-rich oligonucleotide (ARON) has the sequence shown in SEQ ID NO: 10-16.
In another preferred embodiment, the adenine phosphorothioate-rich oligonucleotide (ARON) has the sequence shown in SEQ ID NO:11, or a pharmaceutically acceptable salt thereof.
In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is SEQ ID NO: 12.
In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide consisting of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof.
In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide consisting of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.
In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide consisting of SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.
In another preferred embodiment, the adenine-rich phosphorothioate oligonucleotide is an optionally modified oligonucleotide consisting of SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof.
In another preferred embodiment, at least one 2' sugar group of said adenine-rich phosphorothioate oligonucleotide is modified.
In another preferred embodiment, the 2' sugar group of said adenine-rich phosphorothioate oligonucleotide is modified.
In another preferred embodiment, the modification of the 2' sugar group of the adenine phosphorothioate-rich oligonucleotide is selected from the group consisting of: 2' -O-alkyl modification, 2' -hydroxy modification, 2' -amino modification, 2' halogen modification or 2' -O-methoxyethyl (2' MOE) modification, preferably 2' O-methyl modification.
In another preferred embodiment, in the adenine-rich phosphorothioate oligonucleotide, the sugar group is deoxyribosyl.
In another preferred embodiment, one or more cytosines in said adenine-rich phosphorothioate oligonucleotide are 5-methylcytosine.
In another preferred embodiment, the cytosines in the adenine-rich phosphorothioate oligonucleotide are all 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, a pharmaceutically acceptable salt, hydrate or solvate thereof; and a pharmaceutically acceptable adjuvant, diluent or carrier.
In a third aspect of the present invention, there is provided a use of the compound of the first aspect, or an optical isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, or a use of the pharmaceutical composition of the second aspect, for preparing a pharmaceutical composition for treating and/or preventing a disease associated with viral infection.
In another preferred embodiment, the disease includes, 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 diseases are diseases associated with HBV infection and HBV/HDV co-infection.
In another preferred embodiment, the disease is selected from: 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 to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the antiviral effect of different lengths of phosphorothioate oligonucleotides in a mouse model of AAV-HBV; wherein
FIGS. 1A,1B show the antiviral activity of phosphorothioate oligonucleotides of varying lengths administered to AAV-HBV infected C57 mice intraperitoneally once a week, assessed by qPCR detection of serum HBV-DNA at the end of treatment;
FIGS. 1C,1D show the antiviral activity of phosphorothioate oligonucleotides of varying lengths administered to AAV-HBV infected C57 mice intraperitoneally once a week, assessed by ELISA detection of serum HBsAg at the end of treatment;
FIGS. 1E,1F show the antiviral activity of phosphorothioate oligonucleotides of varying length administered to AAV-HBV infected C57 mice intraperitoneally twice a week, assessed at the end of treatment by qPCR detection of serum HBV-DNA;
FIGS. 1G,1H show the antiviral activity of phosphorothioate oligonucleotides of varying lengths administered intraperitoneally twice a week to AAV-HBV infected C57 mice, evaluated by ELISA detection of serum HBsAg at the end of treatment.
FIG. 2 shows the changes in serum biochemical indicators Albumin (ALB), Creatinine (CRE) given by injection of phosphorothioate oligonucleotides of varying lengths to AAV-HBV infected C57 mice.
FIG. 3 shows the antiviral effect of phosphorothioate oligonucleotides of different base composition in a mouse model of AAV-HBV, wherein
FIGS. 3A,3B show the antiviral activity of phosphorothioate oligonucleotides composed of different bases administered intraperitoneally to AAV-HBV infected C57 mice once a week, evaluated by qPCR detection of serum HBV-DNA at the end of treatment;
FIGS. 3C,3D show the antiviral activity of phosphorothioate oligonucleotides composed of different bases administered intraperitoneally to AAV-HBV infected C57 mice once a week, evaluated by detecting serum HBsAg by ELISA at the end of treatment.
FIG. 4 antiviral Effect of different doses of adenine-rich phosphorothioate oligonucleotides in a mouse model of AAV-HBV, wherein
FIGS. 4A,4B show antiviral activity of PA0028 given to AAV-HBV infected C57 mice at various doses by intraperitoneal injection once a week, assessed by qPCR detection of serum HBV-DNA at the end of treatment;
FIGS. 4C,4D show the antiviral activity of PA0028 administered to AAV-HBV infected C57 mice at various doses by intraperitoneal injection once a week, assessed by ELISA for serum HBsAg at the end of treatment.
FIG. 5 antiviral effect of adenine-rich phosphorothioate oligonucleotides of varying lengths in a mouse model of AAV-HBV, wherein,
FIGS. 5A,5B show the antiviral activity of different lengths of adenine-rich phosphorothioate oligonucleotides (ARON) administered intraperitoneally to AAV-HBV infected C57 mice once a week, evaluated by qPCR detection of serum HBV-DNA at the end of treatment;
FIGS. 5C,5D show the antiviral activity of different lengths of adenine-rich phosphorothioate oligonucleotides (ARON) administered intraperitoneally to AAV-HBV infected C57 mice once a week, assessed by ELISA for serum HBsAg at the end of treatment.
Detailed Description
Through long-term and intensive research, the inventor firstly verifies the antiviral activity of the phosphorothioate oligonucleotide compound in a rodent HBV persistent infection animal model through exploring and optimizing experimental conditions; and the activity of the oligonucleotide with the length of 20-40nt against hepatitis B surface antigen is found to have no significant difference; it is found for the first time that the phosphorothioate oligonucleotides composed of different bases have different activities against hepatitis B surface antigen, and the adenine-rich phosphorothioate oligonucleotide (ARON) with adenine content of more than 50% (such as 75%) has the best activity for inhibiting hepatitis B virus replication and against hepatitis B surface antigen; it is also found that the adenine-rich phosphorothioate oligonucleotide has the minimum length of 16nt with anti-hepatitis B virus activity. Based on the above findings, the inventors have completed the present invention.
In the present invention, "AmAAC 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 mode" CAmAA', m is 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' -halo, or 2' -O-methoxyethyl (2' -MOE).
As used herein, 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 "comprising.
In the present invention, the term "pharmaceutically acceptable" ingredient 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), i.e., at 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 target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend upon 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 not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.
As used herein, 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 pharmaceutical.
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 moieties. Such modified or substituted oligonucleotides may be preferred over the native form due to desirable properties such as: for example, decreased immunoreactivity, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and/or increased 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, and double-stranded molecules such as small interfering rnas (sirnas) or small hairpin rnas (shrnas). Optionally modified oligonucleotides described herein may include various modifications, such as stabilizing 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 fully modified to contain all linkages or sugars or bases having such 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 'halo 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 locked nucleic acids. Specifically, the oligonucleotides have modified linkages throughout or each linkage modified, e.g., phosphorothioates; having a 3 '-cap and/or a 5' -cap: including the terminal 3 '-5' linkage. Base modifications may include 5 'methylation of cytosine bases (5' methylcytosine) and/or 4 'thioation of uracil bases (4' thiouracil). When the synthesis conditions are chemically compatible, different chemically compatible modified linkages can be combined, for example, oligonucleotides having modified bases with phosphorothioate linkages, 2' ribose modifications (e.g., 2' O-methylation), and modified bases (e.g., 5 ' methylcytosine). Oligonucleotides can be further fully modified with all of these different modifications (e.g., each phosphorothioate linkage, each 2' modified ribose, and each modified base).
The term "phosphorothioate nucleotide" refers to a nucleotide having an altered phosphate backbone in which the sugar moieties are linked by phosphorothioate linkages. In the phosphate backbone of the oligonucleotide sequence, the phosphorothioate linkages contain a sulfur atom as a substitute for a non-bridging oxygen atom.
Said phosphorothioate means that the phosphorothioate internucleoside linkages of the oligonucleotide are replaced by phosphorothioate internucleoside linkages.
Illustrative phosphorothioate linkages in the present invention are shown below:
3 ', 5' -phosphorothioate diester linkages
As used herein, the term "adenine-Rich phosphorothioate oligonucleotides" or its acronym "ARON" (Adenosine Rich oligonucleotide oligonucleotides) refers to phosphorothioate oligonucleotides containing more than 50% (e.g., 75%, 80%, 85%, 90%) of adenine over the entire base, whose antiviral activity is not dependent on Toll-like receptor recognition, hybridization to target RNA or DNA, or sequence-dependent aptamer interaction to form secondary or tertiary structures.
Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.
Some of the compounds of the present invention may be crystallized or recrystallized using water or various organic solvents, in which case various solvates may be formed. Solvates of the 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 method.
Active ingredient
The active component of the invention is an adenine-rich phosphorothioate oligonucleotide (ARON), or an optical isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof,
the adenine-rich phosphorothioate oligonucleotide comprises (A)mAAC)nA repeat sequence;
wherein each m is independently 0 or 1; n is a positive number not less than 4; and the length of the adenine-rich phosphorothioate oligonucleotide is 15-40 nucleotides;
80-100% of the phosphate ester in the adenine-rich phosphorothioate oligonucleotide is phosphorothioate.
Preferably, the phosphate esters in the adenine-rich phosphorothioate oligonucleotide are all phosphorothioate.
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 sequence of the adenine-rich phosphorothioate oligonucleotide is (A)mAAC)n1(AAAC)
(AmAAC)n2Wherein 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 sequencesA of the integer partmAAC fragments are selected from the group consisting of: AAAC, AAC, CAAA, CAA.
Preferably, in each of the above sequences, when n is a non-integer, A of the non-integer partmAAC fragment (e.g., (AAAC)
n3 is 1, 2 or 3) selected from one or more oligonucleotides (such as A, C, AA, AC, AAA, AAC, CAA) in AAAC fragment.
Preferably, in each of the above sequences, A is a non-integer partmAAC fragment (e.g., (AAAC)
n3 is 1, 2 or 3) is based on the non-integer part of n (e.g.,
) E.g., when the non-integer portion is 0.25 (i.e., n3 is 1), the number of oligonucleotides of the non-integer portion is 1, i.e., a or C; for example, 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 fragments are selected from the group consisting of: none, 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 to 36 nucleotides in length.
Preferably, in each of the above sequences, the adenine-rich phosphorothioate oligonucleotide is 17 to 33 nucleotides in length.
Preferably, the adenine-rich phosphorothioate oligonucleotide is an AAAC repeat 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 'sugar group of the adenine-rich phosphorothioate oligonucleotide (ARON) is modified, and 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 and 2' -O-methoxyethyl modification.
Preferably, in each of the above sequences, the sugar group of the adenine-rich phosphorothioate oligonucleotide is deoxyribosyl.
Preferably, in each of the above sequences, one or more cytosines in the adenine-rich phosphorothioate oligonucleotide are 5-methylcytosine, and preferably, the cytosines are all 5-methylcytosine.
Preferably, the sequence of said adenine-rich phosphorothioate oligonucleotide is shown in Table A
Preferably, the sequence of the adenine-rich phosphorothioate oligonucleotide is as shown in SEQ ID NO: 10-16.
Preferably, the adenine-rich phosphorothioate oligonucleotide is represented by SEQ ID NO:11, or a pharmaceutically acceptable salt thereof.
Preferably, at least one 2 'sugar group of the adenine-rich phosphorothioate oligonucleotide is modified, and more preferably, all 2' sugar groups of the adenine-rich phosphorothioate oligonucleotide are modified.
Preferably, the modification of the 2' sugar group of the adenine-rich phosphorothioate oligonucleotide is selected from the group consisting of: 2' -O-alkyl modification, 2' amino modification, 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, the phosphodiester bond is a phosphorothioate diester bond.
It is understood that the compounds of the present invention may be prepared in a variety of thermodynamically stable isomers, such as tautomers, conformers, meso compounds, and optical isomers in enantiomeric or diastereomeric relationships, and the like, and that such modifications will be apparent to those skilled in the art upon reading the present disclosure.
Preparation of phosphorothioate oligonucleotides
The adenine-rich phosphorothioate oligonucleotide of the present invention can be prepared and synthesized by conventional synthetic methods in the oligonucleotide industry. For example, phosphorothioate linkages can be converted to phosphorothioate linkages by solid phase phosphoramidite synthesis using the sulfur transfer reagent diphenylacetyl disulfide (PADS) in place of iodine (iododine), under the control of an apparatus such as GE OP 100.
Pharmaceutical compositions and methods of administration
The phosphorothioate oligonucleotides (ARONs) may be used in combination with other drugs known to treat or ameliorate similar conditions. When administered in combination, the mode of administration and dosage of the original drug may be maintained while the phosphorothioate oligonucleotide is administered concurrently or subsequently. When the phosphorothioate 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 phosphorothioate oligonucleotide. The combination also includes administration of the phosphorothioate oligonucleotide with one or more other known drugs at overlapping time periods. When the phosphorothioate oligonucleotide is administered in combination with one or more other drugs, the dosage of the phosphorothioate oligonucleotide or known drugs may be lower than the dosage of the drugs administered alone.
For an suggestive effective dosing regimen for administering the adenine phosphorothioate oligonucleotide of the invention to humans, once weekly (QW) as described in example III, the single dose is 3mg/kg (based on body surface area); follow the dosing regimen commonly used for other phosphorothioate oligonucleotides (e.g., antisense oligonucleotide ASO); the conventional use of 100-500mg compounds administered parenterally weekly is well established in the art.
In accordance with the disclosure presented herein, it is useful to treat subjects having HBV infection or HBV/HDV co-infection with a pharmaceutically acceptable adenine phosphorothioate-rich oligonucleotide formulation.
The compositions described herein may be administered by any suitable means, e.g., oral ingestion; inhalation through mouth; by subcutaneous, intravenous injection or infusion; may be in dosage unit formulations containing non-toxic pharmaceutically acceptable carriers or diluents. For example, the present compositions may be administered in formulations suitable for immediate or sustained release.
When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 50 to 1000 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The invention has the main advantages that:
1. the antiviral activity of the phosphorothioate oligonucleotide compound is verified in a rodent model of HBV persistent infection for the first time; and the activity of the oligonucleotide with the length of 20-40nt against hepatitis B surface antigen is found to have no significant difference;
2. the phosphorothioate oligonucleotides composed of different bases have different activities of resisting hepatitis B surface antigen for the first time, and adenine-rich phosphorothioate oligonucleotides (ARON) with adenine content of more than 50 percent (such as 75 percent) have better activities of inhibiting hepatitis B virus replication and resisting hepatitis B surface antigen;
3. the adenine-rich phosphorothioate oligonucleotide with a length of 16nt is the shortest oligonucleotide with significant anti-hepatitis B virus activity.
4. The oligonucleotide rich in adenine phosphorothioate has higher antiviral activity and a sufficiently large safety window.
5. Because of the commonness of the HBV virus and HIV with HPV, the present invention may be equally applicable to the treatment of infections caused by HIV and HPV.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
EXAMPLE I antiviral Effect of different Length phosphorothioate oligonucleotides in AAV-HBV mouse model
Phosphorothioate oligonucleotides of varying lengths were tested in c57 mice infected with adeno-associated virus carrying 1.3 fold of HBV and continued to replicate HBV-DNA and express HBV antigen to establish their antiviral activity. These phosphorothioate oligonucleotides were 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 ═ deoxyribonucleotide
By 5X1010A male C57BL/6 mouse was injected with rAAV8-1.3HBV (Acanthopanax senticosus and Araliaceae) via tail vein to prepare a persistent hepatitis B infection mouse model. After determining stable replication of HBV virus, the HBV virus was randomly divided into 10 groups (4/group) by body weight, and the groups 1, 3,5, 7 and 9 were divided into QW (QW) groupsThe vehicle (10ml/kg) (blank control group) and 90mg/kg of PA0001, PA0005, PA0008 and PA0028 were intraperitoneally injected, and the vehicle group served as the control group. Groups 2, 4,6, 8, and 10 were injected intraperitoneally twice weekly (BIW) with vehicle (10ml/kg) and 90mg/kg of PA0001, PA0005, PA0008, and PA0028, respectively, with the vehicle group serving as a control group. After 6 weeks of administration, blood is taken twice a week, the hepatitis B virus titer in the serum is analyzed by a qPCR method, the surface antigen concentration in the serum is analyzed by an ELISA method, and a curve chart is drawn.
Results
Serum hepatitis B virus titer (see fig. 1A,1B, 1E, 1F), serum surface antigen concentration (see fig. 1C,1D, 1G, 1H), 2 animals died in group 5 (PA0005 QW), 1 animal died in group 4 (PA0001 BIW), 1 animal died in group 6 (PA0005 BIW), no animal died in other groups, significant splenic enlargement in group 3,5,4,6 (PA0001 QW, PA0005 QW, PA0001 BIW, PA0005 BIW) animals, no significant splenic changes in other groups, and splenic enlargement may be associated with immune activation by excess oligonucleotides.
FIGS. 2A and 2B show the changes in serum biochemical indicators Albumin (ALB), Creatinine (CRE) of different lengths of phosphorothioate oligonucleotides injected into AAV-HBV infected C57 mice, respectively. At the end of the experiment, animal serum Albumin (ALB) was reduced in all of the groups administered with phosphorothioate oligonucleotides PA0001, PA0005, PA0008 and PA0028, relative to the control group, and the reduction was greater for the twice weekly administration (BIW) than for the once weekly administration (QW). The PA0001 group showed the greatest reduction in serum albumin in animals given twice weekly. At the end of the experiment, the serum creatinine concentration of each animal group was not increased and slightly decreased relative to the control group.
The results are summarized in table 2 below:
TABLE 2
# NO 1-4 twice weekly 5-8 weekly
PA0005, NO 6 mice die 8 months and 2 days NO 1,4 mice die 8 months and 20 days
PA0001, NO 6 mice died 8 months and 16 days
The above results show that:
a. all oligonucleotides lead to a reduction of serum HBsAg and HBV-DNA.
b. The oligonucleotides (PA0001, PA0005 and PA0008) with the same repetitive sequence and the length of 20-40nt have a considerable antiviral effect, the toxicity of the oligonucleotides is positively correlated with the length, the longer the length is, the greater the toxicity is, the more serious the spleen enlargement is also positively correlated with the length of the oligonucleotides, and the longer the length is, the more remarkable the spleen enlargement is.
c. Antiviral comparison of oligonucleotides of equivalent length (20nt), different sequences (PA0008 and PA 0028): no hepatitis B virus titer of PA0008 with sequence ACAC repetitive sequence for BIW administration<4log10IU/ml (0%), and PA0028 with sequence AAAC repeat, hepatitis B virus titer of all 4 animals<4log10IU/ml (100%); comparison in terms of reducing blood surface antigens: BIW administration, PA0008 and PA0028 with the same HBsAg>1log, and QW administration, HBsAg of PA0008>1log 25% (1/4), HBsAg of PA0028>1log is 75% (3/4). Therefore, the sequence with the AAAC repetitive sequence has better drug effect and higher safety.
EXAMPLE II antiviral Effect of phosphorothioate oligonucleotides composed of different bases in AAV-HBV mouse model
The antiviral activity was evaluated by testing phosphorothioate oligonucleotides having the same length but different base composition in c57 mice infected with adeno-associated virus carrying 1.3-fold HBV and continuously replicating HBV-DNA and expressing HBV antigen. These phosphorothioate oligonucleotides are PA0008(SEQ ID NO:6), PA0027(SEQ ID NO:17), PA0028(SEQ ID NO:11), PA0029(SEQ ID NO:2), PA0030(SEQ ID NO:3) and PA0031(SEQ ID NO:4), the chemical descriptions of which are provided in Table 3.
TABLE 3
dA ═ deoxyriboadenosine
dC ═ deoxyribonucleotide
dT-dT
By 5X1010A male C57BL/6 mouse was injected with rAAV8-1.3HBV (Acanthopanax senticosus and Araliaceae) via tail vein to prepare a persistent hepatitis B infection mouse model. After determining that HBV virus is stably replicated, the HBV virus is randomly divided into 7 groups (5 groups/group) according to the body weight, and the solvent (10ml/kg) and 90mg/kg of PA0008, PA00027, PA0028, PA0029, PA0030 and PA0031 are respectively injected into the abdominal cavity once per week (QW), and the solvent group is used as a control group. After 12 weeks of administration, blood is taken twice a week, the hepatitis B virus titer in the serum is analyzed by a qPCR method, the surface antigen content in the serum is analyzed by an ELISA method, and a curve chart is drawn.
Results
The titers of hepatitis B virus in serum (see FIGS. 3A and 3B), the concentrations of surface antigens in serum (see FIGS. 3C and 3D), and the results are summarized in Table 4 below:
TABLE 4
The HBsAg of the serum of 2 animals, 2 animals and 1 animal in PA0008, PA0028 and PA0031 groups respectively has reduced amplitude>1log10These animals are also accompanied by a reduction in serum HBV-DNA, in particular PA0028, 100% of animal DNA>2log10(ii) a decrease; possibly related to the formation of secondary structures thereof, PA0027, PA0029 and PA0030 have weak or no antiviral activity; in addition, the weight gain of the animals in the PA0008 group is significantly lower than that of the control group during the administration period, and the weight gain of the animals in the PA0028 group is not significantly different from that of the control group.
The above results show that:
a. the antiviral effect of oligonucleotides of the same length is related to the base composition.
b. PA0008 with 50% of adenine ratio and PA0028 with 75% of adenine ratio both show remarkable antiviral activity, and the HBV-DNA of the PA0028 group is reduced by the largest number of animals, and is reduced by a larger extent, and is more advantageous.
EXAMPLE III antiviral Effect of different doses of adenine-rich phosphorothioate oligonucleotides in the mouse model of AAV-HBV
Different doses of adenine-rich phosphorothioate oligonucleotide PA0028 were tested in c57 mice infected with adeno-associated virus (AAV-HBV, acanthopanax and) carrying HBV1.3 times and continuously replicating HBV-DNA and expressing HBV antigen to evaluate the dose dependence of its antiviral activity, and table 5 provides a chemical description of PA 0028.
TABLE 5
dA ═ deoxyriboadenosine
dC ═ deoxyribonucleotide
By 5X1010A male C57BL/6 mouse was injected with rAAV8-1.3HBV (Acanthopanax senticosus and Araliaceae) via tail vein to prepare a persistent hepatitis B infection mouse model. After determining that HBV virus is stably replicated, the HBV virus is randomly divided into 4 groups (5 in each group) according to the body weight, PA0028 with the dose of 0 (solvent), 10mg/kg, 30mg/kg and 90mg/kg is intraperitoneally injected once a week (QW), and the solvent group is used as a control group (blank). After 12 weeks of administration, blood is taken twice a week, the hepatitis B virus titer in the serum is analyzed by a qPCR method, the surface antigen content in the serum is analyzed by an ELISA method, and a curve chart is drawn.
Results
The serum hepatitis B virus titers (see FIGS. 4A and 4B), the serum surface antigen concentrations (see FIGS. 4C and 4D), and the results are summarized in Table 6 below:
TABLE 6
HBsAg and HBV-DNA in the serum of animals in the Vehicle control group (Vehicle) show smooth fluctuation, and HBV-DNA in the serum of animals in three different dose groups of PA0028>1log10The HBV-DNA of some animals is reduced to the lower limit of quantitation (LLOQ), and 60% of the DNA of the animals (3/5) is present>2log10Decreased, so 10mg/kg is considered to be an effective dose causing significant decrease in HBV-DNA.
The results show that:
serum HBsAg appeared in 80% of animals (4/5) in both the 30mg/kg and 90mg/kg dose groups>1.5log10And the antiviral effects of the 30mg/kg and 90mg/kg doses are not significantly different, so 30mg/kg can be taken as the effective dose for causing the reduction of HBsAg.
EXAMPLE IV antiviral Effect of adenine-rich phosphorothioate oligonucleotides of varying lengths in AAV-HBV mouse models
Adenine-rich phosphorothioate oligonucleotides of varying lengths were tested in c57 mice infected with adeno-associated virus (AAV-HBV, Acanthopanax senticosus and Araliaceae) carrying 1.3-fold HBV and continuously replicating HBV-DNA and expressing HBV antigen to establish their antiviral activity. These phosphorothioate oligonucleotides are PA00017(SEQ ID NO:9), PA0018(SEQ ID NO:10) and PA0028(SEQ ID NO:11), the chemical descriptions of these oligonucleotides being provided in Table 7.
TABLE 7
dA ═ deoxyriboadenosine
dC ═ deoxyribonucleotide
By 5X1010A continuous hepatitis B infected mouse is prepared by injecting rAAV8-1.3HBV (Acanthopanax senticosus and Araliaceae) into male C57BL/6 mouse via tail veinAnd (4) modeling. After determining that HBV virus is stably replicated, the HBV virus is randomly divided into 4 groups according to the body weight, and the solvents (10ml/kg) and 90mg/kg of PA00017, PA00018 and PA0028 are respectively injected into the abdominal cavity once per week (QW), and the solvent group is used as a control group. After 12 weeks of administration, blood is taken twice a week, the hepatitis B virus titer in the serum is analyzed by a qPCR method, the surface antigen content in the serum is analyzed by an ELISA method, and a curve chart is drawn.
Results
The serum hepatitis B virus titers (see FIGS. 5A and 5B), and the serum surface antigen concentrations (see FIGS. 5C and 5D), together with the results shown in Table 8 below:
TABLE 8
| Name of Compound | PA0017 | PA0018 | PA0028 |
| Length of sequence | 12nt | 16nt | 20nt |
| HBsAg>1.5log10Number of drops of | 0/4 | 2/4 | 3/4 |
| DNA maximization>1log10Number of |
0/4 | 4/4 | 4/4 |
HBsAg and HBV-DNA in the serum of animals in the Vehicle group and PA0017 group are not obviously reduced, and HBV-DNA in the serum of animals in the PA0018 group and PA0028 group is shown>1log10The trend of the fluctuation of HBV-DNA in some animals was reduced to the lower limit of quantitation (LLOQ), and 50% (2/4) and 75% (3/4) of HBsAg appeared in the serum of these two groups, respectively>1.5log10Is reduced.
The results show that:
in the oligonucleotides (PA0017, PA0018 and PA0028) with the same repetitive sequence and the length of 12-20 nt, the activity is positively correlated with the length, and the longer the length is, the better the activity is.
Discussion of the related Art
In each of the above examples, the phosphorothioate oligonucleotide of >20nt in length at a dose of 90mg/kg in example I caused toxicity resulting in death of animals, and example III showed that the anti-HBV activity was not significantly increased at a dose of > 30mg/kg, and the 90mg/kg dose of each compound in example IV was a higher dose, and thus, it can be seen that 16nt was the shortest length having significant anti-HBV activity as an adenine-rich phosphorothioate oligonucleotide.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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Claims (10)
1. A compound, or an optical isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, wherein the compound is an adenine-rich phosphorothioate oligonucleotide, wherein the adenine-rich phosphorothioate oligonucleotide comprises (A)mAAC)nA repeat sequence;
wherein each m is independently 0 or 1; n is a positive number not less than 4; and the length of the adenine-rich phosphorothioate oligonucleotide is 15-40 nucleotides;
80-100% of the phosphate ester in the adenine-rich phosphorothioate oligonucleotide is phosphorothioate.
2. The compound of claim 1, or an optical isomer, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the adenine phosphorothioate-rich oligonucleotide has the sequence (A)mAAC)
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 in claim 1.
3. The compound of claim 1 or 2, or an optical isomer, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein each amAAC fragments are independently selected from the group consisting of: AAAC, AAC, CAAA, CAA.
4. The compound of claim 1, or an optical isomer, a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the adenine-rich phosphorothioate oligonucleotide is a nucleotide sequence as set forth in SEQ ID NO: 10-16 and SEQ ID NO: 7-8.
5. The compound of claim 1, or an optical isomer, a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the adenine-rich phosphorothioate oligonucleotide (ARON) is SEQ ID NO:11, or a pharmaceutically acceptable salt thereof.
6. The compound of claim 1, or an optical isomer, a pharmaceutically acceptable salt, hydrate or solvate 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.
7. The compound of claim 1, or an optical isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, wherein the sugar group of the adenine phosphorothioate-rich oligonucleotide is a deoxyribosyl group.
8. The compound of claim 1, or an optical isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, wherein one or more cytosines in the adenine-rich phosphorothioate oligonucleotide are 5-methylcytosines.
9. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claims 1-8, or an optical isomer, pharmaceutically acceptable salt, hydrate or solvate thereof; and a pharmaceutically acceptable adjuvant, diluent or carrier.
10. Use of a compound according to any one of claims 1 to 8, or an optical isomer, a pharmaceutically acceptable salt, hydrate or solvate thereof, or use of a pharmaceutical composition according to claim 9 for the preparation of a pharmaceutical composition for the treatment and/or prevention of a disease associated with a viral infection.
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| PCT/CN2021/101064 WO2022022158A1 (en) | 2020-07-30 | 2021-06-18 | Adenine-rich phosphorothioate oligonucleotide and anti-hepatitis virus application thereof |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101084232A (en) * | 2004-10-19 | 2007-12-05 | 里普利科股份有限公司 | Antiviral oligonucleotides |
| WO2008139262A2 (en) * | 2006-10-26 | 2008-11-20 | Coley Pharmaceutical Gmbh | Oligoribonucleotides and uses thereof |
| WO2013039855A1 (en) * | 2011-09-12 | 2013-03-21 | Idenix Pharmaceuticals, Inc. | Compounds and pharmaceutical compositions for the treatment of viral infections |
| WO2014179446A2 (en) * | 2013-05-01 | 2014-11-06 | Regulus Therapeutics Inc. | Microrna compounds and methods for modulating mir-122 |
| CN105061534A (en) * | 2010-09-22 | 2015-11-18 | 艾丽奥斯生物制药有限公司 | Substituted nucleotide analogs |
| CN106659730A (en) * | 2014-07-10 | 2017-05-10 | 里普利科股份有限公司 | Methods for the treatment of hepatitis b and hepatitis d virus infections |
| WO2018053185A1 (en) * | 2016-09-14 | 2018-03-22 | Alios Biopharma, Inc. | Modified oligonucleotides and methods of use |
-
2020
- 2020-07-30 CN CN202010753174.0A patent/CN114057816A/en active Pending
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2021
- 2021-06-18 WO PCT/CN2021/101064 patent/WO2022022158A1/en active Application Filing
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101084232A (en) * | 2004-10-19 | 2007-12-05 | 里普利科股份有限公司 | Antiviral oligonucleotides |
| WO2008139262A2 (en) * | 2006-10-26 | 2008-11-20 | Coley Pharmaceutical Gmbh | Oligoribonucleotides and uses thereof |
| CN105061534A (en) * | 2010-09-22 | 2015-11-18 | 艾丽奥斯生物制药有限公司 | Substituted nucleotide analogs |
| WO2013039855A1 (en) * | 2011-09-12 | 2013-03-21 | Idenix Pharmaceuticals, Inc. | Compounds and pharmaceutical compositions for the treatment of viral infections |
| WO2014179446A2 (en) * | 2013-05-01 | 2014-11-06 | Regulus Therapeutics Inc. | Microrna compounds and methods for modulating mir-122 |
| CN106659730A (en) * | 2014-07-10 | 2017-05-10 | 里普利科股份有限公司 | Methods for the treatment of hepatitis b and hepatitis d virus infections |
| WO2018053185A1 (en) * | 2016-09-14 | 2018-03-22 | Alios Biopharma, Inc. | Modified oligonucleotides and methods of use |
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