Combination Therapy for Treating Hepatitis B Virus Infection - Patent application

Abstract

The present disclosure relates to a method for treating Hepatitis B infection, the method comprising administering to a subject in need thereof a PAPD5 / 7 inhibitor and a modified ASO targeting HBV mRNA.

Description

[Technical field]

[0001] The present disclosure relates to a method for treating Hepatitis B virus (HBV) infection, comprising administering to a subject in need thereof an RNA polymerase associated domain-containing protein 5 and 7 (PAPD5 / 7) inhibitor and administering a modified antisense oligonucleotide (ASO). [Background technology]

[0002] Hepatitis B virus (HBV) is a double-stranded DNA-containing virus with strict liver tropism. DNA is the genetic material, but the replication cycle involves a reverse transcription step that copies pregenomic RNA into DNA. Primary infection with HBV causes acute hepatitis, with symptoms of organ inflammation, fever, jaundice, and increased hepatic transaminases in the blood. Patients who are unable to overcome the viral infection suffer chronic disease progression over many years and are at increased risk of developing cirrhosis or liver cancer.

[0003] HBV infection results in the production of two distinct particles: 1) the HBV virus itself (or Dane particle), which contains a viral capsid assembled from HBV core antigen proteins (HBcAg), is coated with hepatitis B surface antigen (HBsAg), and is capable of reinfecting cells; and 2) subviral particles (or SVPs), which are high-density lipoprotein-like particles composed of lipids, cholesterol, cholesterol esters, and small and medium-sized non-infectious hepatitis B surface antigens (HBsAg). For each viral particle produced, 1,000 to 10,000 SVPs are released into the blood. Thus, SVPs (and the HBsAg protein they carry) make up the overwhelming majority of viral proteins in the blood. HBV-infected cells also secrete a soluble proteolytic product of the pre-core protein, called HBVe antigen (HBeAg).

[0004] Therapies currently recommended by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) for chronic HBV infection include interferon alpha (INFα), pegylated interferon alpha-2a (Peg-IFN2α), entecavir, and tenofovir. However, typical interferon therapy is 48 weeks long, resulting in significant and unpleasant side effects, and HBeAg seroconversion ranges from only 27 to 36% 24 weeks after therapy is stopped. HBsAg seroconversion is even lower, observed in only 3% immediately after treatment cessation, increasing to more than 12% after 5 years.

[0005] Nucleoside and nucleotide therapies, entecavir and tenofovir, have been successful in reducing viral load, but the seroconversion rates of HBeAg and clearance rates of HBsAg are lower than those obtained using IFNα therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT) and adefovir, are also used, but with nucleoside / nucleotide therapies, the emergence of resistance generally limits therapeutic efficacy. Summary of the Invention [Problem to be solved by the invention]

[0006] There is therefore a need to discover and develop new anti-HBV therapies, more particularly therapies that can increase HBeAg and / or HBsAg seroconversion rates. [Means for solving the problem]

[0007] In one aspect, the present disclosure provides a method for treating a Hepatitis B virus (HBV) infection in a subject in need thereof, comprising: The method is: The following structure: [ka] or a pharma- ceutical acceptable salt thereof; and administering to the subject a therapeutically effective amount of a single-stranded modified oligonucleotide comprising 20 linked nucleosides having the nucleobase sequence 5'-GCAGAGGTGAAGCGAAGTGC-3' (SEQ ID NO: 1); Including, The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, Each cytosine is a 5-methylcytosine (see FIG. 1).

[0008] Compound A described herein is a PAPD5 / 7 inhibitor disclosed as compound number 220 in WO2019 / 069293, the entirety of which is incorporated herein by reference.

[0009] The single-stranded modified oligonucleotide described herein is an antisense oligonucleotide (ASO) disclosed in WO2012 / 145697, the entirety of which is incorporated herein by reference. In one embodiment, the ASO is the compound ISIS number 505358 disclosed in WO2012 / 145697, which is also referred to as bepirovirsen.

[0010] In one embodiment, the disclosure provides a method for treating chronic hepatitis B (CHB) in a human in need thereof, comprising administering to a subject a therapeutically effective amount of Compound A and a therapeutically effective amount of bepivirsen.

[0011] In another aspect, the disclosure provides a method for reducing serum HBsAg levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharma- ceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a single-stranded modified oligonucleotide as described herein.

[0012] In another aspect, the disclosure provides a method for reducing serum HBV DNA levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A or a pharma- ceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a single-stranded modified oligonucleotide described herein.

[0013] In another embodiment, there is provided a combination for use in the treatment of chronic hepatitis B, comprising: The combination is The following structure: [ka] or a pharma- ceutically acceptable salt thereof; a single-stranded modified oligonucleotide comprising 20 linked nucleosides having the nucleobase sequence of SEQ ID NO:1; Including, The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, Combinations are provided in which each cytosine is a 5-methylcytosine.

[0014] In one embodiment, the combination is for use in a method of treating chronic hepatitis B in a subject, the method comprising administering simultaneously to the subject Compound A and a single-stranded modified oligonucleotide.

[0015] In one embodiment, the combination is for use in a method of treating chronic hepatitis B (CHB) in a human in need thereof, comprising administering to a subject a therapeutically effective amount of Compound A and a therapeutically effective amount of bepivirsen.

[0016] In another embodiment, the combination is for use in a method of reducing serum HBsAg levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A or a pharma- ceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a single-stranded modified oligonucleotide as described herein.

[0017] In a further embodiment, the combination is for use in a method of reducing serum HBV DNA levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A or a pharma- ceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a single-stranded modified oligonucleotide described herein. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the structure of a chimeric 2'-MOE phosphorothioate oligonucleotide (MOE gapmer). [Diagram 2] FIG. 2 shows the enzyme activities of PAPD5 and PAPD7 in biochemical assays for Compound A and RG7834. [Diagram 3] FIG. 3 shows the effect of single treatment with Compound A on HBsAg levels in AAV-HBV mice. [Figure 4]FIG. 4 shows the effect of combined treatment with Compound A and bepilovirsen on HBsAg levels in AAV-HBV mice. [Diagram 5] FIG. 5 shows the effect of sequential treatment with Compound A and bepilovirsen on HBsAg levels in AAV-HBV mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.For example, certain terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B and Klbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0020] Throughout this specification and the claims which follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" are understood to imply the inclusion of a stated element, integer or step, or a group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0021] Unless otherwise indicated, the following terms have the following meanings.

[0022] "2'-O-Methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furanose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0023] "2'-O-methoxyethyl nucleoside" (also 2'-MOE nucleoside) refers to a nucleoside that includes a 2'-O-methoxyethyl modified sugar moiety.

[0024] "5-methylcytosine" refers to a cytosine modified with a methyl group attached to position 5. 5-methylcytosine is a modified nucleobase.

[0025] "About" as used herein is intended to qualify the numerical value it modifies and to present such value as a variable within a margin of error. If no specific margin of error (e.g., standard deviation) is stated for the average value given in a chart or table of data, the term "about" should be understood to mean that a range encompassing ±10% of the stated value is included with the stated range.

[0026] "Active pharmaceutical agent" refers to one or more substances in a pharmaceutical composition that provide a therapeutic benefit when administered to a subject. For example, in some embodiments, an antisense oligonucleotide targeting HBV is an active pharmaceutical agent. In some embodiments, a PAPD5 / 7 inhibitor is an active pharmaceutical agent.

[0027] "Administered simultaneously" (or co-administration) refers to the co-administration of two drug substances to a subject over a period of time, in any form such that the first drug substance administered is still present in the subject in a therapeutically effective amount when the second drug substance is administered. Concurrent administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents may overlap over a period of time and need not be to the same extent.

[0028] "Administered sequentially" refers to administering a first drug substance a sufficient time after administering a second drug substance such that the first drug substance administered is not present in the subject in a therapeutically effective amount when the second drug substance is administered. The period between two successive administrations can be from 1 week to 24 weeks, e.g., from 2 weeks to 12 weeks, e.g., 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks.

[0029] "Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0030] "Antisense compound" refers to oligomeric compound that can undergo hybridization to target nucleic acid via hydrogen bond.Examples of antisense compounds include single-stranded and double-stranded compounds, such as antisense oligonucleotides, siRNA, shRNA, snoRNA, miRNA and satellite repeats.

[0031] "Antisense oligonucleotide" refers to a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

[0032] "Chemically distinct region" refers to a region of an antisense compound that is in some way chemically different from another region of the same antisense compound. For example, a region having 2'-O-methoxyethyl nucleosides or 2'-O-methoxyethyl nucleos(t)ides is chemically distinct from a region having nucleosides or nucleotides that lack the 2'-O-methoxyethyl modification.

[0033] "Chimeric antisense compound" refers to an antisense compound that has at least two chemically distinct regions, each region having multiple subunits.

[0034] "Chronic Hepatitis B (CHB) infection" occurs when a person initially suffers from acute infection but is then unable to fight off the infection. Approximately 90% of infants infected at birth progress to chronic disease. However, as people get older, the risk of chronic infection decreases, such that only 20%-50% of people infected as children and less than 10% of people infected as adults progress from acute to chronic infection.

[0035] "Deoxyribonucleoside" refers to a nucleoside that has a hydrogen at the 2' position of the sugar portion of the nucleoside. Deoxyribonucleosides can be modified with any of a variety of substituents.

[0036] "Dose" refers to a specified quantity of a pharmaceutical agent provided in a single administration or in a specified period of time. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, when subcutaneous administration is desired, the desired dose requires a volume that is not easily accommodated in a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reactions in individuals.

[0037] A "dosing regimen" is a combination of doses designed to achieve one or more desired effects.

[0038] "Duration" refers to the period during which an activity or event continues. In certain embodiments, the duration of treatment is the period during which doses of a pharmaceutical agent are administered.

[0039] An "effective amount" in the context of modulating an activity or treating or preventing a condition refers to the amount of active ingredient administered to a subject in need of such modulation, treatment or prevention, either in a single dose or as part of a series, that is effective for modulating that activity, or treating or preventing or ameliorating that condition.

[0040] "Gapmer" refers to a chimeric antisense compound in which an internal region having multiple nucleosides that support RNase H cleavage is positioned between the external regions, and the nucleosides that comprise the internal region are chemically distinct from the nucleosides that comprise the external regions. The internal region may be referred to as a "gap," "gap region," or "gap segment," and the external regions may be referred to as "wings," "wing regions," or "wing segments."

[0041] "HBV" refers to mammalian hepatitis B virus, including human hepatitis B virus. The term encompasses hepatitis B virus, particularly human hepatitis B virus geographic genotypes, and variant lineages of hepatitis B virus geographic genotypes. Human geographic genotypes of HBV include genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean region, Middle East, India); E (Africa); F (Native American, Polynesian); G (United States, France); and H (Central America).

[0042] "HBV antigen" refers to any hepatitis B viral antigen or protein, including the core protein, e.g., "hepatitis B core antigen" or "HBcAg," "hepatitis B e antigen" or "HBeAg," and the envelope protein, e.g., "HBV surface antigen" or "HBsAg."

[0043] "Hepatitis B e antigen" or "HBeAg" is a secreted non-particulate form of the HBV core protein. The HBV antigens HBeAg and HBcAg share a common primary amino acid sequence and therefore exhibit cross-reactivity at the T cell level. HBeAg is not required for viral assembly or replication, although studies suggest that it may be required to establish chronic infection.

[0044] "HBV surface antigen" or "HBsAg" is the envelope protein of the infectious HBV virion (Dane particle), which is also secreted as a non-infectious subviral particle (SVP) whose serum levels are 1000 times higher than those of the HBV virion. Serum levels of HBsAg in infected humans or animals can be as high as 1000 μg / mL (Kann and Gehrlich (1998) Topley & Wilson's Microbiology and Microbial Infections, 9th ed. 745).

[0045] "Hepatitis B associated condition" or "HBV associated condition" refers to any disease, biological state, medical condition or event exacerbated by, caused by, associated with, related to, or resulting from infection, exposure or disease with hepatitis B. The term hepatitis B associated condition includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, hepatitis, liver fibrosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, HBV viremia, transplant-related liver disease, and conditions with symptoms that may include any or all of the following: flu-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain over the liver area of ​​the body, clay-colored or gray stools, itching throughout the body, and dark urine.

[0046] "Induce," "inhibit," "enhance," "elevate," "increase," "decrease," and the like generally refer to a quantitative difference between two states. Such terms can refer to a statistically significant difference between two states. For example, "an amount effective to inhibit HBV activity or expression" refers to a level of HBV activity or expression in treated cells that is quantitatively different from, and can be statistically significant, from, the level of HBV activity or expression in untreated cells. Such terms apply, for example, to levels of expression and levels of activity.

[0047] "Internucleoside linkage" refers to the chemical bond between nucleosides. Phosphodiester and phosphorothioate are examples of internucleoside linkages.

[0048] "Linked nucleosides" refers to adjacent nucleosides linked to each other by an internucleoside bond.

[0049] A "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage), for example, a phosphorothioate internucleoside linkage.

[0050] "Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine and uracil. "Unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include methyl-cytosine.

[0051] A "modified nucleoside" refers to a nucleoside having, independently, a modified sugar moiety and / or a modified nucleobase.

[0052] A "modified nucleotide" refers to a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0053] "Modified oligonucleotide" refers to an oligonucleotide that contains at least one modified internucleoside linkage, modified sugar and / or modified nucleobase. According to some embodiments of the present disclosure, the modified oligonucleotide may be a chimeric antisense compound that contains an internal region having multiple nucleosides that support RNase H cleavage, located between external regions, for example, between two external regions, for example, a 5' external region and a 3' external region, each external region having one or more nucleosides, and the nucleosides contained in the internal region are chemically different from the nucleosides contained in the external regions. The modified oligonucleotide may be in the form of a free acid or a pharma- ceutically acceptable salt thereof (e.g., sodium salt), or a mixture thereof.

[0054] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering ribonucleic acid (siRNA) and microRNA (miRNA). Nucleic acids can include, but are not limited to, modified and / or unmodified nucleotides.

[0055] "Nucleobase" refers to a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0056] "Nucleobase sequence" refers to the order of contiguous nucleobases without regard to any sugar, linkage and / or nucleobase modifications.

[0057] "Oligonucleotide" refers to a polymer of linked nucleosides, each of which, independently of the others, may be modified or unmodified.

[0058] "Pharmaceutically acceptable salt" refers to a physiologically and pharma- ceutical acceptable salt of a compound, i.e., a salt that retains the desired biological activity of the parent active ingredient and does not impart undesired toxicological effects thereto.

[0059] "Drug" refers to a substance that provides a therapeutic benefit when administered to a subject. For example, a PAPD5 / 7 inhibitor and / or an antisense oligonucleotide targeting HBV RNA is a drug.

[0060] "Phosphorothioate linkage" refers to a linkage between nucleosides in which a phosphodiester linkage is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[0061] "Region" is defined, for example, as a part of a nucleic acid having at least one identifiable structure, function, or characteristic.

[0062] "Segment" can refer to, for example, a smaller region within a nucleic acid or a subportion of a region within a nucleic acid.

[0063] "Serum clearance" refers to HBsAg and / or HBV DNA levels below the lower limit of quantification (i.e., <LLOQ) in CHB patients. In testing of human samples, when serum HBsAg levels are measured by a sandwich immunoassay with COBAS HBsAg quant II (Roche), the LLOQ is 0.05 IU / mL. In testing of human samples, when serum HBV DNA levels are measured with COBAS Ampliprep / COBAS Taqman HBV test v.2.0 (Roche), the LLOQ is 20 IU / mL.

[0064] "Subject" refers to a human or non-human animal selected for treatment or therapy. In one embodiment, the subject is human.

[0065] "Therapeutically effective amount" is the amount of a drug that, when administered alone or as part of a pharmaceutical composition, and either in a single dose or as part of a series of doses, to a subject, results in the administration of an amount of the drug that can have any detectable positive effect on any symptom, aspect, or characteristic of a disease or condition.

[0066] "Treatment" refers to administering a composition to a subject to affect a change or improvement in a disease or condition. The term "treat" as used herein in the context of chronic hepatitis B infection refers to administering a suitable composition intended to reduce symptoms of CHB, prevent the progression of CHB, or reduce the level of one or more detectable markers of CHB.

[0067] method The present disclosure provides methods for treating Hepatitis B virus (HBV) infection. In some embodiments, the HBV infection is chronic hepatitis B (CHB).

[0068] In some embodiments, a method for treating an HBV infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a PAPD5 / 7 inhibitor and an antisense oligonucleotide.

[0069] In some embodiments, the method for treating an HBV infection in a subject in need thereof comprises administering to a subject a compound having the following structure: [ka] The method includes administering to a subject a therapeutically effective amount of Compound A having the formula:

[0070] In another embodiment, the method comprises: administering a therapeutically effective amount of Compound A to a subject; It contains 20 linked nucleosides and has the nucleobase sequence: 5'-GCAGAGGTGAAGCGAAGTGC-3' (SEQ ID NO: 1) administering to a subject a single-stranded modified oligonucleotide having the formula: Including, The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, Each cytosine is a 5-methylcytosine.

[0071] In one embodiment, Compound A is administered as the free acid. In another embodiment, Compound A is administered as a pharma- ceutically acceptable salt thereof.

[0072] In some embodiments, the modified oligonucleotide consists of 20 linked nucleosides and has the nucleobase sequence of SEQ ID NO:1. In some embodiments, the modified oligonucleotide is bepilovirsen. In some embodiments, bepilovirsen is administered as a free acid, a pharma- ceutically acceptable salt thereof (e.g., the sodium salt), or a combination thereof. In some embodiments, bepilovirsen is administered as a free acid. In some embodiments, bepilovirsen is administered as a pharma- ceutically acceptable salt thereof (e.g., the sodium salt). In some embodiments, bepilovirsen is administered as a combination of the free acid and the sodium salt.

[0073] In some embodiments, the method for treating HBV infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of Compound A and a therapeutically effective amount of bepilovirsen.The therapeutically effective amount of bepilovirsen is calculated based on the amount of bepilovirsen free acid.The subject may be a human.

[0074] In one embodiment, the disclosure provides a method for treating chronic hepatitis B (CHB) in a human in need thereof, comprising administering to a subject a therapeutically effective amount of Compound A and a therapeutically effective amount of bepivirsen.

[0075] In one embodiment, the subject is on stable nucleoside or nucleotide analogue (NA) therapy (e.g., tenofovir disoproxil, tenofovir alafenamide, or entecavir). "Stable" is defined as no changes to the nucleoside or nucleotide regimen for at least six months prior to treatment and no planned changes to the regimen for the duration of treatment. In some embodiments, the NA therapy is lamivudine, adefovir, adefovir dipivoxil, telbivudine, entecavir, tenofovir, tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof. In some embodiments, the NA therapy is entecavir, tenofovir, tenofovir disoproxil fumarate or tenofovir alafenamide. In some embodiments, the NA therapy is entecavir. In some embodiments, the NA therapy is tenofovir. In some embodiments, the NA therapy is tenofovir disoproxil fumarate. In some embodiments, the NA therapy is tenofovir alafenamide.

[0076] In another embodiment, the subject is not on NA therapy. In some embodiments, the subject is treatment naive.

[0077] In some embodiments, Compound A is administered orally. In one embodiment, Compound A is administered as a capsule. In one embodiment, Compound A is administered as a tablet. In some embodiments, Compound A is administered to a subject under fasting conditions. In some embodiments, Compound A is administered to a subject under fed conditions. In some embodiments, Compound A is administered for 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, Compound A is administered for 4 weeks, 8 weeks, or 12 weeks. In one embodiment, Compound A is administered for 4 weeks.

[0078] In some embodiments, the modified oligonucleotide is administered by subcutaneous injection. In some embodiments, the modified oligonucleotide is administered by subcutaneous injection in the form of an aqueous solution. In some embodiments, the modified oligonucleotide is administered once a week at a dose of about 150 mg or about 300 mg. In one embodiment, the modified oligonucleotide is administered once a week at a dose of about 150 mg. In another embodiment, the modified oligonucleotide is administered once a week at a dose of about 300 mg. In some embodiments, the modified oligonucleotide is administered every week with additional loading doses on days 4 and 11 of the first two weeks. In another embodiment, the modified oligonucleotide is administered once a week at a dose of about 300 mg with additional loading doses on days 4 and 11.

[0079] In some embodiments, the modified oligonucleotide is administered for about 12 to about 24 weeks. In some embodiments, the modified oligonucleotide is administered for 12, 14, 16, 18, 20, 22 or 24 weeks. In one embodiment, the modified oligonucleotide is administered for 12 weeks. In one embodiment, the modified oligonucleotide is administered for 24 weeks. In one embodiment, the modified oligonucleotide is bepivirsen and is administered for 12 or 24 weeks with additional loading doses on days 4 and 11.

[0080] In some embodiments, compound A and modified oligonucleotide are administered simultaneously. In some embodiments, compound A and modified oligonucleotide are administered simultaneously during the first treatment period. In some embodiments, the first treatment period is 2-12 weeks, e.g., 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks. In some embodiments, the first treatment period is 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, the first treatment period is 4-12 weeks. In one embodiment, compound A and bepilovirsen are administered simultaneously during the first treatment period (e.g., 4 weeks, 8 weeks, or 12 weeks). In one embodiment, compound A and bepilovirsen are administered simultaneously for 4 weeks. In one embodiment, compound A and bepilovirsen are administered simultaneously for 8 weeks. In one embodiment, compound A and bepilovirsen are administered simultaneously for 12 weeks.

[0081] In some embodiments, after the co-administration of the first treatment period, the modified oligonucleotide is administered alone for a second treatment period. In some embodiments, the second treatment period is 8 to 20 weeks. In some embodiments, the second treatment period is 8 to 48 weeks. In some embodiments, the second treatment period is 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, or 20 weeks. In one embodiment, the second treatment period is 8 weeks. In one embodiment, the second treatment period is 20 weeks.

[0082] In other embodiments, after the co-administration of the first treatment period, Compound A is administered alone for a second treatment period. In some embodiments, the second treatment period is 4 to 12 weeks. In some embodiments, the second treatment period is 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.

[0083] In some embodiments, compound A and modified oligonucleotide are administered simultaneously for a first treatment period, and then modified oligonucleotide is administered alone for a second treatment period. In some embodiments, the first treatment period is 4 weeks, and the second treatment period is 8-20 weeks. In some embodiments, compound A and bepilovirsen are administered simultaneously for 4 weeks, and then bepilovirsen is administered alone for 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks. In one embodiment, compound A and bepilovirsen are administered simultaneously for 4 weeks, and then bepilovirsen is administered alone for 8 weeks. In one embodiment, compound A and bepilovirsen are administered simultaneously for 4 weeks, and then bepilovirsen is administered alone for 20 weeks.

[0084] In some embodiments, compound A and modified oligonucleotides are administered simultaneously for a first treatment period, and then compound A is administered alone for a second treatment period. In some embodiments, the first treatment period is 12 weeks, and the second treatment period is 4-12 weeks. In some embodiments, compound A and bepirovirsen are administered simultaneously for 12 weeks, and then compound A is administered alone for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0085] The present disclosure provides that the amount of Compound A and / or single-stranded oligonucleotide administered to a subject as described herein is a therapeutically effective amount. In some embodiments, Compound A is administered twice daily. In some embodiments, the amount of Compound A administered is about 0.02 to about 20 mg per dose, or about 0.02 mg, about 0.05 mg, about 0.1 mg, about 0.3 mg, about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 20 mg per dose, or within a range of any two of the foregoing values ​​(e.g., 0.02 to 0.05 mg, 0.02 to 0.1 mg). , 0.02-0.3mg, 0.02-1mg, 0.02-3mg, 0.02-10mg, 0.05-0.1mg, 0.05-0.3mg, 0.05-1mg, 0.05-3mg, 0.05-10mg, 0.1-0.3mg, 0.1-1mg, 0.1-3mg, 0.1-10mg, 0.3-1mg, 0.3-3mg, 0.3-10mg, 0.5-1mg, 0.5-3mg, 0.5-10mg, 1-3mg, 1-10mg or 3-10mg). In some embodiments, compound A is administered at about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg per dose, or within a range of any two of the aforementioned values, for example, about 0.5 to about 1 mg, about 0.5 to about 2 mg, about 1 to about 10 mg, about 2 to about 6 mg, or about 3 to about 5 mg per dose. In some embodiments, compound A is administered at about 0.5 mg, about 1 mg, about 3 mg, or about 10 mg per dose. In some embodiments, compound A is administered at about 0.5 mg per dose. In some embodiments, compound A is administered at about 1 mg per dose. In some embodiments, compound A is administered at about 3 mg per dose.

[0086] In some embodiments, compound A is administered twice daily at a dose of about 1 to about 10 mg or about 1 to about 20 mg. In some embodiments, compound A is administered twice daily at a dose of about 3 mg. In some embodiments, compound A is administered twice daily at a dose of about 0.5 mg or about 1 mg. In some embodiments, compound A is administered twice daily at a dose of about 0.5 mg. In some embodiments, compound A is administered twice daily at a dose of about 1 mg.

[0087] In certain embodiments, administration of Compound A and modified oligonucleotides reduces blood HBV RNA, HBV DNA, HBV protein, HBsAg or HBeAg levels by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%. In some embodiments, administration of Compound A and bepirovirsen reduces serum HBV DNA and / or serum HBsAg levels by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%.

[0088] In some embodiments, the Hepatitis B virus infection is caused by any of the following human geographic genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean region, Middle East, India); E (Africa); F (Native American, Polynesia); G (United States, France); or H (Central America). In some embodiments, the subject has chronic hepatitis B (CHB).

[0089] In some embodiments, the subject achieves serum clearance of Hepatitis B surface antigen (HBsAg) at the end of treatment. In some embodiments, the subject maintains serum clearance of HBsAg 24 weeks after the end of treatment. "End of treatment" refers to administration of the last dose of Compound A or the last dose of modified oligonucleotide, whichever is later, for the methods described herein.

[0090] In some embodiments, the subject achieves serum clearance of HBV DNA at the end of treatment, hi some embodiments, the subject maintains serum clearance of HBV DNA 24 weeks after the end of treatment.

[0091] In some embodiments, the subject achieves serum clearance of HBsAg at the end of treatment, hi some embodiments, the subject maintains serum clearance of HBsAg 24 weeks after the end of treatment.

[0092] In some embodiments, the subject achieves serum clearance of HBsAg and HBV DNA at the end of treatment, hi some embodiments, the subject maintains serum clearance of HBsAg and HBV DNA 24 weeks after the end of treatment.

[0093] The methods described herein provide a higher response rate for subjects receiving combination therapy (Compound A and bepilovirsen) compared to subjects receiving monotherapy (Compound A or bepilovirsen) to achieve serum clearance of HBV DNA at the end of treatment or 24 weeks after the end of treatment. In some embodiments, the response rate for subjects receiving combination therapy is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% higher than subjects receiving monotherapy.

[0094] The methods described herein also provide a higher response rate for subjects receiving combination therapy (Compound A and bepilovirsen) compared to subjects receiving monotherapy (Compound A or bepilovirsen) to achieve serum clearance of HBsAg at the end of treatment or 24 weeks after the end of treatment. In some embodiments, the response rate for subjects receiving combination therapy is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% higher than subjects receiving monotherapy.

[0095] The methods described herein further provide a higher response rate for subjects receiving combination therapy (Compound A and bepilovirsen) compared to subjects receiving monotherapy (Compound A or bepilovirsen) to achieve serum clearance of HBsAg and HBV DNA at the end of treatment or 24 weeks after the end of treatment. In some embodiments, the response rate for subjects receiving combination therapy is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% higher than subjects receiving monotherapy.

[0096] In some embodiments, the subject achieves a reduction in HBsAg levels from baseline of at least 1, at least 1.5, at least 2, at least 2.5 or at least 3 log10 IU / mL at the end of treatment, hi some embodiments, the subject maintains a reduction in HBsAg levels from baseline of at least 1, at least 1.5, at least 2, at least 2.5 or at least 3 log10 IU / mL 24 weeks after the end of treatment.

[0097] In another embodiment, the present disclosure provides a combination for use in treating an HBV infection, comprising: The combination is The following structure: [ka] or a pharma- ceutically acceptable salt thereof; a single-stranded modified oligonucleotide comprising 20 linked nucleosides and having the nucleobase sequence of SEQ ID NO:1; Including, The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, Combinations are provided in which each cytosine is a 5-methylcytosine.

[0098] In another aspect, the disclosure provides the use of compound A in the manufacture of a medicament for the treatment of HBV infection, comprising: Compound A is administered together with a single-stranded modified oligonucleotide comprising 20 linked nucleosides and having the nucleobase sequence of SEQ ID NO:1; The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, In accordance with the present invention, each cytosine is a 5-methylcytosine.

[0099] In another aspect, the disclosure provides a use of a single stranded modified oligonucleotide in the manufacture of a medicament for the treatment of HBV infection, comprising: The single-stranded modified oligonucleotide comprises 20 linked nucleosides and has the nucleobase sequence of SEQ ID NO:1, a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, In accordance with the present invention, each cytosine is a 5-methylcytosine.

[0100] In another embodiment, the disclosure provides the use of a combination of Compound A and a single-stranded modified oligonucleotide in the manufacture of a medicament for the treatment of HBV infection, comprising: The single-stranded modified oligonucleotide comprises 20 linked nucleosides and has the nucleobase sequence of SEQ ID NO:1, a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, In accordance with the present invention, each cytosine is a 5-methylcytosine.

[0101] In some embodiments, the combination of Compound A and a modified oligonucleotide (eg, bepilovirsen) is for use in the treatment of chronic hepatitis B (CHB) in a human in need thereof.

[0102] In some embodiments, the combination of Compound A and a modified oligonucleotide (eg, bepilovirsen) is for use in the manufacture of a medicament for the treatment of CHB in a human in need thereof.

[0103] The doses and administration intervals described above in connection with the method of the invention are also suitable for the medical uses of the invention.

[0104] Combination The present disclosure also provides a pharmaceutical combination for treating Hepatitis B virus (HBV) infection. In some embodiments, the HBV infection is chronic hepatitis B (CHB). In addition to the features described below, the combination disclosed herein can be used in any of the aforementioned methods.

[0105] The combination comprises two compositions: a first composition comprising a first pharmaceutical ingredient and a second composition comprising a second pharmaceutical ingredient. For example, in one embodiment, the first composition comprises a PAPD5 / 7 inhibitor, such as Compound A, and / or the second composition comprises an antisense oligonucleotide, such as bepivirsen.

[0106] In some embodiments, the combination comprises a PAPD5 / 7 inhibitor and an antisense oligonucleotide. In particular, the combination comprises a first composition comprising a PAPD5 / 7 inhibitor and a second composition comprising an antisense oligonucleotide. In these embodiments, the combination may be for use in treating HBV infection in a subject in need thereof, and the treatment comprises administering to the subject a therapeutically effective amount of a PAPD5 / 7 inhibitor and an antisense oligonucleotide.

[0107] In some embodiments, the combination has the following structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0108] In another embodiment, the combination comprises: Compound A, Nucleotide sequence: 5'-GCAGAGGTGAAGCGAAGTGC-3' (SEQ ID NO: 1) and a single stranded modified oligonucleotide comprising 20 linked nucleosides, Including, The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and A 3' wing segment consisting of five linked nucleosides Including, the gap segment is located between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage, Each cytosine is a 5-methylcytosine.

[0109] In one embodiment, Compound A is the free acid. In another embodiment, Compound A is a pharma- ceutically acceptable salt thereof.

[0110] In some embodiments, the modified oligonucleotide consists of 20 linked nucleosides having the nucleobase sequence of SEQ ID NO:1. In some embodiments, the modified oligonucleotide is bepilovirsen. In some embodiments, bepilovirsen is administered as a free acid, a pharma- ceutically acceptable salt thereof (e.g., the sodium salt), or a combination thereof. In some embodiments, bepilovirsen is administered as a free acid. In some embodiments, bepilovirsen is administered as a pharma- ceutically acceptable salt thereof (e.g., the sodium salt). In some embodiments, bepilovirsen is administered as a combination of the free acid and the sodium salt.

[0111] In some embodiments, the combination is for use in a method of treating HBV infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A and a therapeutically effective amount of bepilovirsen.

[0112] In one embodiment, the disclosure provides a combination for use in a method of treating chronic hepatitis B (CHB) in a human in need thereof, the method comprising administering to the human a therapeutically effective amount of Compound A and a therapeutically effective amount of bepivirsen.

[0113] In one embodiment, the subject is on stable nucleoside or nucleotide analog (NA) therapy (e.g., tenofovir disoproxil, tenofovir alafenamide, or entecavir). In some embodiments, the NA therapy is lamivudine, adefovir, adefovir dipivoxil, telbivudine, entecavir, tenofovir, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof. In some embodiments, the NA therapy is entecavir, tenofovir, tenofovir disoproxil fumarate or tenofovir alafenamide. In some embodiments, the NA therapy is entecavir. In some embodiments, the NA therapy is tenofovir. In some embodiments, the NA therapy is tenofovir disoproxil fumarate. In some embodiments, the NA therapy is tenofovir alafenamide.

[0114] In another embodiment, the subject has not received NA therapy prior to administration of Compound A or a modified oligonucleotide. In some embodiments, the subject is treatment naive.

[0115] In some embodiments, Compound A is for oral administration. In one embodiment, Compound A is formulated as a capsule. In one embodiment, Compound A is formulated as a tablet.

[0116] In some embodiments, the modified oligonucleotide is formulated for delivery by subcutaneous injection. In some embodiments, the modified oligonucleotide is in an aqueous solution. In some embodiments, the combination comprises 150 mg or 300 mg of the modified oligonucleotide.

[0117] In some embodiments, the combination is for use in a method in which compound A and the modified oligonucleotide are administered simultaneously. In some embodiments, this simultaneous administration occurs during a first treatment period. In some embodiments, the first treatment period is 2 to 12 weeks, for example, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, the first treatment period is 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In one embodiment, the first treatment period is 4 weeks. In one embodiment, the combination comprises compound A and bepilovirsen for use in a method in which compound A and bepilovirsen are administered simultaneously during a first treatment period (e.g., 4 weeks, 8 weeks, or 12 weeks). In one embodiment, the combination comprises compound A and bepilovirsen for use in a method in which compound A and bepilovirsen are administered simultaneously for 4 weeks.

[0118] The present disclosure provides a combination comprising a therapeutically effective amount of compound A and a single-stranded modified oligonucleotide. In some embodiments, the combination comprises 0.02-20 mg of compound A, or 0.02 mg, 0.05 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1 mg, 3 mg, 10 mg, or 20 mg of compound A, or an amount within a range of any two of the aforementioned values ​​(e.g., 0.02-0.05 mg, 0.02-0.1 mg, 0.02-0.3 mg, 0.02-1 mg, 0.02-3 mg, 0.02-4 mg, 0.02-5 mg, 0.02-6 mg, 0.02-7 mg, 0.02-8 mg, 0.02-9 mg, 0.02-10 mg, 0.02-11 mg, 0.02-12 mg, 0.02-13 mg, 0.02-14 mg, 0.02-15 mg, 0.02-16 mg, 0.02-17 mg, 0.02-18 mg, 0.02-20 mg, 0.02-25 mg, 0.02-26 mg, 0.02-27 mg, 0.02-28 mg, 0.02-29 mg, 0.02-30 mg, 0.02-31 mg, 0.02-32 mg, 0.02-33 mg, 0.02-34 mg, 0.02-35 mg, 0.02-36 mg, 0.02-37 mg, 0.02-38 mg, 0.02-39 mg, 0.02-39 mg, 0.02-39 mg, 0.02-39 mg, 0.02 mg, 0.02-10 mg, 0.05-0.1 mg, 0.05-0.3 mg, 0.05-1 mg, 0.05-3 mg, 0.05-10 mg, 0.1-0.3 mg, 0.1-1 mg, 0.1-3 mg, 0.1-10 mg, 0.3-1 mg, 0.3-3 mg, 0.3-10 mg, 0.5-1 mg, 0.5-3 mg, 0.5-10 mg, 1-3 mg, 1-10 mg, or 3-10 mg). In some embodiments, the combination contains 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of compound A, or an amount between any two of the aforementioned values, for example, about 0.5 to about 1 mg, about 1 to about 10 mg, about 2 to about 6 mg, or about 3 to about 5 mg. In some embodiments, the combination comprises 0.5 mg, 1 mg, 3 mg, or 10 mg of Compound A. In some embodiments, the combination comprises 3 mg of Compound A. In some embodiments, the combination comprises 0.5 mg of Compound A. In some embodiments, the combination comprises 1 mg of Compound A.

[0119] In one embodiment, the combination comprises 150 mg of the modified oligonucleotide and 3 mg of compound A. In another embodiment, the combination comprises 300 mg of the modified oligonucleotide and 3 mg of compound A. In one embodiment, the combination comprises 150 mg of the modified oligonucleotide and 0.5-1 mg of compound A. In another embodiment, the combination comprises 300 mg of the modified oligonucleotide and 0.5-1 mg of compound A. In one embodiment, the combination comprises 150 mg of the modified oligonucleotide and 0.5 mg of compound A. In another embodiment, the combination comprises 300 mg of the modified oligonucleotide and 0.5 mg of compound A. In one embodiment, the combination comprises 150 mg of the modified oligonucleotide and 1 mg of compound A. In another embodiment, the combination comprises 300 mg of the modified oligonucleotide and 0.5 mg of compound A. In one embodiment, the combination comprises 150 mg of the modified oligonucleotide and 1 mg of compound A. In another embodiment, the combination comprises 300 mg of the modified oligonucleotide and 1 mg of compound A.

[0120] kit In one aspect of the present invention, the drug substance disclosed herein, particularly in the form of a pharmaceutical composition, is included in a kit together with instructions for use. In one embodiment, the kit includes an antisense oligonucleotide targeting HBV, i.e., a modified oligonucleotide disclosed herein, and a PAPD5 / 7 inhibitor disclosed herein, each in separate containers. In one embodiment, the kit includes bepilovirsen. In one embodiment, the kit includes Compound A or a pharmaceutically acceptable salt thereof. In one embodiment, the kit includes bepilovirsen and Compound A or a pharmaceutically acceptable salt thereof.

[0121] For convenience, the kit can include a predetermined amount of drug substance together with instructions for use. In one embodiment, the predetermined amount is as disclosed in the combination section above. In one embodiment, the kit includes 300 mg of modified oligonucleotide. In one embodiment, the kit includes 150 mg of modified oligonucleotide.

[0122] In one embodiment, the kit comprises Compound A formulated for oral administration. In one embodiment, the kit comprises one or more capsules or tablets comprising Compound A. In one embodiment, the kit comprises one or more capsules or tablets comprising 0.02-20 mg of Compound A. In one embodiment, each capsule or tablet comprises 0.5-1 mg of Compound A. In one embodiment, each capsule or tablet comprises 0.5 mg of Compound A. In one embodiment, each capsule or tablet comprises 1 mg of Compound A. In one embodiment, Compound A is formulated as a tablet. In one embodiment, Compound A is formulated as a capsule.

[0123] The kit can also include a device used to administer the pharmaceutical composition.

[0124] In another embodiment, the kit comprises another pharmaceutical agent. EXAMPLES

[0125] Aspects of the disclosure provided herein have been described in greater detail according to several embodiments, however the following examples, which highlight certain features and characteristics of exemplary embodiments of the disclosure described herein, serve only to illustrate, and are not intended to be limiting of, the disclosure described herein.

[0126] Example 1: In vitro antiviral activity of compound A Efficacy of Compound A in reducing HBV HBsAg (pIC 50 ) was studied in primary human hepatocytes (PHH) from three donors. HBV-infected PHH samples were treated with Compound A for 21 days and secreted HBsAg was measured using an ELISA assay to determine % inhibition and mean potency of replicates (two per PHH donor). Compound A showed similar potency in HBsAg reduction across three different PHH donors with a mean pIC50 of 9.08. No signs of cytotoxicity were detected by visual inspection.

[0127] Example 2: Toxicity evaluation of PAPD5 / 7 inhibitors Studies were conducted to assess toxicity in rats and monkeys, including additional endpoints studying the neurotoxicity of Compound A after 28 days of dosing followed by a 6-week washout period. No adverse effect dose was established for Compound A in either species. Another PAPD5 / 7 inhibitor, RG7834 (structure shown below), was terminated in Phase 1 clinical trials due to preclinical toxicity and the inability to establish an adverse effect-free dose.

[0128] [ka]

[0129] Example 3: Comparison of antiviral activity of PAPD5 / 7 inhibitors The potency of Compound A, RG7834 and Compound B (structures shown below) to reduce HBV HBsAg was studied in HepAD38 cell line. Compound A showed higher potency compared to RG7834 and Compound B (Table 1).

[0130] [ka]

[0131] HepAD38 cells were maintained in cell culture medium (DMEM / F12 with 10% fetal bovine serum (FBS), GlutaMax-1, penicillin / streptomycin, non-essential amino acids, Na pyruvate, 250 μg / mL geneticin, and 1 μg / mL doxycycline) in collagen-coated flasks. Compound solutions were prepared in DMSO and compounds were serially diluted to final concentrations of 4000, 1000, 250, 62.5, 15.6, 3.91, 0.977, 0.244, 0.061, and 0.015 nM. Cells were then trypsinized and cells were plated at 10,000 cells per well. Plates were incubated at 37° C., 5% CO2 for 4 days. Medium was replaced with fresh medium with compound treatment. Plates were then incubated at 37° C., 5% CO2 for an additional 3 days, making the total treatment time 7 days. For antiviral responses, HBsAg was measured using an HBsAg ELISA kit (International Immuno-diagnostics) with accompanying instructions. 100 μL of cell media samples were used for ELISA. Absorbance at 450 nm was read on a SPECTRAMAX384 plate reader (Molecular Devices). For cytotoxicity, cells were used in CELLTITER-GLO Luminescent Cell Viability Assay reagent (Promega). Luminescence was read on an Envision Multilabel Reader (Perkin Elmer). Data were analyzed relative to DMSO controls.

[0132] % Inhibition = (1-(Unknown / High Control))*100

[0133] The mean % inhibition values ​​from duplicate assay plates were then plotted in GraphPad Prism to determine EC values: a 4-parameter logistic curve with the formula: Y=bottom+(top-bottom) / (1+10^((LogEC50-X)*Hillslope)).

[0134] [Table 1]

[0135] Example 4: Comparison of biochemical enzyme inhibition of PAPD5 / 7 inhibitors Dose-response studies were performed on compound A and RG7834 in the PAPD5 and PAPD7 A15 RNA SPA format. Four different batches of compound A and two different batches of RG7834 were used in both assays. Reactions contained 5 μM ATP, 0.0025 μCi / μL 3H-ATP, 50 nM A15 RNA, 10 nM enzyme in assay buffer (20 mM Tris 7.5, 3 mM CHAPS, 0.05% dBSA, 10 mM MgCl2, 1 mM DTT, 25 mM KCl). Reaction products were detected using an SPA bead mixture with final concentrations of 1 mg / mL PEI-PS, 1.25 mM ATP, 11.25 mM EDTA. Endpoint signal detection correlated with the slope of the reaction time course. Curve fitting was performed using 4-parameter fitting in ActivityBase software. The average pIC50 values ​​for Compound A in the PAPD5 and PAPD7 assays were 7.9 and 7.7, respectively. The data show that Compound A was >0.5 log more potent than RG7834 (see Figure 2).

[0136] Example 5: In vivo antiviral activity in the AAV-HBV mouse model A. Single treatment After 28 days of infection with AAV-HBV, single treatment of infected male C57BL / 6 mice with either Compound A or bepivirsen was initiated. Combined vehicle and the lowest dose of Compound A (0.3 mg / kg) showed minimal reduction in HBsAg over the course of treatment. Animals receiving either the medium dose (3 mg / kg) or high dose (30 mg / kg) of Compound A showed statistically significant reduction in HBsAg compared to the combined vehicle group. Reduction in HBsAg for the 3 and 30 mg / kg Compound A dose groups was approximately 1 log and 1 log, respectively, within 1 week of treatment, and the reduction level was sustained throughout the duration of treatment. After 1 week of treatment cessation, HBsAg levels rebounded to baseline. In measuring the effect on HBeAg levels, there was a dose-dependent reduction during treatment with Compound A that stabilized after day 7 and rebounded within 1 week after the end of treatment. The maximum observed reduction in HBeAg was 0.5 log at 30 mg / kg Compound A. There was a small reduction in HBV DNA (0.26-0.40 log) at the 3 and 30 mg / kg Compound A dose levels compared to the combined vehicle group. The change in HBsAg with Compound A treatment is shown in Figure 3.

[0137] With either 20 mg / kg or 40 mg / kg bepilovirsen treatment, maximum reductions in HBsAg of over 1 log and 2 logs, respectively, were achieved 2 weeks after the start of treatment. Return to baseline was progressive until the end of the study for the 20 mg / kg bepilovirsen group, while the 40 mg / kg bepilovirsen group had a reduction in HBsAg at the end of the study. Reductions in HBeAg in the 20 mg / kg and 40 mg / kg bepilovirsen groups stabilized within 2 weeks of treatment at 1.35 log and 1.85 log, respectively, compared to the combined vehicle group. After the end of treatment, HBeAg slowly rebounded, but reductions of 0.34 log and 0.80 log were maintained in the 20 mg / kg bepilovirsen and 40 mg / kg dose groups, respectively. Based on weight profiles, all treatments were well tolerated.

[0138] B. Combination Treatment After 28 days of infection with AAV-HBV, combination treatment of infected male C57BL / 6 mice was initiated with Compound A and bepilovirsen. For groups receiving both compounds, an enhanced effect on HBsAg reduction was observed for Compound A at 3 mg / kg or 30 mg / kg with bepilovirsen at 20 mg / kg or 40 mg / kg. The maximum observed reduction in HBsAg was greater than 2 logs and nearly 3 logs for the 30 mg / kg Compound A + 20 mg / kg bepilovirsen and 30 mg / kg Compound A + 40 mg / kg bepilovirsen groups, respectively. The effect of combination treatment on the levels of HBsAg for the highest dose of Compound A with and without bepilovirsen is shown in Figure 4. The effect on HBeAg reduction was measured, with HBeAg reduced by 0.15 to 0.44 logs in the 3 or 30 mg / kg Compound A + 20 or 40 mg / kg bepilovirsen dose groups. HBeAg rebound in these combination groups occurred similarly to the bepilovirsen monotherapy dose groups. In all bepilovirsen dose groups (with or without Compound A), there was a robust reduction in HBV DNA of >2.5 logs, which stabilized by day 14 of treatment. At the end of the study, HBV DNA was maintained at 0.84-1.35 logs in groups receiving 20 mg / kg bepilovirsen and 1.72-2.01 logs in groups receiving 40 mg / kg bepilovirsen compared to the combined vehicle groups. Based on weight profiles, all treatments were well tolerated.

[0139] C. Sequential Treatment After 35 days of infection with AAV-HBV, sequential treatment of infected male C57BL / 6 mice was initiated first with Compound A followed by bepilovirsen. Within 1 week of treatment with 30 mg / kg Compound A alone, a statistically significant, approximately 1 log reduction in HBsAg was observed compared to the group receiving Compound A vehicle, which remained stable for the duration of lead-in treatment with Compound A (up to 28 days). Within 1 week of treatment cessation, HBsAg rebounded to baseline and was indistinguishable from levels in the combined vehicle group.

[0140] Within one week of initiating treatment with bepilovirsen administered with Compound A vehicle at either 20 or 40 mg / kg, a reduction in HBsAg was observed, reaching a maximum reduction of more than 1 log and nearly 2 logs, respectively, by week 2. This reduction was statistically significant compared to the combined vehicle group. In groups administered Compound A first, followed by bepilovirsen (30 mg / kg Compound A, then either 20 or 40 mg / kg bepilovirsen), a further reduction in HBsAg was observed by week 2 of bepilovirsen treatment, compared to the reduction maintained during the induction treatment of Compound A.

[0141] On the last day of treatment with bepilovirsen and one week after the last day, the groups treated with 30 mg / kg Compound A followed by either 20 or 40 mg / kg bepilovirsen failed to show further reduction in HBsAg compared to the groups treated with bepilovirsen alone, suggesting that there is no added benefit of induction treatment with Compound A on the HBsAg-reducing effect of bepilovirsen when following a sequential format. Furthermore, HBsAg levels rebounded similarly in groups that did or did not receive 30 mg / kg Compound A. Based on the body weight profile, all treatment regimens were well tolerated. The effect of sequential treatment at the highest dose of Compound A and bepilovirsen is shown in Figure 5.

[0142] Example 6: Studies for predicting human dose A. Prediction of human pharmacokinetics

[0143] A physiologically based PK (PBPK) model with an advanced compartmental absorption and transit (ACAT) model coupled to a compartmental disposition model using GastroPlus was established using in silico, in vitro and in vivo preclinical data. The ACAT model was developed using PK data from rats, dogs, minipigs and monkeys, with default gastrointestinal physiology settings used for all species. Overall, the established PBPK model matched the shape of the preclinical PK profiles and adequately explained the PK parameters AUC and Cmax, with less than a two-fold difference between predicted and observed values. PK predictions were performed assuming an average human body weight of 70 kg.

[0144] The antiviral activity of compound A was evaluated in vitro in HBV systems: (1) HepAD38, a hepatoma cell line with inducible expression of HBV, (2) primary human hepatocytes (PHH) infected with HBV, and in vivo in male C57BL / 6 mice infected with AAV-HBV. Overall, there is a correlation between in vitro potency (HepAD38&PHH) and HBsAg inhibition in the AAV-HBV in vivo model.

[0145] The IC90 values ​​calculated based on the IC50s from the three experiments above are shown in Table 2.

[0146] [Table 2]

[0147] B. Therapeutic Dose Rationale The minimum therapeutic dose is defined as the dose that results in a Cmin at steady state associated with greater than 90% of the predicted maximal pharmacological activity (Cmin>IC90).

[0148] Based on a predicted half-life of approximately 13 hours and consistent with optimized dosing with safety margins, a twice-daily dosing frequency was selected for Compound A. Furthermore, the analysis described in the therapeutic dose rationale supports that this regimen provides pharmacological activity over the dosing interval (i.e., at predicted Cmin concentrations at 3 mg BID dosing, predicted effective doses are estimated to provide 91.9%, 87.0% and 99.1% activity in HepAD38 cells, PHH, AAV HBV mice experiments, respectively). Twice-daily dosing also minimizes the exposure (Cmax) associated with maintaining concentrations above target (EC90), thus ensuring a robust safety margin.

[0149] C. Rationale for Maximum Dose The maximum single dose is defined based on: The dose that produces a concentration 24 hours after administration (C24) associated with approximately 99% of the predicted maximum pharmacological activity (limit of pharmacological activity). · Dose associated with higher exposure than predicted at the highest planned dose in a multiple dose study (PK criteria).

[0150] Based on predicted human pharmacokinetics, including plasma C24 and Cmax data, a maximum dose of Compound A of 20 mg administered as a single dose is proposed for human trials (see Table 3). Considering that the planned highest dose in multiple doses is 3 x the therapeutic BID dose (3 x 3 mg = 9 mg to 10 mg), the maximum single dose of 20 mg is expected to cover, by PK criteria, the predicted PK of the planned highest dose (10 mg) in multiple dose studies (see Tables 3 and 4 below).

[0151] [Table 3]

[0152] [Table 4]

[0153] Example 7: A four-part, randomized, double-blind, multicenter, placebo-controlled study to evaluate the safety, tolerability, PK, and PD of Compound A monotherapy in healthy participants and CHB patients; and Compound A in combination with bepirovirsen in CHB patients The first two parts of the study will evaluate the safety, tolerability and pharmacokinetics (PK) of single (Part 1) and repeated doses (Part 2A) of Compound A, as well as the effect of a tablet / food containing single dose (Part 2B) in healthy participants. Part 3 will evaluate the ability of Compound A to lower HBsAg in participants living with chronic hepatitis B infection (PLWCHB). Part 4 will evaluate the safety and tolerability of combination therapy with Compound A and bepirovirsen, as well as its potential to produce a sustained virologic response in PLWCHB.

[0154] Part 4 is a 12-week repeat-dose single-dose level study of Compound A in combination with bepilovirsen in PLWCHB on stable NA therapy who are not participating in Part 3 of the study. Sixty participants are randomized in a 3:1 ratio (45 participants to active and 15 participants to placebo) to receive either Compound A or placebo as an oral tablet twice daily (approximately 12-hour dosing interval) for 28 days. Compound doses will be determined based on the results of Parts 1-3 of the study. In addition, all participants in this cohort will also receive open-label bepilovirsen (300 mg subcutaneous [SC], weekly loading doses on days +4 and 11) for 28 days simultaneously. Bepilovirsen administration will continue for an additional 8 weeks after Compound A administration is completed (for a total of 12 weeks). Patients will be monitored with follow-up for 24 weeks after the last bepilovirsen dose.

[0155] The primary efficacy endpoint is a sustained virological response, which is a composite endpoint defined as HBsAg <LLOQ (0.05 IU / mL) and HBV DNA <LLOQ (20 IU / mL) at the end of the bepirovirsen treatment, which persists for 24 weeks after the bepirovirsen treatment. Serum HBsAg levels are measured by a sandwich immunoassay on the COBAS HBsAg quant II (Roche). Serum HBV DNA levels are measured by the COBAS Ampliprep / COBAS Taqman HBV test v.2.0 (Roche). Serum clearance in this study refers to participants with HBsAg and HBV DNA <LLOQ (regardless of the presence or absence of HBs antibody formation). Seroconversion refers to participants with <LLOQ HBsAg and HBV DNA + HBs antibody formation. Both terms are used to assess efficacy. In this study, a sustained response is defined as 24 consecutive weeks from the end of the bepirovirsen treatment, during which the level of HBsAg in the serum is maintained below the LLOQ and the HBV DNA is maintained below the LLOQ.

[0156] The objectives and endpoints of parts 3 and 4 of this study are listed below.

[0157]

Table 5

[0158] Example 8: In Vitro Evaluation of the Antiviral Effect of a Combination of Bepirovirsen and Compound A HBV-infected primary human hepatocytes (PHH) are used to evaluate the combined antiviral effects of bepilovirsen and compound A in vitro. PHH are left for 3 days after plating in collagen-coated 96-well plates to establish a monolayer and assimilate into in vitro culture, and are subsequently infected with HBV at an MOI of 200-500 genome equivalents (GE) / cell. HBV-infected (or uninfected control) PHH are treated on day 7 post-HBV infection and continue treatment at the pre-determined dosing frequency until the end of the study on day 21 post-treatment / day 28 post-infection. Dosing is performed in a checkerboard matrix layout where 8 serial dilutions of bepilovirsen and 8 serial dilutions of compound A are combined. The highest evaluated concentration of each treatment is serially diluted 3-, 4-, and 5-fold to include concentrations expected to result in 0 and maximum % inhibition. The serial dilution scheme places the EC50 of each treatment, determined in a prior in vitro study of HBV-infected PHH, near the center of the expected dose-response curve. In parallel, monotherapy is studied as a comparison when combined with vehicle control. Each treatment in a single 96-well plate is tested alone, but each plate is performed with 2-3 replicates to assess assay and biological variability. At time points such as 7, 14, and 21 days after treatment, culture supernatants are collected and stored at -80°C until analysis. The level of HBsAg secreted into the culture supernatant serves as the primary efficacy readout for the activity of bepilovirsen and Compound A. Additional viral endpoints can be evaluated to determine the antiviral effect of the combination of bepilovirsen and Compound A on the production of other HBV antigens, HBV RNA, and HBV DNA. Statistical analysis is performed using Synergy software to determine whether synergistic, antagonistic, or other effects are observed when bepilovirsen and Compound A are combined.

[0159] Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The particular embodiments described herein are intended to be representative and illustrative and are not intended as limitations on the scope of the invention. Modifications therein and other uses will be apparent to those skilled in the art and are encompassed within the spirit of the invention as defined by the scope of the claims.

[0160] All patent applications, patents, and printed publications cited herein are incorporated herein by reference in their entirety, except to the extent that the incorporated material conflicts with the disclosure expressly set forth herein, except for any definitions, disclaimers or disclaimers of subject matter, in which case the language of the present disclosure will control.

Claims

1. 1. A combination for use in the treatment of chronic hepatitis B in a human, comprising: The combination is The following structure: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof; a single-stranded modified oligonucleotide comprising 20 linked nucleosides and having the nucleobase sequence of SEQ ID NO: 1; Including, The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of five linked nucleosides; and a 3' wing segment consisting of five linked nucleosides Including, the gap segment is positioned between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; The combination wherein each cytosine is a 5-methylcytosine.

2. The combination of claim 1, wherein compound A is administered as a free acid.

3. The combination described in claim 1, wherein the single-stranded modified oligonucleotide is bepivirsen.

4. A combination described in any one of claims 1 to 3, wherein the subject is undergoing stable nucleoside or nucleotide analog (NA) therapy.

5. A combination described in any one of claims 1 to 3, wherein the NA therapy is lamivudine, adefovir, adefovir dipivoxil, telbivudine, entecavir, tenofovir, tenofovir disoproxil fumarate or tenofovir alafenamide or a pharmaceutically acceptable salt thereof.

6. A combination described in any one of claims 1 to 3, wherein compound A is administered orally.

7. The combination of claim 6, wherein compound A is administered twice daily at a dose of about 0.5 mg or about 1 mg.

8. A combination described in any one of claims 1 to 3, wherein the single-stranded modified oligonucleotide is administered by subcutaneous injection.

9. A combination described in any one of claims 1 to 3, wherein the single-stranded modified oligonucleotide is administered once a week at a dose of about 150 mg or about 300 mg.

10. A combination described in any one of claims 1 to 3, wherein compound A and the single-stranded modified oligonucleotide are administered simultaneously.

11. The combination of claim 10, wherein compound A and the single-stranded modified oligonucleotide are administered simultaneously for approximately four weeks.

12. The combination described in claim 10, wherein after simultaneous administration, the modified oligonucleotide is administered alone during a second treatment period.

13. The combination of claim 12, wherein the second treatment period is from about 8 to about 20 weeks.

14. 1. Use of Compound A, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of chronic hepatitis B in a human, comprising: The compound A has the following structure: 【Chemistry 2】 and Compound A is administered in combination with a single-stranded modified oligonucleotide comprising 20 linked nucleosides and having the nucleobase sequence of SEQ ID NO: 1; The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of five linked nucleosides; and a 3' wing segment consisting of five linked nucleosides Including, the gap segment is positioned between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; The above use, wherein each cytosine is a 5-methylcytosine.

15. 1. Use of a single-stranded modified oligonucleotide comprising 20 linked nucleosides and having the nucleobase sequence of SEQ ID NO: 1 in the manufacture of a medicament for the treatment of chronic hepatitis B in a human, comprising: The single-stranded modified oligonucleotide is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of five linked nucleosides; and a 3' wing segment consisting of five linked nucleosides Including, the gap segment is positioned between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; each cytosine is a 5-methylcytosine, The single-stranded modified oligonucleotide has the following structure: 【Transformation 3】 or a pharmaceutically acceptable salt thereof.

16. 1. Use of a combination of Compound A, or a pharmaceutically acceptable salt thereof, and a single-stranded modified oligonucleotide in the manufacture of a medicament for the treatment of chronic hepatitis B in a human, comprising: The compound A has the following structure: 【Chemistry 4】 and the single-stranded modified oligonucleotide comprises 20 linked nucleosides and has the nucleobase sequence of SEQ ID NO: 1; and a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of five linked nucleosides; and a 3' wing segment consisting of five linked nucleosides Including, the gap segment is positioned between the 5' wing segment and the 3' wing segment; each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; The above use, wherein each cytosine is a 5-methylcytosine.

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