WO2022078479A1 - Combination therapy for treating hepatitis b virus infection - Google Patents

Combination therapy for treating hepatitis b virus infection Download PDF

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Publication number
WO2022078479A1
WO2022078479A1 PCT/CN2021/123990 CN2021123990W WO2022078479A1 WO 2022078479 A1 WO2022078479 A1 WO 2022078479A1 CN 2021123990 W CN2021123990 W CN 2021123990W WO 2022078479 A1 WO2022078479 A1 WO 2022078479A1
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Prior art keywords
methyl
phenyl
thiazol
oxo
fluoro
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PCT/CN2021/123990
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French (fr)
Inventor
Ying Tan
Ren ZHU
Jan Martin Berke
George Kukolj
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Janssen Pharmaceuticals, Inc.
Johnson & Johnson (China) Investment Ltd.
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Publication of WO2022078479A1 publication Critical patent/WO2022078479A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to the combination of an RNA interference (RNAi) component and a heteroaryldihydropyrimidine (HAP) compound for treating hepatitis B virus infection or inhibiting the expression of at least one hepatitis B virus gene.
  • RNAi RNA interference
  • HAP heteroaryldihydropyrimidine
  • the hepatitis B virus is a hepatotrophic, double-stranded DNA containing virus. Although DNA is the genetic material, the replication cycle involves a reverse transcription step to copy a pregenomic RNA into DNA.
  • Hepatitis B virus is classified as one member of the Hepadnaviruses and belongs to the family of Hepadnaviridae. The primary infection of adult humans with hepatitis B virus causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood. Those patients that are not able to overcome the virus infection suffer a chronic disease progression over many years with increased risk of developing cirrhotic liver or liver cancer. Perinatal transmission from hepatitis B virus-infected mothers to newborns also leads to chronic hepatitis.
  • cccDNA serves as a template for transcription of five major viral mRNAs, which are 3.5, 3.5, 2.4, 2.1 and 0.7 kb long. All mRNAs are 5′-capped and polyadenylated at the 3′-end. There is sequence overlap at the 3′-end between all five mRNAs.
  • One 3.5 kb mRNA serves as template for core protein and polymerase production.
  • the same transcript serves as a pre-genomic replication intermediate and allows the viral polymerase to initiate the reverse transcription into DNA.
  • Core protein is needed for nucleocapsid formation.
  • the other 3.5 kb mRNA encodes pre-core, the secretable e-antigen (HBeAg) .
  • HBeAg secretable e-antigen
  • the 2.4 and 2.1 kb mRNAs carry the open reading frames ( “ORF” ) pre-S1, pre-S2 and S for expression of viral large, medium and small surface antigen.
  • the s-antigen is associated with infectious, complete particles.
  • blood of infected patients also contain non-infectious particles derived from s-antigen alone, free of genomic DNA or polymerase. The function of these particles is not fully understood.
  • the complete and lasting depletion of detectable s-antigen in blood is considered as a reliable indicator for hepatitis B virus clearance.
  • the 0.7 kb mRNA encodes the X protein. This gene product is important for efficient transcription of viral genes and also acts as a transactivator on host gene expression. The latter activity seems to be important for hepatocyte transformation during development of liver cancer.
  • Chronic HBV infection can be classified into five phases: (I) HBeAg-positive chronic infection, (II) HBeAg-positive chronic hepatitis, (III) HBeAg-negative chronic infection, (IV) HBeAg-negative chronic hepatitis and (V) HBsAg-negative phase.
  • HCC cirrhosis and hepatocellular carcinoma
  • Nucleoside analogs as inhibitors of reverse transcriptase activity are typically the first treatment option for many patients.
  • Long term administration of lamivudine, tenofovir, and/or entecavir has been shown to suppress hepatitis B virus replication, sometimes to undetectable levels, with improvement of liver function and reduction of liver inflammation typically seen as the most important benefits.
  • only few patients achieve complete and lasting remission after the end of treatment.
  • the hepatitis B virus develops drug resistance with increasing duration of treatment. This is especially difficult for patients co- infected with hepatitis B and human immunodeficiency virus (HIV) . Both viruses are susceptible to nucleoside analogue drugs and may co-develop resistance.
  • Pegylated interferon-alpha has been used to treat mild to moderate chronic hepatitis B patients.
  • current treatment of chronic hepatitis B has limited efficacy (Erha et al., Gut. 2005 Jul; 54 (7) : 1009–1013) .
  • the Asian genotype B gives very poor response rates.
  • Co-infection with hepatitis D virus (HDV) or human immunodeficiency virus has been shown to render interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy.
  • RNAi agents have been previously shown to inhibit expression of HBV gene expression.
  • RNAi hepatitis B virus-specific RNA interference
  • U.S. Patent Application Publication No. 2013/0005793, to Chin et al., and WO2018027106A1, to Li et al., which are incorporated herein by reference in its entirety disclose certain double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of hepatitis B virus gene.
  • dsRNA double-stranded ribonucleic acid
  • HBV inhibitors such as capsid assembly modulator (CAM)
  • CAM capsid assembly modulator
  • HBV capsids can bind to hepatitis B core protein and interferes with the viral capsid assembly process, thereby preventing the polymerase-bound pgRNA encapsidation. This results in the formation of HBV capsids, devoid of HBV DNA or RNA (non-functional capsids) , and ultimately in the inhibition of HBV replication. See e.g., WO2015/132276A1 and WO2020/125730A1.
  • HAP heteroaryldihydropyrimidine
  • HBV therapy that can overcome at least one of the disadvantages of existing treatment options, such as are toxicity, mutagenicity, lack of selectivity, poor efficacy, poor bioavailability, and difficulty of synthesis, while providing additional benefits such as increased potency or an increased safety window.
  • RNAi component RNAi component
  • HAP heteroaryldihydropyrimidine
  • RNAi component comprises
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; and
  • the heteroaryldihydropyrimidine (HAP) compound is a compound of Formula (Ia) or a compound of Formula (Ib) or a stereoisomer or a tautomeric form thereof or a pharmaceutically acceptable salt or a solvate thereof:
  • R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4 alkyl, halogen and CN;
  • R 2 is C 1-4 alkyl
  • R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine and C 1-6 alkyl; n is an integer of 0 or 1;
  • R 4 and R 5 are independently selected from H and -COOH;
  • R 7 is CN
  • R 8 is CF 3 ;
  • R 9 is selected from the group consisting of H, -C 1-6 alkyl, -C 1-6 alkyl-R 10 , -C 1-6 alkoxy-C 1- 6 alkyl-R 10 , - (CH 2 ) p -C (R 11 R 12 ) -R 10 and - (CH 2 ) p -Q-R 10 ;
  • p is an integer of 0, 1, 2, or 3;
  • R 11 and R 12 together with carbon atom to which they are attached form a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with one or more substituents selected from the group consisting of with H, -C 1-6 alkyl, -C 1-6 alkoxy-C 1-6 alkyl and -C 1-6 alkylcarbonyl;
  • Q is selected from the group consisting of aryl, heteroaryl, and a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6 alkyl, -C 1-6 alkoxy-C 1-6 alkyl and -C 1-6 alkylcarbonyl;
  • R 1 is hydrogen, halogen or C 1-6 alkyl
  • R 2 is hydrogen or halogen
  • R 3 is hydrogen or halogen
  • R 4 is C 1-6 alkyl
  • R 5 is hydrogen, hydroxy C 1-6 alkyl, aminocarbonyl, C 1-6 alkoxycarbonyl or carboxy;
  • R 6 is hydrogen, C 1-6 alkoxycarbonyl or carboxy-C m H 2m -;
  • X is carbonyl or sulfonyl
  • Y is -CH 2 -, -O-or -N (R 7 ) -,
  • R 7 is hydrogen, C 1-6 alkyl, haloC 1-6 alkyl, C 3-7 cycloalkyl-C m H 2m -, C 1-6 alkoxycarbonyl-C m H 2m -, -C t H 2t -COOH, -haloC 1-6 alkyl-COOH, - (C 1-6 alkoxy) C 1-6 alkyl-COOH, -C 1-6 alkyl-O-C 1-6 alkyl-COOH, -C 3 - 7 cycloalkyl-C m H 2m -COOH, -C m H 2m -C 3-7 cycloalkyl-COOH, hydroxy-C t H 2t -, carboxyspiro [3.3] heptyl or carboxyphenyl-C m H 2m -, carboxypyridinyl-C m H 2m -;
  • W is -CH 2 -, -C (C 1-6 alkyl) 2 -, -O-or carbonyl;
  • n 0 or 1
  • n 0-7;
  • t 1-7.
  • a pharmaceutical combination for a use selected from the group consisting of:
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B Virus
  • hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection
  • HBV Hepatitis B Virus
  • RNAi component RNAi component
  • HAP heteroaryldihydropyrimidine
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B Virus
  • hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection
  • HBV Hepatitis B Virus
  • RNAi component and the heteroaryldihydropyrimidine (HAP) compound and the pharmaceutical combination are as above defined.
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B Virus
  • hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection
  • HBV Hepatitis B Virus
  • RNAi component RNAi component
  • HAP heteroaryldihydropyrimidine
  • RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
  • RNAi component and a heteroaryldihydropyrimidine (HAP) compound for a use selected from the group consisting of:
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B Virus
  • hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection
  • HBV Hepatitis B Virus
  • RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
  • RNAi component for a use selected from the group consisting of:
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B Virus
  • hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection
  • HBV Hepatitis B Virus
  • RNAi component is to be administered in combination therapy with a heteroaryldihydropyrimidine (HAP) compound;
  • RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
  • heteroaryldihydropyrimidine (HAP) compound for a use selected from the group consisting of:
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B viral
  • HBV Hepatitis B Virus
  • hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection
  • HBV Hepatitis B Virus
  • HAP heteroaryldihydropyrimidine
  • RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
  • kits comprising an effective amount of a RNAi component and a heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
  • HAP heteroaryldihydropyrimidine
  • a first option refers to the applicability of the first element without the second.
  • a second option refers to the applicability of the second element without the first.
  • a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or. ”
  • any numerical value such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about. ”
  • a numerical value typically includes ⁇ 10%of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL.
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • ethyl is “optionally” substituted by halogen means the ethyl is unsubstituted (CH 2 CH 3 ) , mono-substituted (e.g., CH 2 CH 2 F) , poly-substituted (e.g., CHFCH 2 F, CH 2 CHF 2 , or the like) or completely substituted (CF 2 CF 3 ) .
  • CH 2 CH 3 unsubstituted
  • mono-substituted e.g., CH 2 CH 2 F
  • poly-substituted e.g., CHFCH 2 F, CH 2 CHF 2 , or the like
  • CF 2 CF 3 completely substituted
  • C m-n used herein means that it has m-n carbon atoms.
  • C 3- 10 cycloalkyl means said cycloalkyl has 3-10 carbon atoms.
  • C 0-6 alkylene means said alkylene has 0-6 carbon atoms, wherein the alkylene is a bond when it has 0 carbon atoms.
  • the numerical range herein refers to each of the integers therein.
  • C 1-10 means said group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms or 10 carbon atoms.
  • substituted means that one or more hydrogen atoms on a given atom are replaced by a substituent, provided that the valence of the particular atom is normal and the compound after substitution is stable.
  • two hydrogen atoms are replaced, and the ketone substitution will not occur at an aromatic group.
  • any variable e.g., R
  • R any variable
  • substituents and/or the variants thereof are allowed only if such a combination will result in a stable compound.
  • hetero means heteroatom or heteroatom radical (i.e., a radical containing heteroatom) , i.e. the atoms beyond carbon and hydrogen atoms or the radical containing such atoms, wherein the heteroatom is independently selected from the group consisting O, N, S, P, Si, Ge, Al and B.
  • the two or more heteroatoms can be the same, or part or all of the two or more heteroatoms can be different.
  • halo or “halogen, ” alone or as part of another substituent means, unless otherwise stated, a fluorine (F) , chlorine (Cl) , bromine (BR) , or iodine (I) atom, preferably F, Cl, or Br, more preferably F or Cl.
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C 1 -C 4 alkyl or C 1-4 alkyl means an alkyl having one to four carbon) and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl.
  • aryl refers to monocyclic or fused polycyclic aromatic cyclic group which has conjugated ⁇ electronic system and all the ring atoms are carbon.
  • an aryl can have 6-20 carbon atoms, 6-14 carbon atoms or 6-12 carbon atoms.
  • Non-limiting examples of aryls include, but are not limited to, phenyl, naphthyl, anthryl, or the like.
  • heteroaryl refers to monocyclic or fused polycyclic system containing at least one ring atom selected from the group consisting of N, O and S with other ring atoms being C and containing at least one aromatic ring.
  • Non-limiting examples of heteroaryls include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, or the like.
  • heteroaryl or “heteroaromatic” refers to a heterocycle having aromatic character.
  • capsid assembly modulator refers to a compound that disrupts or accelerates or inhibits or hinders or delays or reduces or modifies normal capsid assembly (e.g., during maturation) or normal capsid disassembly (e.g., during infectivity) or perturbs capsid stability, thereby inducing aberrant capsid morphology and function.
  • a capsid assembly modulator accelerates capsid assembly or disassembly, thereby inducing aberrant capsid morphology.
  • a capsid assembly modulator interacts (e.g.
  • a capsid assembly modulator causes a perturbation in structure or function of CA (e.g., ability of CA to assemble, disassemble, bind to a substrate, fold into a suitable conformation, or the like) , which attenuates viral infectivity or is lethal to the virus.
  • the half maximal effective concentration (EC 50 ) is intended in accordance with its general meaning in the field. It may more particularly refer to the concentration of a compound which induces a response halfway between the baseline and maximum, typically after a specified exposure time.
  • the EC 50 value is commonly used as a measure of a compound’s potency, with a lower value generally indicating a higher potency.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
  • pharmaceutically acceptable salt refers to a salt of any of the compounds herein which are known to be non-toxic and are commonly used in the pharmaceutical literature.
  • the pharmaceutically acceptable salt of a compound retains the biological effectiveness of the compounds described herein and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66 (1) , 1-19.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethylsulfonic acid, p-toluenesulfonic acid, stearic acid and salicylic acid.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.
  • the pharmaceutically acceptable salts according to the invention may be prepared from the parent compound containing acidic or basic group through conventional chemical procedures.
  • such salts can be prepared through the reaction of the compounds in the form of free acid or base with stoichiometric appropriate base or acid in water, organic solvent or the mixture thereof.
  • nonaqueous medium like ether, ethyl acetate, ethanol, isopropanol, acetonitrile etc. are preferable.
  • Some compounds according to the invention can exist in unsolvated or solvated forms, including hydrate form. In general, the solvated forms are equivalent to unsolvated forms and both of them are encompassed within the scope of the invention. Some compounds according to the invention can exist in polymorphic or amorphous forms.
  • Some compounds according to the invention can have asymmetric carbon atom (optical center) or double bond. Racemate, diastereomer, geometric isomer and individual isomer are encompassed within the scope of the invention.
  • the compound according to the invention can have special geometric isomer or stereoisomer form.
  • Such compounds are encompassed by the invention, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomer, (D) -isomer, (L) -isomer, and racemic mixture or other mixture thereof, such as the mixture enriched in enantiomer or diastereomer, and all the mixtures are encompassed within the scope of the invention.
  • the substituent like alkyl can have other asymmetric carbon atom. All the isomers and the mixture thereof are encompassed within the scope of the invention.
  • Optical (R) -and (S) -isomers as well as D and L isomers can be prepared through chiral synthesis or chiral agent or other conventional technology.
  • An enantiomer of the compound according to the invention can be prepared through asymmetric synthesis or derivatization with chiral auxiliary, wherein the resultant diastereomer mixture is separated and the desired pure enantiomer is obtained by cleavage of the auxiliary group.
  • basic functional group e.g. amino
  • acidic functional group e.g.
  • the diastereomeric salt can be formed with appropriate optical acid or base and then the diastereomeric resolution is performed with fractional crystallization or chromatography which is well-known in the art so as to recover the pure enantiomer. Additionally, separation of enantiomer from diastereomer is generally performed with chromatography, which uses chiral stationary phase and is optionally combined with chemical derivatization (for example, carbamate formed from amine) .
  • the compound according to the invention can contain atomic isotope in non-natural ratio at one or more atoms constituting said compound.
  • the compound may be labeled with radioisotope, such as Tritium ( 3 H) , Iodine-125 ( 125 I) or C-14 ( 14 C) . Alternation of all the radioisotopes of the compound, either radioactive or not, is encompassed within the scope of the invention.
  • patient refers to an animal, such as a mammal, bird, or fish.
  • patient or subject is a mammal.
  • mammal encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human.
  • the patient or subject is a human, for example a human that has been or will be the object of treatment, observation or experiment.
  • the compounds, compositions, and methods described herein can be useful in both human therapy and veterinary applications.
  • a “solvate” is formed by the interaction of a solvent and a compound.
  • suitable solvents include, for example, water and alcohols (e.g., ethanol) .
  • Solvates include hydrates having any ratio of compound to water, such as monohydrates, dihydrates and hemi-hydrates.
  • therapeutically effective amount refers to that amount of a compound disclosed and/or described herein that is sufficient to affect treatment, as defined herein, when administered to a subject in need of such treatment.
  • the therapeutically effective amount will vary depending upon, for example, the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound, the dosing regimen to be followed, timing of administration, the manner of administration, all of which can readily be determined by one of ordinary skill in the art.
  • the therapeutically effective amount can be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability by one of ordinary skill in the art in view of the present invention.
  • a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or (xii
  • a therapeutically effective amount can also be an amount of the compound sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; achieve sustained HBsAg clearance associated with reduction of infected hepatocytes by a subject’s immune system; induce HBV-antigen specific activated T-cell populations; and/or achieve persistent loss of HBsAg within 12 months.
  • a target index include lower HBsAg below a threshold of 500 copies of HBsAg international units (IU) and/or higher CD8 counts.
  • target indexes include, but are not limited to, serum HBV DNA levels lower than the lower limit of quantification (LLoQ) or lower than 20 IU/mL, more particularly lower than 15 IU/mL, more particularly lower than 10 IU/mL; serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; HBeAg-negative serum; serum HBsAg level of 100 IU/mL or lower, more particularly of 10 IU/mL or lower; and/or HBs seroconversion.
  • LLoQ lower limit of quantification
  • RNAi component described herein may contain particular components arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in RNAi of the application.
  • the application contemplates use of any of the applicable components in any combination that can be used the application, whether or not a particular combination is expressly described.
  • the invention generally relates to a therapeutic combination comprising one or more HBV RNAi component and a heteroaryldihydropyrimidine (HAP) compound.
  • HAP heteroaryldihydropyrimidine
  • RNAi component RNAi component
  • HAP heteroaryldihydropyrimidine
  • the pharmaceutical combination is in the form of a product, e.g a composition or a kit.
  • the pharmaceutical combination is in the form of a product, where the therapeutic agents may be administered independently at the same time or separately within time intervals that allow that the therapeutic agents show a cooperative effect.
  • the RNAi component comprises a first RNAi agent and a second RNAi agent.
  • Each RNAi agent disclosed herein includes at least a sense strand and an antisense strand.
  • the sense strand and the antisense strand can be partially, substantially, or fully complementary to each other.
  • the length of the RNAi agent sense and antisense strands described herein each can be 16 to 30 nucleotides in length.
  • the sense and antisense strands are independently 17 to 26 nucleotides in length.
  • the sense and antisense strands are independently 19 to 26 nucleotides in length.
  • the sense and antisense strands are independently 21 to 26 nucleotides in length.
  • the sense and antisense strands are independently 21 to 24 nucleotides in length.
  • the sense and antisense strands can be either the same length or different lengths.
  • the HBV RNAi agents disclosed herein have been designed to include antisense strand sequences that are at least partially complementary to a sequence in the HBV genome that is conserved across the majority of known serotypes of HBV.
  • the RNAi agents described herein upon delivery to a cell expressing HBV, inhibit the expression of one or more HBV genes in vivo or in vitro.
  • An RNAi agent includes a sense strand (also referred to as a passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence.
  • a sense strand of the HBV RNAi agents described herein includes a core stretch having at least about 85%identity to a nucleotide sequence of at least 16 consecutive nucleotides in an HBV mRNA.
  • the sense strand core nucleotide stretch having at least about 85%identity to a sequence in an HBV mRNA is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • An antisense strand of an HBV RNAi agent comprises a nucleotide sequence having at least about 85%complementary over a core stretch of at least 16 consecutive nucleotides to a sequence in an HBV mRNA and the corresponding sense strand.
  • the antisense strand core nucleotide sequence having at least about 85%complementarity to a sequence in an HBV mRNA or the corresponding sense strand is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, or a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
  • the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
  • the first and the second RNAi agents disclosed herein comprise any of the sequences in Table 1.
  • the RNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art.
  • Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates (DPCs) (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, each of which is incorporated herein by reference) .
  • DPCs Dynamic Polyconjugates
  • an HBV RNAi agent is delivered to target cells or tissues by covalently linking the RNAi agent to a targeting group.
  • the targeting group can include a cell receptor ligand, such as an asialoglycoprotein receptor (ASGPr) ligand.
  • ASGPr asialoglycoprotein receptor
  • an ASGPr ligand includes or consists of a galactose derivative cluster.
  • a galactose derivative cluster includes an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.
  • a galactose derivative cluster is an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.
  • a targeting group can be linked to the 3′ or 5′ end of a sense strand or an antisense strand of an HBV RNAi agent. In some embodiments, a targeting group is linked to the 3′ or 5′ end of the sense strand. In some embodiments, a targeting group is linked to the 5’ end of the sense strand. In some embodiments, a targeting group is linked to the RNAi agent via a linker.
  • the RNAi component comprises a combination or cocktail of a first and a second RNAi agent having different nucleotide sequences.
  • the first and the second RNAi agents are each separately and independently linked to targeting groups.
  • the first and the second RNAi agents are each linked to targeting groups comprised of N-acetyl-galactosamines.
  • each of the RNAi agents is linked to the same targeting group.
  • each of the RNAi agents is linked to different targeting groups, such as targeting groups having different chemical structures.
  • targeting groups are linked to the first and the second RNAi agents without the use of an additional linker.
  • the targeting group is designed having a linker readily present to facilitate the linkage to the first or the second RNAi agent.
  • the first and the second RNAi agents may be linked to the targeting groups using the same linkers.
  • the first and the second RNAi agents are linked to the targeting groups using different linkers.
  • the non-nucleotide group can be covalently linked to the 3′ and/or 5′ end of either the sense strand and/or the antisense strand.
  • the first or second RNAi agent contains a non-nucleotide group linked to the 3′ and/or 5′ end of the sense strand.
  • a non-nucleotide group is linked to the 5′ end of the first or second RNAi agent sense strand.
  • a non-nucleotide group may be linked directly or indirectly to the first or second RNAi agent via a linker/linking group.
  • a non-nucleotide group is linked to the first or second RNAi agent via a labile, cleavable, or reversible bond or linker.
  • Targeting groups and linking groups include the following, for which their chemical structures are provided below in Table 2: (PAZ) , (NAG13) , (NAG13) s, (NAG18) , (NAG18) s, (NAG24) , (NAG24) s, (NAG25) , (NAG25) s, (NAG26) , (NAG26) s, (NAG27) , (NAG27) s, (NAG28) , (NAG28) s, (NAG29) , (NAG29) s, (NAG30) , (NAG30) s, (NAG31) , (NAG31) s, (NAG32) , (NAG32) s, (NAG33) , (NAG33) s, (NAG34) , (NAG34) s, (NAG35) , (NAG35) s, (NAG36) , (NAG36) s, (
  • the first or the second RNAi agent contains one or more modified nucleotides.
  • a “modified nucleotide” is a nucleotide other than a ribonucleotide (2′-hydroxyl nucleotide) .
  • at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides.
  • modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (represented herein as Ab) , 2′-modified nucleotides, 3′ to 3′ linkages (inverted) nucleotides (represented herein as invdN, invN, invn, invAb) , non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs) , 2′, 3′-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as NUNA or NUNA) , locked nucleotides (represented herein as NLNA or NLNA) , 3′-O-methoxy (2′ internucleoside linked) nucleotides (represented herein as 3′-OMen) , 2'-F-Arabino nucleotides (represented herein
  • 2′-modified nucleotides include, but are not limited to, 2′-O-methyl nucleotides (represented herein as a lower case letter 'n' in a nucleotide sequence) , 2′-deoxy-2′-fluoro nucleotides (represented herein as Nf, also represented herein as 2′-fluoro nucleotide) , 2′-deoxy nucleotides (represented herein as dN) , 2′-methoxyethyl (2′-O-2-methoxylethyl) nucleotides (represented herein as NM or 2′-MOE) , 2′-amino nucleotides, and 2′-alkyl nucleotides.
  • 2′-O-methyl nucleotides represented herein as a lower case letter 'n' in a nucleotide sequence
  • 2′-deoxy-2′-fluoro nucleotides represented herein as Nf, also represented herein as
  • RNAi agent sense strands and antisense strands may be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.
  • Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine) , 5-methylcytosine (5-me-C) , 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil
  • all or at least 90%of the nucleotides of the first or the second RNAi agent are modified nucleotides.
  • an RNAi agent wherein at least 90%of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides.
  • a sense strand wherein at least 90%of the nucleotides present are modified nucleotides, is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides.
  • an antisense sense strand wherein at least 90%of the nucleotides present are modified nucleotides, is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides.
  • one or more nucleotides of an RNAi agent is a ribonucleotide.
  • one or more nucleotides of the first or the second RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones) .
  • a modified internucleoside linkage is a non-phosphate-containing covalent internucleoside linkage.
  • Modified internucleoside linkages or backbones include, but are not limited to, 5’-phosphorothioate groups (represented herein as a lower case “s” ) , chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3′-alkylene phosphonates) , chiral phosphonates, phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates) , thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of boranophosphates, or boranophosphates having inverted polarity
  • a modified internucleoside linkage or backbone lacks a phosphorus atom.
  • Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages.
  • modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH 2 components.
  • a sense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages.
  • a sense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
  • the first or the second RNAi agent sense strand contains at least two phosphorothioate internucleoside linkages. In some embodiments, the at least two phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6- 8 from the 5' end of the sense strand. In some embodiments, the first or the second RNAi agent antisense strand contains four phosphorothioate internucleoside linkages.
  • the four phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the sense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end.
  • the first or the second RNAi agent contains at least two phosphorothioate internucleoside linkages in the sense strand and three or four phosphorothioate internucleoside linkages in the antisense strand.
  • the first or the second RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2′-modified nucleoside is combined with modified internucleoside linkage. In some embodiments, the first and the second RNAi agents disclosed herein comprise any of the modified sequences in Table 3.
  • A adenosine-3′-phosphate
  • G guanosine-3′-phosphate
  • n any 2′-OMe modified nucleotide
  • Nf any 2′-fluoro modified nucleotide
  • Tfs 2′-fluoro-5′-methyluridine-3′-phosphorothioate
  • dN any 2′-deoxyribonucleotide
  • N UNA 2′, 3′-seco nucleotide mimics (unlocked nucleobase analogs)
  • N LNA locked nucleotide
  • Nf ANA 2'-F-Arabino nucleotide
  • NM 2′-methoxyethyl nucleotide
  • AMs 2′-methoxyethyladenosine-3′-phosphorothioate
  • TMs 2′-methoxyethylthymidine-3′-phosphorothioate
  • the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO: 1 and a sense strand comprising SEQ ID NO: 10; an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11; an antisense strand comprising SEQ ID NO: 3 and a sense strand comprising SEQ ID NO: 11; an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12; an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16; an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 17; an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 13; and an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO:
  • the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11; an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12; an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16; an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 13; and an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 18.
  • the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1: 2 to about 5: 1.
  • the ratio of the first RNAi agent to the second RNAi agent by weight is about 2: 1.
  • the first and the second RNAi agents are each independently conjugated to (NAG37) s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11, and the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16.
  • the first RNAi agent comprises SEQ ID NO: 5 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 6 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 7 and SEQ ID NO: 15. In some embodiments, the first RNAi agent comprises SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 3 and SEQ ID NO: 10, 11, or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 4 and SEQ ID NO: 12. In some embodiments, the second RNAi agent comprises SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the second RNAi agent comprises SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 5 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 6 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 7 and SEQ ID NO: 15 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 3 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 4 and SEQ ID NO: 12 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 11 and the second RNAi agent comprising SEQ ID NO: 16 and SEQ ID NO: 8.
  • the RNAi component comprises a first and a second RNAi agent in a ratio of about 1: 1, 2: 1, 3: 1, 4: 1 or 5: 1.
  • the two HBV RNAi agents are administered in a ratio of about 2: 1.
  • the first or the second RNAi agent comprises at least one modified nucleotide and/or at least one modified internucleoside linkage
  • the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and /or
  • the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1: 2 to about 5: 1.
  • the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
  • the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and
  • the targeting ligand comprises N-acetyl-galactosamine
  • NAG13 preferably selected from the group consisting of (NAG13) , (NAG13) s, (NAG18) , (NAG18) s, (NAG24) , (NAG24) s, (NAG25) , (NAG25) s, (NAG26) , (NAG26) s, (NAG27) , (NAG27) s, (NAG28) , (NAG28) s, (NAG29) , (NAG29) s, (NAG30) , (NAG30) s, (NAG31) , (NAG31) s, (NAG32) , (NAG32) s, (NAG33) , (NAG33) s, (NAG34) , (NAG34) , (NAG34) s, (NAG35) , (NAG35) s, (NAG36) , (NAG36) s, (NAG37) , (NAG
  • NAG25 particularly preferably selected from the group consisting of (NAG25) , (NAG25) s, (NAG31) , (NAG31) s, (NAG37) , and (NAG37) s.
  • the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and
  • the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent, preferably the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent.
  • the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
  • HAP Heteroaryldihydropyrimidine
  • the heteroaryldihydropyrimidine (HAP) compound is a compound of Formula (Ia) or a stereoisomer or a tautomeric form thereof or a pharmaceutically acceptable salt or a solvate thereof:
  • R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4 alkyl, halogen and CN;
  • R 2 is C 1-4 alkyl
  • R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine and C 1-6 alkyl;
  • n is an integer of 0 or 1;
  • R 4 and R 5 are independently selected from H and -COOH;
  • R 7 is CN
  • R 8 is CF 3 ;
  • R 9 is selected from the group consisting of H, -C 1-6 alkyl, -C 1-6 alkyl-R 10 , -C 1-6 alkoxy-C 1- 6 alkyl-R 10 , - (CH 2 ) p -C (R 11 R 12 ) -R 10 and - (CH 2 ) p -Q-R 10 ;
  • p is an integer of 0, 1, 2, or 3;
  • R 11 and R 12 together with carbon atom to which they are attached form a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with one or more substituents selected from the group consisting of with H, -C 1-6 alkyl, -C 1-6 alkoxy-C 1-6 alkyl and -C 1-6 alkylcarbonyl;
  • Q is selected from the group consisting of aryl, heteroaryl, and a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6 alkyl, -C 1-6 alkoxy-C 1-6 alkyl and -C 1-6 alkylcarbonyl;
  • R 1 of Formula (Ia) is phenyl substituted with one or more substituents selected from halogens and C 1-6 alkyl.
  • R 2 of Formula (Ia) is methyl or ethyl.
  • R 3 of Formula (Ia) is thiazolyl.
  • R 4 and R 5 of Formula (Ia) are H.
  • Z of Formula (Ia) is CH 2
  • R 9 of Formula (Ia) is -C 1- 6 alkyl-CO 2 H, - (CH 2 ) p -C (R 11 R 12 ) -R 10 or - (CH 2 ) p -Q-R 10 .
  • Q of Formula (Ia) is phenyl
  • Q of Formula (Ia) is a C 3- 6 cycloalkyl, or R 11 and R 12 of Formula (Ia) together with carbon atom to which they are attached form a C 3-6 cycloalkyl.
  • Q of Formula (Ia) is a 3-to 6-saturated membered ring containing an oxygen, or R 11 and R 12 of Formula (Ia) together with carbon atom to which they are attached form a 3-to 6-saturated membered ring containing an oxygen.
  • the compound of Formula (Ia) is selected from the group consisting of the compounds having the following formulae:
  • the heteroaryldihydropyrimidine (HAP) compound is a compound of Formula (Ib) or a stereoisomer or a tautomeric form thereof or a pharmaceutically acceptable salt or a solvate thereof:
  • R 1 is hydrogen, halogen or C 1-6 alkyl
  • R 2 is hydrogen or halogen
  • R 3 is hydrogen or halogen
  • R 4 is C 1-6 alkyl
  • R 5 is hydrogen, hydroxy C 1-6 alkyl, aminocarbonyl, C 1-6 alkoxycarbonyl or carboxy;
  • R 6 is hydrogen, C 1-6 alkoxycarbonyl or carboxy-C m H 2m -;
  • X is carbonyl or sulfonyl
  • Y is -CH 2 -, -O-or -N (R 7 ) -,
  • R 7 is hydrogen, C 1-6 alkyl, haloC 1-6 alkyl, C 3-7 cycloalkyl-C m H 2m -, C 1-6 alkoxycarbonyl-C m H 2m -, -C t H 2t -COOH, -haloC 1-6 alkyl-COOH, - (C 1-6 alkoxy) C 1-6 alkyl-COOH, -C 1-6 alkyl-O-C 1-6 alkyl-COOH, -C 3 - 7 cycloalkyl-C m H 2m -COOH, -C m H 2m -C 3-7 cycloalkyl-COOH, hydroxy-C t H 2t -, carboxyspiro [3.3] heptyl or carboxyphenyl-C m H 2m -, carboxypyridinyl-C m H 2m -;
  • W is -CH 2 -, -C (C 1-6 alkyl) 2 -, -O-or carbonyl;
  • n 0 or 1
  • n 0-7;
  • t 1-7.
  • the compound of formula (Ib) is selected from the group consisting of the compounds:
  • heteroaryldihydropyrimidine (HAP) compound is selected from the group consisting of:
  • Compounds of Formula (Ia) or (Ib) are capable of capsid assembly modulation.
  • Compounds of Formula (Ia) or (Ib) may modulate (e.g., accelerate, delay, inhibit, disrupt or reduce) normal viral capsid assembly or disassembly, bind to HBV core protein and capsid or alter metabolism of cellular polyproteins and precursors. The modulation may occur during the assembly process when the capsid protein is mature, or during viral infectivity.
  • Compounds of Formula (Ia) or (Ib) may be used in methods of modulating the activity or properties of HBV cccDNA, or the generation or release of HBV RNA particles from within an infected cell.
  • Compounds of Formula (Ia) or (Ib) may accelerate the kinetics of HBV capsid assembly, thereby preventing or competing with the encapsidation of the Pol-pgRNA complex and thus blocking the reverse transcription of the pgRNA.
  • the compounds of Formula (Ia) or (Ib) may possess one or more stereocenters, and each stereocenter may exist independently in either R or S configuration.
  • the stereochemical configuration may be assigned at indicated centers as (*R) , (*S) , (R*) or (S*) when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure.
  • Compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
  • a stereoisomeric form of a compound refers to all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which are not interchangeable.
  • Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a mixture of one or more isomers can be utilized as the disclosed compound described herein.
  • Compounds described herein may contain one or more chiral centers. These compounds can be prepared by any means, including stereoselective synthesis, enantioselective synthesis or separation of a mixture of enantiomers or diastereomers. Resolution of compounds and isomers thereof can be achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • described herein are methods for therapeutic and/or prophylactic treatment of diseases/disorders which are associated with HBV infection or inhibition of expression of one or more HBV genes comprising administering a pharmaceutical composition comprising one or more HBV RNAi agents that can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted device) , and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.
  • methods described herein comprise one or more HBV RNAi agents, wherein the one or more HBV agents are prepared as pharmaceutical compositions or formulations.
  • pharmaceutical compositions include at least one HBV RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism.
  • the pharmaceutical compositions can be used to treat a subject having a disease or disorder that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene.
  • the pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene.
  • the method includes administering an HBV RNAi agent linked to a targeting ligand as described herein, to a subject to be treated.
  • one or more pharmaceutically acceptable excipients are added to the pharmaceutical compositions including an HBV RNAi agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a human.
  • compositions that include an HBV RNAi agent and methods disclosed herein may decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, or subject, including: administering to the subject a therapeutically effective amount of a herein described HBV RNAi agent, thereby inhibiting the expression of a target mRNA in the subject.
  • the described pharmaceutical compositions including an HBV RNAi agent are used for treating or managing clinical presentations associated with HBV infection.
  • a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment, prevention or management.
  • administration of any of the disclosed HBV RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
  • the described pharmaceutical compositions including an HBV RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of HBV mRNA.
  • the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions including an HBV RNAi agent thereby treating the symptom.
  • the subject is administered a prophylactically effective amount of one or more HBV RNAi agents, thereby preventing the at least one symptom.
  • the route of administration is the path by which an HBV RNAi agent is brought into contact with the body.
  • methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein.
  • the HBV RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route.
  • herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some embodiments, there herein described pharmaceutical compositions via subcutaneous injection.
  • compositions including an HBV RNAi agent described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery technologies known in the art.
  • any suitable method recognized in the art for delivering a nucleic acid molecule in vitro or in vivo can be adapted for use with a herein described compositions.
  • delivery can be by local administration, (e.g., direct injection, implantation, or topical administering) , systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal) , intramuscular, transdermal, airway (aerosol) , nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • the compositions are administered by subcutaneous or intravenous infusion or injection.
  • the herein described pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients.
  • the pharmaceutical compositions described herein can be formulated for administration to a subject.
  • a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., HBV RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage.
  • Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use.
  • a pharmaceutically acceptable excipient may or may not be an inert substance.
  • Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol) , and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension.
  • Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
  • the active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • the HBV RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • a pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions.
  • additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc. ) .
  • anti-pruritics e.g., antihistamine, diphenhydramine, etc.
  • anti-inflammatory agents e.g., antihistamine, diphenhydramine, etc.
  • cells, tissues or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions. ”
  • “pharmacologically effective amount, ” “therapeutically effective amount, ” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic or preventive result.
  • an effective amount of an active compound will be in the range of from about 0.1 to about 100 mg/kg of body weight/day, e.g., from about 1.0 to about 50 mg/kg of body weight/day. In some embodiments, an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg/kg of body weight per dose. In some embodiments, an effective amount of an active compound will be in the range of 25-400 mg per 1-18 weeks or 1-6 months. In some embodiments, an effective amount of an active compound will be in the range of 50-125 mg per 4 weeks or per one month. In some embodiments, an effective amount of an active ingredient will be in the range of from about 0.5 to about 3 mg/kg of body weight per dose.
  • an effective amount of an active ingredient will be in the range of from about 25-400 mg per dose. In some embodiments, an effective amount of an active ingredient will be in the range of from about 50-125 mg per dose.
  • the amount administered will also likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.
  • an effective amount of the RNAi component is in the range of about 25-600 mg per dose. In some embodiments, an effective amount of the RNAi component is in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose.
  • an effective amount of the RNAi component is about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, an effective amount of the RNAi component is about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
  • the one or more (e.g., at least two) HBV RNAi agents described herein can be formulated into one single composition or separate individual compositions.
  • the HBV RNAi agents in separate individual compositions can be formulated with the same or different excipients and carriers.
  • the HBV RNAi agents in separate individual compositions agents can be administered through same or different administration routes.
  • the HBV RNAi agents are administered subcutaneously.
  • compositions described herein including an HBV RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and/or a vaccine.
  • HBV RNAi agents when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.
  • the pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
  • the composition comprises an effective amount of an RNAi component in the range of about 25-600 mg and an effective amount of a heteroaryldihydropyrimidine (HAP) compound in the range of about 75-600 mg per dose. In some embodiments, the composition comprises an effective amount of an RNAi component in the range of about 25-300 mg and an effective amount of a heteroaryldihydropyrimidine (HAP) compound in the range of about 75-300 mg per dose.
  • HAP heteroaryldihydropyrimidine
  • the composition comprises an effective amount of an RNAi of about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg and an effective amount of a heteroaryldihydropyrimidine (HAP) compound of about 100 mg, about 150 mg or about 250 mg per dose.
  • HAP heteroaryldihydropyrimidine
  • HAP Heteroaryldihydropyrimidine
  • an effective amount of the heteroaryldihydropyrimidine (HAP) compound is in the range of about 75-600 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is in the range of about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose.
  • an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 150 mg or about 250 mg per dose.
  • heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, or any subgroup thereof may be formulated into various pharmaceutical forms for administration purposes.
  • compositions there may be cited all compositions usually employed for systemically administering drugs.
  • an effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • a pharmaceutically acceptable carrier which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration orally, rectally, percutaneously, or by parenteral injection.
  • any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets.
  • solid pharmaceutical carriers are employed.
  • the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included.
  • Injectable solutions may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution.
  • Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations intended to be converted, shortly before use, to liquid form preparations.
  • the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin.
  • the compounds of the present invention may also be administered via oral inhalation or insufflation in the form of a solution, a suspension or a dry powder using any art-known delivery system.
  • Unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • unit dosage forms are tablets (including scored or coated tablets) , capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions and the like, and segregated multiples thereof.
  • the first and the second RNAi agents are each independently conjugated to (NAG25) or (NAG37) s
  • the first RNAi agent comprises an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11
  • the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16
  • the heteroaryldihydropyrimidine (HAP) compound is
  • the pharmaceutical combination further comprises another agent for treating infection caused by hepatitis B virus HBV.
  • the other agent can be a nucleoside analog.
  • the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
  • the RNAi component and the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof included in the combinations described herein are provided in separate containers. In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof included in the combinations described herein are provided in the same container. In some embodiments, the RNAi component includes a first RNAi agent in a first container and a second RNAi agent in a second container.
  • the first RNAi agent is in a first container
  • the second RNAi agent is in a second container
  • the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof is in a third container.
  • the RNAi component includes the first RNAi agent and the second RNAi agent in the same container.
  • the first RNAi agent and the second RNAi agent are in a first container
  • the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof is in a second container.
  • Exemplary containers include vials, bags, tubes, or other suitable containers. In some embodiments, the contents of the container are sterile.
  • Also provided herein is a method for treating a Hepatitis B viral (HBV) infection in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided herein is a method for enhancing an immune response in a subject with an HBV infection, wherein the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided herein is a method for decreasing viral replication in a subject with a HBV infection, wherein the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound.
  • HAP Hepatitis B viral
  • HAP heteroaryldihydropyrimidine
  • a method for modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject in need thereof comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound.
  • HAP heteroaryldihydropyrimidine
  • a method for inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound.
  • the HBV infection is a chronic HBV infection.
  • administering the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound decreases the immune tolerogenicity of the subject to HBV, more particularly the immune tolerogenicity of the liver of the subject to HBV. Decreasing the immune tolerogenicity of the subject to HBV or the immune tolerogenicity of the liver of the subject to HBV infection is characterized by the reactivation of the immune system in the subject with the chronic HBV infection.
  • Reactivation of the immune system can occur after administration of the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound, which serves to decrease or reduce the level of HBsAg in the subject, which allows for the reactivation of the immune system.
  • HAP heteroaryldihydropyrimidine
  • the at least one of, at least two of, at least three of, at least four of, or five of features (i) , (ii) , (iii) , (iv) , and (v) are still met six (6) months after the end of treatment.
  • LLoQ
  • the serum HBsAg level in the subject is reduced at a faster rate, is reduced at a greater level, and/or is reduced for a greater time period than in a subject in which only an effective amount of the RNAi component is administered or an effective amount of the heteroaryldihydropyrimidine (HAP) compound is administered.
  • HAP heteroaryldihydropyrimidine
  • enhancing an immune response in the subject results in an increase in an innate immune system and/or an adaptive immune system.
  • the increase in the innate immune system results in an increase in the expression of one or several from among an interferon stimulated gene (ISG) , an interferon gamma-induced protein 10 (IP10) , an interferon alpha (IFN ⁇ ) , an interleukin 12 (IL12) , or an interleukin-6 (IL-6) .
  • ISG interferon stimulated gene
  • IP10 interferon gamma-induced protein 10
  • IFN ⁇ interferon alpha
  • IL12 interleukin 12
  • IL-6 interleukin-6
  • the increase in the adaptive immune system results in an increase in the level of HBV-specific T cell responses and/or an increased activation of the HBV-specific T cells.
  • Activation of HBV-specific T cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., an interferon gamma ELISPOT) .
  • the increase in expression of the at least one of ISG, IP10, IFN ⁇ , IL12, or IL-6; the increase in the level of HBV-specific T cell responses; and/or the increased activation of HBV-specific T cells can be compared to a control.
  • the control can be an expression level of ISG, IP10, IFN ⁇ , IL12, or IL-6; the level of HBV-specific T cell responses; the level of activation of HBV-specific T cells from a sample from the subject prior to administration of the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound.
  • HAP heteroaryldihydropyrimidine
  • control can be the expression level of ISG, IP10, IFN ⁇ , IL12, or IL-6; the level of HBV-specific T cell responses; and/or the level of activation of HBV-specific T cells from a subject administered the effective amount of the RNAi component, but not the effective amount of the heteroaryldihydropyrimidine (HAP) compound; or from a subject administered the effective amount of the heteroaryldihydropyrimidine (HAP) compound, but not the effective amount of the RNAi component.
  • HAP heteroaryldihydropyrimidine
  • a person skilled in the art will understand the proper control for determining an increase in the expression level of ISG, IP10, IFN ⁇ , IL12, or IL-6; an increase in the level of HBV-specific T cell responses; and/or an increased activation level of HBV-specific T cells in the subject.
  • enhancing an immune response in the subject results in an increase in the immunocompetence of the subject.
  • An increase in the immunocompetence of the subject can, for example, result in an increase in the level of HBV-specific T cells, B cells, or NK cells in the liver and/or an increased activation of HBV-specific T cells, B cells, or NK cells in the liver.
  • An increase in the immunocompetence of the subject can, for example, result in an increase in NK cells, T cells, or B cells in a peripheral immune cell compartment and/or an increased activation of NK cells, T cells, or B cells in the peripheral immune cell compartment.
  • An increase in the immunocompetence of the subject can, for example, result in a decrease of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or in a peripheral immune cell compartment; and/or a decreased immunosuppressive activity of MDSCs in the lever and/or in a peripheral immune cell compartment.
  • MDSCs Myeloid Derived Suppressive Cells
  • the increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the increase in the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or the decrease in the level of or decreased immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment can, for example, be determined by comparing to a control.
  • MDSCs Myeloid Derived Suppressive Cells
  • the control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment from a sample from the subject prior to administration of the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound.
  • MDSCs Myeloid Derived Suppressive Cells
  • control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment from a subject administered the effective amount of the RNAi component, but not the effective amount of the heteroaryldihydropyrimidine (HAP) compound; or from a subject administered the effective amount of the heteroaryldihydropyrimidine (HAP) compound, but not the effective amount of the RNAi component.
  • MDSCs Myeloid Derived Suppressive Cells
  • a person skilled in the art will understand the proper control for determining an increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or a decrease in the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment in the subject.
  • MDSCs Myeloid Derived Suppressive Cells
  • HBV-specific NK cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., interferon gamma ELISPOT) .
  • Activation of HBV-specific B cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by anti-HBs antibody production (e.g., anti-HBs ELISPOT) .
  • the immunosuppressive activity of MDSCs can be measured by any means that the person of average skill in the art may find appropriate, for example, by arginase expression.
  • decreasing the viral replication in the subject results in a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 IU/mL, more particularly is lower than 15 IU/mL, more particularly is lower than 10 IU/mL.
  • LLoQ lower limit of quantification
  • the targeted hepatocytes comprise cccDNA. In some embodiments, the targeted hepatocytes comprise integrated HBV DNA.
  • the RNAi component comprises: (i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; and (ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
  • the first RNAi agent comprises SEQ ID NO: 5 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 6 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 7 and SEQ ID NO: 15. In some embodiments, the first RNAi agent comprises SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 3 and SEQ ID NO: 10, 11, or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 4 and SEQ ID NO: 12. In some embodiments, the second RNAi agent comprises SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the second RNAi agent comprises SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 5 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 6 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 7 and SEQ ID NO: 15 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 3 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 4 and SEQ ID NO: 12 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
  • the two HBV RNAi agents are administered in a ratio of about 1: 1, 2: 1, 3: 1, 4: 1 or 5: 1. In some embodiments, the two HBV RNAi agents are administered in a ratio of about 2: 1.
  • the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2.
  • the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2.
  • the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-100 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2.
  • the two HBV RNAi agents are administered in a combined amount of about 25-400 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2: 1.
  • the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 125-225 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2: 1.
  • the two HBV RNAi agents are administered in a combined amount of about 100 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 40 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50 mg per dose administration and in the ratio of about 2: 1.
  • the two HBV RNAi agents are administered in a combined amount of about 75 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200 mg per dose administration and in the ratio of about 2: 1.
  • the first RNAi agent is administered in an amount of about 3-650 mg per dose administration, and the second RNAi agent is administered in an amount of about 2-325 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-150 mg per dose administration, and the second RNAi agent is administered in an amount of about 5-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 35-265 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 50-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-75 mg per dose administration.
  • the second RNAi agent is administered in an amount of about 20-125 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 25-50 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 5-40 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 17 mg per dose administration, and the second RNAi agent is administered in an amount of about 8 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 23 mg per dose administration, and the second RNAi agent is administered in an amount of about 12 mg per dose administration.
  • the first RNAi agent is administered in an amount of about 27 mg per dose administration, and the second RNAi agent is administered in an amount of about 13 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 33 mg per dose administration, and the second RNAi agent is administered in an amount of about 17 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 67 mg per dose administration, and the second RNAi agent is administered in an amount of about 33 mg per dose administration.
  • two RNAi agents are administered at a combined dose of 25-400 mg per dose administration. In some embodiments, two RNAi agents are administered at a combined dose of 25-400 mg, and the first RNAi agent is administered with the second RNAi agent at a ratio of 1: 1. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 12 mg for a combined dose of about 25 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 17 mg for a combined dose of about 35 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 20 mg for a combined dose of about 40 mg.
  • the dose of each of the first and second RNAi agents is in an amount of about 25 mg for a combined dose of about 50 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 50 mg for a combined dose of about 100 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 100 mg for a combined dose of about 200 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 150 mg for a combined dose of about 300 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 200 mg for a combined dose of about 400 mg.
  • two RNAi agents are administered at a combined dose of 25-400 mg per dose, and the first RNAi agent is administered with the second RNAi agent at a ratio of 2: 1.
  • the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg.
  • the dose of the first RNAi agent is in an amount of about 24 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 35 mg.
  • the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 65 mg, and the dose of the second RNAi agent is in an amount of about 35 mg for a combined dose of about 100 mg.
  • the dose of the first RNAi agent is in an amount of about 133 mg, and the dose of the second RNAi agent is in an amount of about 67 mg for a combined dose of about 200 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 200 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 300 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 270 mg, and the dose of the second RNAi agent is in an amount of about 135 mg for a combined dose of about 400 mg.
  • two RNAi agents are administered at a combined dose of 25-400 mg per dose, the first RNAi agent is administered with the second RNAi agent at a ratio of 3: 1.
  • the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg.
  • the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 9 mg for a combined dose of about 35 mg.
  • the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 75 mg, and the dose of the second RNAi agent is in an amount of about 25 mg for a combined dose of about 100 mg.
  • the dose of the first RNAi agent is in an amount of about 150 mg, and the dose of the second RNAi agent is in an amount of about 50 mg for a combined dose of about 200 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 225 mg, and the dose of the second RNAi agent is in an amount of about 75 mg for a combined dose of about 300 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 300 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 400 mg.
  • the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275-300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 25-75 mg, 50-100 mg, 100-150 mg, 150-200 mg, 200-250 mg, 250-300 mg, 300-350 mg, 350-400 mg, 25-100 mg, 50-150 mg, 100-200 mg, 150-250 mg, 200-300 mg, 300-400 mg, 25-200 mg, or 200-400 mg per dose administration.
  • the first RNAi agent to the second RNAi agent are administered in a combined amount of about 25 mg, about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, or about 400 mg per dose administration.
  • the first RNAi agent and the second RNAi agent are administered in a combined amount of about 50 mg, about 75 mg, about 100 mg, or about 125 mg per dose administration.
  • the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25 mg, about 35 mg, about 40 mg, or about 200 mg per dose administration.
  • the two HBV RNAi agents are administered in a combined amount of about 1-10 mg/kg per dose administration. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-5 mg/kg per dose administration. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-1.5 mg/kg, about 1.5-2.0 mg/kg, about 2.0-2.5 mg/kg, about 2.5-3.0 mg/kg, about 3.0-3.5 mg/kg, about 3.5-4.0 mg/kg, about 4.0-4.5 mg/kg, about 4.5-5.0 mg/kg, about 5.0-5.5 mg/kg, about 5.5-6.0 mg/kg, about 6.0-6.5 mg/kg, about 6.5-7.0 mg/kg, about 7.0-7.5 mg/kg, about 7.5-8.0 mg/kg, about 8.0-8.5 mg/kg, about 8.5-9.0 mg/kg, about 9.0-9.5 mg/kg, about 9.5-10 mg/kg, about 1-2.5 mg/kg, about 2.5-5.0 mg/kg, about
  • the first RNAi agent is administered in an amount of about 0.6-7 mg/kg per dose administration, and the second RNAi agent is administered in an amount of about 0.3-5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.5-2.5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.3-1.5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 0.6-5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 1-2.5 mg/kg per dose administration.
  • the two RNAi agents are administered in about 1-18 week intervals. In some embodiments, the two RNAi agents are administered in about 1-week intervals, about 2-week intervals, about 3-week intervals, about 4-week intervals, about 5-week intervals, about 6-week intervals, about 7-week intervals, about 8-week intervals, about 9-week intervals, about 10-week intervals, about 11-week intervals, about 12-week intervals, about 13-week intervals, about 14-week intervals, about 15-week intervals, about 16-week intervals, about 17-week intervals, or about 18-week intervals. In some embodiments, the two RNAi agents are administered in about 1-6 month intervals.
  • the two RNAi agents are administered in about 1-month intervals, about 2-month intervals, about 3-month intervals, about 4-month intervals, about 5-month intervals, or about 6-month intervals. In some embodiments, the two RNAi agents are administered in about 4-week intervals or 1-month intervals. In some embodiments, the two RNAi agents are administered once per month.
  • the first RNAi agent and the second RNAi agent are administered for a duration of about 1-12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of about 1-18 weeks.
  • the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, at least about 80 weeks, at least about 90 weeks, or at least 96 weeks.
  • the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg, and the first RNAi agent is administered with the second RNAi agent at a ratio of 1: 1. In some embodiments, the dose of the first RNAi agent is administered with the second RNAi agent is in an amount of about 12 mg for a combined dose of about 25 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 17 mg for a combined dose of about 35 mg.
  • the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 20 mg for a combined dose of about 40 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 25 mg for a combined dose of about 50 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 50 mg for a combined dose of about 100 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 100 mg for a combined dose of about 200 mg.
  • the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 150 mg for a combined dose of about 300 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 200 mg for a combined dose of about 400 mg.
  • the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, and the second RNAi agent is administered with the first RNAi agent at a ratio of 1: 2.
  • the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg.
  • the dose of the second RNAi agent is in an amount of about 12 mg, and the dose of the first RNAi agent is in an amount of about 24 mg for a combined dose of about 35 mg.
  • the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 35 mg, and the dose of the first RNAi agent is in an amount of about 65 mg for a combined dose of about 100 mg.
  • the dose of v is in an amount of about 67 mg, and the dose of the first RNAi agent is in an amount of about 133 mg for a combined dose of about 200 mg.
  • the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 200 mg for a combined dose of about 300 mg.
  • the dose of the second RNAi agent is in an amount of about 135 mg, and the dose of the first RNAi agent is in an amount of about 270 mg for a combined dose of about 400 mg.
  • the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, the second RNAi agent is administered with the first RNAi agent at a ratio of 1: 3.
  • the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg.
  • the dose of the second RNAi agent is in an amount of about 9 mg, and the dose of the first RNAi agent is in an amount of about 27 mg for a combined dose of about 35 mg.
  • the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 25 mg, and the dose of the first RNAi agent is in an amount of about 75 mg for a combined dose of about 100 mg.
  • the dose of the second RNAi agent is in an amount of about 50 mg, and the dose of the first RNAi agent is in an amount of about 150 mg for a combined dose of about 200 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 75 mg, and the dose of the first RNAi agent is in an amount of about 225 mg for a combined dose of about 300 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 300 mg for a combined dose of about 400 mg.
  • about 1 mg/kg (mpk) of the first RNAi agent and about 1 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of the first RNAi agent and about 1.5 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof.
  • about 3.2 mg/kg of the first RNAi agent and about 0.8 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.7 mg/kg of the first RNAi agent and about 1.3 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 4.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.3 mg/kg of the first RNAi agent and about 1.7 mg/kg of the second RNAi agent are administered to a subject in need thereof.
  • between about 0.05 and about 5 mg/kg of the first RNAi agent and between about 0.05 and about 5 mg/kg of the second RNAi agent are administered to a subject in need thereof.
  • about the first RNAi agent and about the second RNAi agent are administered separately (e.g., in separate injections) .
  • the respective dose of the first RNAi agent and the respective dose of the second RNAi agent are administered together (e.g., in the same injection) .
  • the respective dose of the first RNAi agent and the respective dose of the second RNAi agent are prepared in a single pharmaceutical composition.
  • the RNAi component is administered to the subject once monthly in a dose of about 40-350 mg, such as about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg.
  • the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt is administered in the amount of about 100-500 mg per dose.
  • an effective amount of the heteroaryldihydropyrimidine (HAP) compound is in the range of about 100-500 mg, about 100-450 mg, about 100-400 mg, about 100-350 mg, about 100-300 mg, about 100-250 mg, about 100-200 mg, about 100-150 mg, about 150-500 mg, about 150-450 mg, about 150-400 mg, about 150-350 mg, about 150-300 mg, about 150-250 mg, about 150-200 mg, about 200-500 mg, about 200-450 mg, about 200-400 mg, about 200-350 mg, about 200-300 mg, about 200-250 mg, about 250-500 mg, about 250-450 mg, about 250-400 mg, about 250-350 mg, about 250-300 mg, about 300-500 mg, about 300-450 mg, about 300-400 mg, about 300-350 mg, about 350-
  • an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg per dose.
  • HAP heteroaryldihydropyrimidine
  • an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg per dose.
  • the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, at least about 80 weeks, at least about 90 weeks, or at least about 96 weeks.
  • the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is administered for a duration of about 24 weeks or 48 weeks. In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is administered daily, every other day, once a week, twice a week, once every 2 weeks, once every 3 weeks, or once every month.
  • the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt is formulated in a solid form, such as a tablet or capsule.
  • the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized.
  • the RNAi component is formulated in a solid form, such as a tablet or capsule.
  • the RNAi component is formulated for subcutaneous injection.
  • the RNAi component is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized.
  • the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are administered simultaneously or intermittently. In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are administered and formulated separately and administered with different dosing frequencies. In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are formulated as one or separate compositions. In some embodiments, the RNAi component is formulated as a solution and administered once per month or once every four weeks via subcutaneous injection. In some embodiments, the heteroaryldihydropyrimidine (HAP) compound is formulated as an oral tablet or oral liquid solution and administered daily.
  • the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, such as any of those described herein is administered to the subject in a daily dose of about 100-500 mg, about 100-400 mg, about 100-300 mg, about 100-200 mg, about 200-500 mg, about 200-400 mg, about 200-300 mg, more particularly of about 200-300 mg, more particularly of about 250 mg.
  • the first and the second RNAi agents are each independently conjugated to (NAG25) or (NAG37) s
  • the first RNAi agent comprises an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11
  • the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16
  • the heteroaryldihydropyrimidine (HAP) compound is
  • the method further comprises administering a nucleoside analog.
  • the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
  • the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.01-5 mg, about 0.01-0.05 mg, about 0.05-0.1 mg, about 0.1-0.5 mg, about 0.5-1 mg, about 1-2 mg, about 2-3 mg, about 3-4 mg or about 4-5 mg.
  • the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.5 mg. In some embodiments, the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 100-500 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, 300-400 mg, about 400-500 mg. In some embodiments, the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 300 mg.
  • the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 5-100 mg, about 5-25 mg, about 25-50 mg, about 50-75 or about 75-100 mg. In some embodiments, the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 25 mg. In some embodiments, the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of about 50-600 mg, about 50-300 mg, about 100-300 mg, about 100-500 mg, about 150-400 mg, about 200-350, or about 250-300 mg.
  • the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of 100 mg, 150 mg, or 300 mg. In some embodiments, the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of about 300-800 mg, about 400-700 mg, about 300-600 mg, about 300-400 mg, about 400-500 mg, or about 500-600 mg. In some embodiments, the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of 600 mg. In some embodiments, the patients have been exposed to the nucleoside analog prior to the combination therapy. In some embodiments, the patients have been administered the nucleoside analog for at least 1 month, at least 3 months, at least 6 months, or at least 1 year prior to receiving the combination therapy.
  • the patients are screened for HBeAg status prior to administration of the combination therapy.
  • the patients are HBeAg positive.
  • the patients are HBeAg negative.
  • the patients are screened for immune tolerance prior to administration of the combination therapy.
  • the HBsAg level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1.
  • the HBeAg level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1.
  • the HBcrAg level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1.
  • the HBV DNA level in the patient is reduced by at least about log 10 0.5, about log 10 1, about log 10 1.5, about log 10 2, about log 10 3, about log 10 4, about log 10 5 or about log 10 7.5 from base line on Day 1.
  • the HBV RNA level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1.
  • kits comprising an effective amount of the RNAi component and an effective amount of the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof.
  • HAP heteroaryldihydropyrimidine
  • HBV RNAi agents and the heteroaryldihydropyrimidine (HAP) compounds may be presented in the form of a kit such as when added to pharmaceutically acceptable excipients or adjuvants, and packaged into kits, containers, packs, or dispensers.
  • the pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
  • the kit can comprise any combinations or compositions described herein.
  • the kit further comprises a package insert including, without limitation, appropriate instructions for preparation and administration of the formulation, side effects of the formulation, and any other relevant information.
  • the instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, optical disc or directions to internet-based instructions.
  • kits for treating an individual who suffers from or is susceptible to the conditions described herein comprising a first container comprising a dosage amount of a composition or formulation as disclosed herein, and a package insert for use.
  • the container can be any of those known in the art and appropriate for storage and delivery of intravenous formulation.
  • the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for preparation of the formulation to be administered to the individual.
  • the kit comprises one or more doses of the heteroaryldihydropyrimidine (HAP) compound in the range of about 100-500 mg per dose. In some embodiments, the kit comprises one or more doses of the heteroaryldihydropyrimidine (HAP) compound in the range of about 100-500 mg, about 100-450 mg, about 100-400 mg, about 100-350 mg, about 100-300 mg, about 100-250 mg, about 100-200 mg, about 100-150 mg, about 150-500 mg, about 150-450 mg, about 150-400 mg, about 150-350 mg, about 150-300 mg, about 150-250 mg, about 150-200 mg, about 200-500 mg, about 200-450 mg, about 200-400 mg, about 200-350 mg, about 200-300 mg, about 200-250 mg, about 250-500 mg, about 250-450 mg, about 250-400 mg, about 250-350 mg, about 250-300 mg, about 300-500 mg, about 300-450 mg, about 300-400 mg, about 300-350 mg, about 300-
  • an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg per dose.
  • HAP heteroaryldihydropyrimidine
  • an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg per dose.
  • the kit comprises one or more doses of the RNAi component in the range of about 25-600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose.
  • the kit comprises one or more doses of the RNAi component of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose.
  • the kit comprises one or more doses of the RNAi component of about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
  • the kit contains an RNAi component useful for the invention, such as those described herein, for once monthly (or every four weeks) administration to a subject in a dose of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg; more particularly 200 mg; and a heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, such as those described herein, for administration to a subject in a dose of about 0.6-3.0 mg, about 0.6-2.5 mg, about 0.6-2.0 mg, more particularly of about 100-500 mg, about 100-400 mg, about 100-300 mg, about 100-200 mg, about 200- 500 mg, about 200-400 mg, about 200-300 mg, or about 250 mg, wherein said dose is administered daily.
  • HAP heteroaryldihydropyrimidine
  • the kit further comprises instructions for using the RNAi component and the heteroaryldihydropyrimidine (HAP) compound contained therein for administration to treat a subject with an HBV infection, in particular, a subject having a chronic HBV infection.
  • HAP heteroaryldihydropyrimidine
  • the kit further comprises instructions for using the RNAi component and the heteroaryldihydropyrimidine (HAP) compound contained therein for administration to a subject with an HBV infection to enhance the immune response, to decrease viral replication, to decrease expression of one or more HBV polypeptide (s) , to modulate Hepatitis B viral (HBV) capsid assembly or disassembly, and/or to increase the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA.
  • HAP heteroaryldihydropyrimidine
  • kits may also be provided that contain sufficient dosages of the compositions described herein (including pharmaceutical compositions thereof) to provide effective treatment for an individual for an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles or more.
  • one cycle of treatment is about 1-24 months, about 1-3 months, about 3-6 months, about 6-9 months, about 9-12 months, about 12-18 months, about 18-21 months or about 21-24 months.
  • one cycle of treatment is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months or about 24 months.
  • kits can also include multiple doses and may be packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • the kits may include a dosage amount of at least one composition as disclosed herein.
  • the application also relates to an effective amount of an RNAi component and a heteroaryldihydropyrimidine (HAP) compound, optionally a nucleoside analog, each of which as that described herein, in the manufacture of a medicament for treating a Hepatitis B viral (HBV) infection in a subject; enhancing an immune response in a subject with an HBV infection; decreasing viral replication in a subject with HBV; decreasing the expression of one or more HBV polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof; modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject with a HBV infection; and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA in a subject with a HBV infection.
  • HAP heteroaryldihydropyrimidine
  • the RNAi component is for once monthly or once every four weeks administration to a subject in a dose of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg;
  • the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is for administration to a subject in a dose of about 100-500 mg, particularly of 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg, more particularly of 250 mg, wherein said dose is administered daily.
  • the medicament is for administering to a subject infected by HBV, in particular, a subject having a chronic HBV infection.
  • Fig. 1 indicates the treatment schedule and the dosage of each animal group of the examples.
  • the combination comprised Compound A having the following structure and an RNAi component comprising a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 11 and a second RNAi agent comprising SEQ ID NO: 16 and SEQ ID NO: 8 in a ratio of 2: 1.
  • Compound A can be prepared according to WO2020125730A1 and the RNAi component can be prepared according to WO2018027106A1.
  • C57BL/6 mice male, approximately 4-5 weeks old, commercially available from Shanghai Lingchang Bio Tech co., Ltd, Shanghai, China) received a single tail vein injection with 200 ⁇ L PBS containing 1x10 11 vg rAAV-HBV virus (this recombinant virus carries 1.3 copies of the HBV genome (genotype D; serotype ayw) and was packaged in AAV serotype 8 (AAV8) capsids, commercially available from Beijing FivePlus Molecular Medicine Institute, China) .
  • Plasma samples were obtained from mice by submandibular vein bleeding at various time point after injection, to monitor HBsAg, HBeAg and HBV genomic DNA. This model can mimic chronic HBV infection. Stable HBV replication and continuous expression of HBV antigens are usually established after 4 weeks of the injection with rAAV-HBV, at which time the model is ready for compound dosing.
  • RNAi component was formulated in PBS to form the RNAi component formulation.
  • Vehicle A (Group 1, vehicle of the Compound A formulation) and the Compound A formulation (Group 2 and Group 4) were dosed once daily by oral gavage with a volume of 10 mL/kg during Dosing Day 0 ⁇ 83.
  • the dosage of Compound A is 12 mg/kg.
  • Vehicle B (Group 1, vehicle of the RNAi component formulation) and the RNAi component formulation (Group 3 and Group 4) were dosed subcutaneously with a volume of 3 mL/kg on Dosing Day 0, 21, 42 and 63.
  • the dosage of the RNAi component is 3 mg/kg.
  • HBeAg levels were determined with Autobio ELISA kit (05150312) as described by the manufacturer.
  • HBcrAg levels were determined with Lumipulse CLEIA (Fujirebio) .
  • HBV DNA was isolated from plasma using QIAamp 96 Virus QIAcube HT Kit (Qiagen, 57731) following the manufacturer’s instructions. The number of copies of HBV DNA was determined by LightCycler 480 Real-Time PCR as described in Antiviral Res, 2017 Jun 21; 144: 205 (doi: 10.1016/j. antiviral. 2017.06.016) .
  • HBeAg reduction was observed in mouse Group 2, 3 and 4 (Table 5) among which Group 4 induced the fastest HBeAg reduction.
  • HBcrAg reduction was observed in mouse Group 2, 3 and 4 (Table 6) among which Group 4 induced the fastest HBcrAg reduction.
  • mice Average HBsAg levels (Mean, Log ng/ml) and standard deviation (SD) of mice in each group following the administration
  • HBV DNA was significantly reduced in mouse Group 2, 3 and 4 (Table 8) . HBV DNA was efficiently reduced under LLOQ in Group 2 and Group 4.

Abstract

Provided are RNA interference (RNAi) agents for inhibiting the expression of Hepatitis B Virus (HBV) used in combination with a heteroaryldihydropyrimidine (HAP) compound (i.e., a capsid assembly modulator (CAM) ), and methods of administering the same. The HBV RNAi agents and the heteroaryldihydropyrimidine (HAP) compound are administered to effectively inhibit HBV gene expression and to treat HBV infections.

Description

COMBINATION THERAPY FOR TREATING HEPATITIS B VIRUS INFECTION
FIELD OF INVENTION
The present invention relates generally to the combination of an RNA interference (RNAi) component and a heteroaryldihydropyrimidine (HAP) compound for treating hepatitis B virus infection or inhibiting the expression of at least one hepatitis B virus gene.
BACKGROUND
The hepatitis B virus (HBV) is a hepatotrophic, double-stranded DNA containing virus. Although DNA is the genetic material, the replication cycle involves a reverse transcription step to copy a pregenomic RNA into DNA. Hepatitis B virus is classified as one member of the Hepadnaviruses and belongs to the family of Hepadnaviridae. The primary infection of adult humans with hepatitis B virus causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood. Those patients that are not able to overcome the virus infection suffer a chronic disease progression over many years with increased risk of developing cirrhotic liver or liver cancer. Perinatal transmission from hepatitis B virus-infected mothers to newborns also leads to chronic hepatitis.
Upon uptake by hepatocytes, the nucleocapsid is transferred to the nucleus and DNA is released. There, the DNA strand synthesis is completed and gaps repaired to give the covalently closed circular (ccc) supercoiled DNA of 3.2kb. The cccDNA serves as a template for transcription of five major viral mRNAs, which are 3.5, 3.5, 2.4, 2.1 and 0.7 kb long. All mRNAs are 5′-capped and polyadenylated at the 3′-end. There is sequence overlap at the 3′-end between all five mRNAs.
One 3.5 kb mRNA serves as template for core protein and polymerase production. In addition, the same transcript serves as a pre-genomic replication intermediate and allows the viral polymerase to initiate the reverse transcription into DNA. Core protein is needed for nucleocapsid formation. The other 3.5 kb mRNA encodes pre-core, the secretable e-antigen (HBeAg) . In the absence of replication inhibitors, the abundance of e-antigen in blood correlates with Hepatitis B Virus replication in liver and serves as an important diagnostic marker for monitoring the disease progression.
The 2.4 and 2.1 kb mRNAs carry the open reading frames ( “ORF” ) pre-S1, pre-S2 and S for expression of viral large, medium and small surface antigen. The s-antigen is associated with infectious, complete particles. In addition, blood of infected patients also contain non-infectious particles derived from s-antigen alone, free of genomic DNA or polymerase. The function of these particles is not fully understood. The complete and lasting depletion of detectable s-antigen in blood is considered as a reliable indicator for hepatitis B virus clearance.
The 0.7 kb mRNA encodes the X protein. This gene product is important for efficient transcription of viral genes and also acts as a transactivator on host gene expression. The latter activity seems to be important for hepatocyte transformation during development of liver cancer.
Patients with detectable s-antigen (HBsAg) , e-antigen (HBeAg) , and/or viral DNA in the blood for more than 6 months are considered chronically infected. Chronic HBV infection can be classified into five phases: (I) HBeAg-positive chronic infection, (II) HBeAg-positive chronic hepatitis, (III) HBeAg-negative chronic infection, (IV) HBeAg-negative chronic hepatitis and (V) HBsAg-negative phase. All patients with chronic HBV infection are at increased risk of progression to cirrhosis and hepatocellular carcinoma (HCC) , depending on host and viral factors (Lampertico et al., J Hepatol., 2017, 67 (2) : 370-398) . At the present, a complete sterilizing cure, i.e., viral eradication from the host, is unlikely to be feasible (Lok et al., J Hepatol., 2017, 67 (4) : 847-861) . The main goal of therapy is to improve survival and quality of life by preventing disease progression, and consequently HCC development. The induction of long-term suppression of HBV replication represents the main endpoint of current treatment strategies, while HBsAg loss is an optimal endpoint.
Nucleoside analogs as inhibitors of reverse transcriptase activity are typically the first treatment option for many patients. Long term administration of lamivudine, tenofovir, and/or entecavir has been shown to suppress hepatitis B virus replication, sometimes to undetectable levels, with improvement of liver function and reduction of liver inflammation typically seen as the most important benefits. However, only few patients achieve complete and lasting remission after the end of treatment. Furthermore, the hepatitis B virus develops drug resistance with increasing duration of treatment. This is especially difficult for patients co- infected with hepatitis B and human immunodeficiency virus (HIV) . Both viruses are susceptible to nucleoside analogue drugs and may co-develop resistance.
Pegylated interferon-alpha (IFN) has been used to treat mild to moderate chronic hepatitis B patients. However, current treatment of chronic hepatitis B has limited efficacy (Erha et al., Gut. 2005 Jul; 54 (7) : 1009–1013) . For example, the Asian genotype B gives very poor response rates. Co-infection with hepatitis D virus (HDV) or human immunodeficiency virus has been shown to render interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy.
Certain hepatitis B virus-specific RNA interference (RNAi) agents have been previously shown to inhibit expression of HBV gene expression. For example, U.S. Patent Application Publication No. 2013/0005793, to Chin et al., and WO2018027106A1, to Li et al., which are incorporated herein by reference in its entirety, disclose certain double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of hepatitis B virus gene.
Additionally, HBV inhibitors, such as capsid assembly modulator (CAM) , can bind to hepatitis B core protein and interferes with the viral capsid assembly process, thereby preventing the polymerase-bound pgRNA encapsidation. This results in the formation of HBV capsids, devoid of HBV DNA or RNA (non-functional capsids) , and ultimately in the inhibition of HBV replication. See e.g., WO2015/132276A1 and WO2020/125730A1. The references WO2015/132276A1 and WO2020/125730A1 are incorporated herein in their entirety, particularly the descriptions of the heteroaryldihydropyrimidine (HAP) compounds as capsid assembly modulators and the method of preparing them.
Several combinations have been tried in the treatment of HBV in various settings (Paul, Curr Hepat Rep. 2011 Jun; 10 (2) : 98–105) . However, various combination therapies for HBeAg-positive chronic HBV or HBeAg-negative hepatitis B have not established a benefit over monotherapy. Id.
There is a need for improved HBV therapy that can overcome at least one of the disadvantages of existing treatment options, such as are toxicity, mutagenicity, lack of  selectivity, poor efficacy, poor bioavailability, and difficulty of synthesis, while providing additional benefits such as increased potency or an increased safety window.
The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.
SUMMARY OF THE INVENTION
Provided herein is a pharmaceutical combination comprising an RNAi component and a heteroaryldihydropyrimidine (HAP) compound,
wherein:
(a) the RNAi component comprises
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; and
(b) the heteroaryldihydropyrimidine (HAP) compound is a compound of Formula (Ia) or a compound of Formula (Ib) or a stereoisomer or a tautomeric form thereof or a pharmaceutically acceptable salt or a solvate thereof:
Figure PCTCN2021123990-appb-000001
wherein in Formula (Ia) :
R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4alkyl, halogen and CN;
R 2 is C 1-4alkyl;
R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine and C 1-6alkyl; n is an integer of 0 or 1;
R 4 and R 5 are independently selected from H and -COOH;
Figure PCTCN2021123990-appb-000002
is a single bond or a double bond;
when X and Y are linked by a single bond, X is selected from the group consisting of C (=S) , C (=NR 6) , C (=CHR 7) and CHR 8, and Y is NR 9;
when X and Y are linked by a double bond, X is C-SR 9 or C-OR 9, and Y is N atom;
Z is selected from the group consisting of CH 2, and C (=O) ;
R 6 is selected from the group consisting of CN, C (=O) CH 3 and SO 2CH 3;
R 7 is CN;
R 8 is CF 3;
R 9 is selected from the group consisting of H, -C 1-6alkyl, -C 1-6alkyl-R 10, -C 1-6alkoxy-C 1- 6alkyl-R 10, - (CH 2)  p-C (R 11R 12) -R 10 and - (CH 2)  p-Q-R 10;
p is an integer of 0, 1, 2, or 3;
R 11 and R 12 together with carbon atom to which they are attached form a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with one or more substituents selected from the group consisting of with H, -C 1-6alkyl, -C 1-6alkoxy-C 1-6alkyl and -C 1-6alkylcarbonyl;
Q is selected from the group consisting of aryl, heteroaryl, and a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6alkyl, -C 1-6alkoxy-C 1-6alkyl and -C 1-6alkylcarbonyl; R 10 is selected from -COOH, -C (=O) NHS (=O)  2-C 1-6alkyl, tetrazolyl, and carboxylic acid bioisosteres;
Figure PCTCN2021123990-appb-000003
wherein in Formula (Ib) :
R 1 is hydrogen, halogen or C 1-6alkyl;
R 2 is hydrogen or halogen;
R 3 is hydrogen or halogen;
R 4 is C 1-6alkyl;
R 5 is hydrogen, hydroxy C 1-6alkyl, aminocarbonyl, C 1-6alkoxycarbonyl or carboxy;
R 6 is hydrogen, C 1-6alkoxycarbonyl or carboxy-C mH 2m-;
X is carbonyl or sulfonyl;
Y is -CH 2-, -O-or -N (R 7) -,
wherein R 7 is hydrogen, C 1-6alkyl, haloC 1-6alkyl, C 3-7cycloalkyl-C mH 2m-, C 1-6alkoxycarbonyl-C mH 2m-, -C tH 2t-COOH, -haloC 1-6alkyl-COOH, - (C 1-6alkoxy) C 1-6alkyl-COOH, -C 1-6alkyl-O-C 1-6alkyl-COOH, -C 3- 7cycloalkyl-C mH 2m-COOH, -C mH 2m-C 3-7cycloalkyl-COOH, hydroxy-C tH 2t-, carboxyspiro [3.3] heptyl or carboxyphenyl-C mH 2m-, carboxypyridinyl-C mH 2m-;
W is -CH 2-, -C (C 1-6alkyl)  2-, -O-or carbonyl;
n is 0 or 1;
m is 0-7; and
t is 1-7.
Provided herein is a pharmaceutical combination for a use selected from the group consisting of:
(1) treating a Hepatitis B viral (HBV) infection in a subject;
(2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
(3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
(4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
(5) increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
(6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
(7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof; wherein the pharmaceutical combination is as above defined.
Provided herein is use of an RNAi component and a heteroaryldihydropyrimidine (HAP) compound in the manufacture of a pharmaceutical combination for a use selected from the group consisting of:
(1) treating a Hepatitis B viral (HBV) infection in a subject;
(2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
(3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
(4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
(5) increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
(6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
(7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof; wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound and the pharmaceutical combination are as above defined.
Provided herein is a method for a use selected from the group consisting of:
(1) treating a Hepatitis B viral (HBV) infection in a subject;
(2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
(3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
(4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
(5) for increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
(6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
(7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof; wherein the method comprises administering to the subject an RNAi component and a heteroaryldihydropyrimidine (HAP) compound;
wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
Provided herein is a RNAi component and a heteroaryldihydropyrimidine (HAP) compound for a use selected from the group consisting of:
(1) treating a Hepatitis B viral (HBV) infection in a subject;
(2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
(3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
(4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
(5) increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
(6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
(7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof; wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
Provided herein is a RNAi component for a use selected from the group consisting of:
(1) treating a Hepatitis B viral (HBV) infection in a subject;
(2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
(3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
(4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
(5) increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
(6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
(7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof; wherein the RNAi component is to be administered in combination therapy with a heteroaryldihydropyrimidine (HAP) compound;
wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
Provided herein is a heteroaryldihydropyrimidine (HAP) compound for a use selected from the group consisting of:
(1) treating a Hepatitis B viral (HBV) infection in a subject;
(2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
(3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
(4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
(5) increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
(6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
(7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof; wherein the heteroaryldihydropyrimidine (HAP) compound is to be administered in combination therapy with the RNAi component;
wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
Provided herein is a kit comprising an effective amount of a RNAi component and a heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined.
Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.
DETAILED DESCRIPTION
The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific compositions, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.
Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.
It must be noted that as used herein and in the appended claims, the singular forms “a, ” “an, ” and “the, ” include plural reference unless the context clearly dictates otherwise.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the  specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise, ” and variations such as “comprises” and “comprising, ” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having. ”
When used herein “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising, ” “containing, ” “including, ” and “having, ” whenever used herein in the context of an aspect or embodiment of the application can be replaced with the term “consisting of” or “consisting essentially of” to vary scopes of the invention.
As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or, ” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or. ”
Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about. ” Thus, a numerical value typically includes ± 10%of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values  within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
By “optional” or “optionally” is meant that the event described subsequent thereto may or may not happen. This term encompasses the cases that the event may or may not happen. For example, the expression that ethyl is “optionally” substituted by halogen means the ethyl is unsubstituted (CH 2CH 3) , mono-substituted (e.g., CH 2CH 2F) , poly-substituted (e.g., CHFCH 2F, CH 2CHF 2, or the like) or completely substituted (CF 2CF 3) . A person skilled in the art will know that with respect to any group containing one or more substitutes, a substitution or substitution mode which cannot exist and/or cannot be synthesized will not be introduced.
The expression C m-n used herein means that it has m-n carbon atoms. For example, “C 3- 10 cycloalkyl” means said cycloalkyl has 3-10 carbon atoms. “C 0-6 alkylene” means said alkylene has 0-6 carbon atoms, wherein the alkylene is a bond when it has 0 carbon atoms. The numerical range herein refers to each of the integers therein. For example, “C 1-10” means said group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms or 10 carbon atoms.
The term “substituted” means that one or more hydrogen atoms on a given atom are replaced by a substituent, provided that the valence of the particular atom is normal and the compound after substitution is stable. When the substituent is a ketone group (i.e. =O) , two hydrogen atoms are replaced, and the ketone substitution will not occur at an aromatic group.
When any variable (e.g., R) occurs at the composition or structure over one time, it is defined independently at each case. Therefore, for example, if a group is substituted by 0-2 R, the group can be optionally substituted by at most two R and R has an independent option at each case. Additionally, a combination of substituents and/or the variants thereof are allowed only if such a combination will result in a stable compound.
Unless stated otherwise, the term “hetero” means heteroatom or heteroatom radical (i.e., a radical containing heteroatom) , i.e. the atoms beyond carbon and hydrogen atoms or the radical containing such atoms, wherein the heteroatom is independently selected from the group consisting O, N, S, P, Si, Ge, Al and B. In some embodiments wherein two or more  heteroatoms are involved, the two or more heteroatoms can be the same, or part or all of the two or more heteroatoms can be different.
As used herein, the term “halo” or “halogen, ” alone or as part of another substituent means, unless otherwise stated, a fluorine (F) , chlorine (Cl) , bromine (BR) , or iodine (I) atom, preferably F, Cl, or Br, more preferably F or Cl.
As used herein, the term “alkyl” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C 1-C 4alkyl or C 1-4alkyl means an alkyl having one to four carbon) and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl.
The term “aryl” refers to monocyclic or fused polycyclic aromatic cyclic group which has conjugated π electronic system and all the ring atoms are carbon. For example, an aryl can have 6-20 carbon atoms, 6-14 carbon atoms or 6-12 carbon atoms. Non-limiting examples of aryls include, but are not limited to, phenyl, naphthyl, anthryl, or the like.
The term “heteroaryl” refers to monocyclic or fused polycyclic system containing at least one ring atom selected from the group consisting of N, O and S with other ring atoms being C and containing at least one aromatic ring. Non-limiting examples of heteroaryls include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, or the like.
As used herein, the term “heteroaryl” or “heteroaromatic” refers to a heterocycle having aromatic character.
Whenever sustituents are represented by chemical structure, “---” represents the bond of attachment to the remainder of the molecule. Lines (such as “--- “) drawn into a particular ring of a ring system indicate that the bond may be attached to any of the suitable ring atoms.
As used herein, the term “capsid assembly modulator” refers to a compound that disrupts or accelerates or inhibits or hinders or delays or reduces or modifies normal capsid assembly  (e.g., during maturation) or normal capsid disassembly (e.g., during infectivity) or perturbs capsid stability, thereby inducing aberrant capsid morphology and function. In some embodiments, a capsid assembly modulator accelerates capsid assembly or disassembly, thereby inducing aberrant capsid morphology. In another embodiment, a capsid assembly modulator interacts (e.g. binds at an active site, binds at an allosteric site, modifies or hinders folding and the like) with the core protein (a capsid assembly protein (CA) ) , thereby disrupting capsid assembly or disassembly. In yet another embodiment, a capsid assembly modulator causes a perturbation in structure or function of CA (e.g., ability of CA to assemble, disassemble, bind to a substrate, fold into a suitable conformation, or the like) , which attenuates viral infectivity or is lethal to the virus.
As used herein, the half maximal effective concentration (EC 50) is intended in accordance with its general meaning in the field. It may more particularly refer to the concentration of a compound which induces a response halfway between the baseline and maximum, typically after a specified exposure time. The EC 50 value is commonly used as a measure of a compound’s potency, with a lower value generally indicating a higher potency.
The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
The term “pharmaceutically acceptable salt” refers to a salt of any of the compounds herein which are known to be non-toxic and are commonly used in the pharmaceutical literature. In some embodiments, the pharmaceutically acceptable salt of a compound retains the biological effectiveness of the compounds described herein and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66 (1) , 1-19. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic  acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethylsulfonic acid, p-toluenesulfonic acid, stearic acid and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.
The pharmaceutically acceptable salts according to the invention may be prepared from the parent compound containing acidic or basic group through conventional chemical procedures. Generally, such salts can be prepared through the reaction of the compounds in the form of free acid or base with stoichiometric appropriate base or acid in water, organic solvent or the mixture thereof. Typically, nonaqueous medium like ether, ethyl acetate, ethanol, isopropanol, acetonitrile etc. are preferable.
Some compounds according to the invention can exist in unsolvated or solvated forms, including hydrate form. In general, the solvated forms are equivalent to unsolvated forms and both of them are encompassed within the scope of the invention. Some compounds according to the invention can exist in polymorphic or amorphous forms.
Some compounds according to the invention can have asymmetric carbon atom (optical center) or double bond. Racemate, diastereomer, geometric isomer and individual isomer are encompassed within the scope of the invention.
The graphic representations of racemic, ambiscalemic and scalemic or enantiomerically pure compounds used herein are taken from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless stated otherwise, solid and broken wedges are used to denote the absolute configuration of a  stereocenter. When the compound according to the invention contains ethylenical double bond (s) or other geometric asymmetry center (s) , unless stated otherwise, E and Z geometric isomer are encompassed. Likewise, all the tautomeric forms are encompassed with the scope of the invention.
The compound according to the invention can have special geometric isomer or stereoisomer form. Such compounds are encompassed by the invention, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomer, (D) -isomer, (L) -isomer, and racemic mixture or other mixture thereof, such as the mixture enriched in enantiomer or diastereomer, and all the mixtures are encompassed within the scope of the invention. The substituent like alkyl can have other asymmetric carbon atom. All the isomers and the mixture thereof are encompassed within the scope of the invention.
Optical (R) -and (S) -isomers as well as D and L isomers can be prepared through chiral synthesis or chiral agent or other conventional technology. An enantiomer of the compound according to the invention can be prepared through asymmetric synthesis or derivatization with chiral auxiliary, wherein the resultant diastereomer mixture is separated and the desired pure enantiomer is obtained by cleavage of the auxiliary group. Alternatively, when there is basic functional group (e.g. amino) or acidic functional group (e.g. carboxyl) in the molecule, the diastereomeric salt can be formed with appropriate optical acid or base and then the diastereomeric resolution is performed with fractional crystallization or chromatography which is well-known in the art so as to recover the pure enantiomer. Additionally, separation of enantiomer from diastereomer is generally performed with chromatography, which uses chiral stationary phase and is optionally combined with chemical derivatization (for example, carbamate formed from amine) .
The compound according to the invention can contain atomic isotope in non-natural ratio at one or more atoms constituting said compound. For example, the compound may be labeled with radioisotope, such as Tritium ( 3H) , Iodine-125 ( 125I) or C-14 ( 14C) . Alternation of all the radioisotopes of the compound, either radioactive or not, is encompassed within the scope of the invention.
The terms “patient” and “subject” refer to an animal, such as a mammal, bird, or fish. In some embodiments, the patient or subject is a mammal. The term “mammal” as used herein,  encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human. In some embodiments, the patient or subject is a human, for example a human that has been or will be the object of treatment, observation or experiment. The compounds, compositions, and methods described herein can be useful in both human therapy and veterinary applications.
A “solvate” is formed by the interaction of a solvent and a compound. Suitable solvents include, for example, water and alcohols (e.g., ethanol) . Solvates include hydrates having any ratio of compound to water, such as monohydrates, dihydrates and hemi-hydrates.
The term “therapeutically effective amount” or “effective amount” refers to that amount of a compound disclosed and/or described herein that is sufficient to affect treatment, as defined herein, when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon, for example, the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound, the dosing regimen to be followed, timing of administration, the manner of administration, all of which can readily be determined by one of ordinary skill in the art. The therapeutically effective amount can be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability by one of ordinary skill in the art in view of the present invention.
In particular embodiments of the application, a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV  replication in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect (s) of another therapy.
A therapeutically effective amount can also be an amount of the compound sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; achieve sustained HBsAg clearance associated with reduction of infected hepatocytes by a subject’s immune system; induce HBV-antigen specific activated T-cell populations; and/or achieve persistent loss of HBsAg within 12 months. Examples of a target index include lower HBsAg below a threshold of 500 copies of HBsAg international units (IU) and/or higher CD8 counts. Additional examples of target indexes include, but are not limited to, serum HBV DNA levels lower than the lower limit of quantification (LLoQ) or lower than 20 IU/mL, more particularly lower than 15 IU/mL, more particularly lower than 10 IU/mL; serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; HBeAg-negative serum; serum HBsAg level of 100 IU/mL or lower, more particularly of 10 IU/mL or lower; and/or HBs seroconversion.
In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections, or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the invention, including the claims, is limited to these examples. For example, while embodiments of RNAi component described herein may contain particular components arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in RNAi of the application. The application contemplates use of any of the applicable components in any combination that can be used the application, whether or not a particular combination is expressly  described. The invention generally relates to a therapeutic combination comprising one or more HBV RNAi component and a heteroaryldihydropyrimidine (HAP) compound.
Pharmaceutical Combination
Provided herein is a pharmaceutical combination comprising an RNAi component and a heteroaryldihydropyrimidine (HAP) compound.
In some embodiments, the pharmaceutical combination is in the form of a product, e.g a composition or a kit.
In some embodiments, the pharmaceutical combination is in the form of a product, where the therapeutic agents may be administered independently at the same time or separately within time intervals that allow that the therapeutic agents show a cooperative effect.
RNAi Component
The RNAi component comprises a first RNAi agent and a second RNAi agent. Each RNAi agent disclosed herein includes at least a sense strand and an antisense strand. The sense strand and the antisense strand can be partially, substantially, or fully complementary to each other. The length of the RNAi agent sense and antisense strands described herein each can be 16 to 30 nucleotides in length. In some embodiments, the sense and antisense strands are independently 17 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. The HBV RNAi agents disclosed herein have been designed to include antisense strand sequences that are at least partially complementary to a sequence in the HBV genome that is conserved across the majority of known serotypes of HBV. The RNAi agents described herein, upon delivery to a cell expressing HBV, inhibit the expression of one or more HBV genes in vivo or in vitro.
An RNAi agent includes a sense strand (also referred to as a passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence. A sense strand of the HBV RNAi agents described herein includes a core stretch having at least about 85%identity to a nucleotide sequence of at least 16 consecutive  nucleotides in an HBV mRNA. In some embodiments, the sense strand core nucleotide stretch having at least about 85%identity to a sequence in an HBV mRNA is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. An antisense strand of an HBV RNAi agent comprises a nucleotide sequence having at least about 85%complementary over a core stretch of at least 16 consecutive nucleotides to a sequence in an HBV mRNA and the corresponding sense strand. In some embodiments, the antisense strand core nucleotide sequence having at least about 85%complementarity to a sequence in an HBV mRNA or the corresponding sense strand is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
In some embodiments, the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, or a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
In some embodiments, the first and the second RNAi agents disclosed herein comprise any of the sequences in Table 1.
Table 1. Exemplary sequences for first and second RNAi agents
Figure PCTCN2021123990-appb-000004
Targeting Group
In some embodiments, the RNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates (DPCs) (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, each of which is incorporated herein by reference) . In some embodiments, an HBV RNAi agent is delivered to target cells or tissues by covalently linking the RNAi agent to a targeting group. In some embodiments, the targeting group can include a cell receptor ligand, such as an asialoglycoprotein receptor (ASGPr) ligand. In some embodiments, an ASGPr ligand includes or consists of a galactose derivative cluster. In some embodiments, a galactose derivative cluster includes an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer. In some embodiments, a galactose derivative cluster is an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.
A targeting group can be linked to the 3′ or 5′ end of a sense strand or an antisense strand of an HBV RNAi agent. In some embodiments, a targeting group is linked to the 3′ or 5′ end of the sense strand. In some embodiments, a targeting group is linked to the 5’ end of the sense strand. In some embodiments, a targeting group is linked to the RNAi agent via a linker.
In some embodiments, the RNAi component comprises a combination or cocktail of a first and a second RNAi agent having different nucleotide sequences. In some embodiments, the first and the second RNAi agents are each separately and independently linked to targeting groups. In some embodiments, the first and the second RNAi agents are each linked to targeting groups comprised of N-acetyl-galactosamines. In some embodiments, when first  and the second RNAi agents are included in a composition, each of the RNAi agents is linked to the same targeting group. In some embodiments, when first and the second RNAi agents are included in a composition, each of the RNAi agents is linked to different targeting groups, such as targeting groups having different chemical structures.
In some embodiments, targeting groups are linked to the first and the second RNAi agents without the use of an additional linker. In some embodiments, the targeting group is designed having a linker readily present to facilitate the linkage to the first or the second RNAi agent. In some embodiments, when the first and the second RNAi agents are included in a composition, the first and the second RNAi agents may be linked to the targeting groups using the same linkers. In some embodiments, when the first and the second RNAi agents are included in a composition, the first and the second RNAi agents are linked to the targeting groups using different linkers.
Examples of targeting groups and linking groups are provided in Table 2. The non-nucleotide group can be covalently linked to the 3′ and/or 5′ end of either the sense strand and/or the antisense strand. In some embodiments, the first or second RNAi agent contains a non-nucleotide group linked to the 3′ and/or 5′ end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5′ end of the first or second RNAi agent sense strand. A non-nucleotide group may be linked directly or indirectly to the first or second RNAi agent via a linker/linking group. In some embodiments, a non-nucleotide group is linked to the first or second RNAi agent via a labile, cleavable, or reversible bond or linker.
Targeting groups and linking groups include the following, for which their chemical structures are provided below in Table 2: (PAZ) , (NAG13) , (NAG13) s, (NAG18) , (NAG18) s, (NAG24) , (NAG24) s, (NAG25) , (NAG25) s, (NAG26) , (NAG26) s, (NAG27) , (NAG27) s, (NAG28) , (NAG28) s, (NAG29) , (NAG29) s, (NAG30) , (NAG30) s, (NAG31) , (NAG31) s, (NAG32) , (NAG32) s, (NAG33) , (NAG33) s, (NAG34) , (NAG34) s, (NAG35) , (NAG35) s, (NAG36) , (NAG36) s, (NAG37) , (NAG37) s, (NAG38) , (NAG38) s, (NAG39) , (NAG39) s. Each sense strand and/or antisense strand can have any targeting groups or linking groups listed above, as well as other targeting or linking groups, conjugated to the 5′ and/or 3′ end of the sequence.
Table 2. Structures Representing Various Modified Nucleotides, Targeting Groups, And Linking Groups
Figure PCTCN2021123990-appb-000005
Figure PCTCN2021123990-appb-000006
Figure PCTCN2021123990-appb-000007
Figure PCTCN2021123990-appb-000008
Figure PCTCN2021123990-appb-000009
Figure PCTCN2021123990-appb-000010
Figure PCTCN2021123990-appb-000011
Figure PCTCN2021123990-appb-000012
Figure PCTCN2021123990-appb-000013
Figure PCTCN2021123990-appb-000014
Figure PCTCN2021123990-appb-000015
Figure PCTCN2021123990-appb-000016
Figure PCTCN2021123990-appb-000017
Figure PCTCN2021123990-appb-000018
Figure PCTCN2021123990-appb-000019
Modified Nucleotides
In some embodiments, the first or the second RNAi agent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2′-hydroxyl nucleotide) . In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (represented herein as Ab) , 2′-modified nucleotides, 3′ to 3′ linkages (inverted) nucleotides (represented herein as invdN, invN, invn, invAb) , non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs) , 2′, 3′-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as NUNA or NUNA) , locked nucleotides (represented herein as NLNA or NLNA) , 3′-O-methoxy (2′ internucleoside linked) nucleotides (represented herein as 3′-OMen) , 2'-F-Arabino nucleotides (represented herein as NfANA or NfANA) , 5'-Me, 2'-fluoro nucleotide (represented herein as 5Me-Nf) , morpholino nucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN) , vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides (cPrpN) . 2′-modified nucleotides (i.e. a nucleotide with a group other than a hydroxyl group at the 2′ position of the five-membered sugar ring) include, but are not limited to, 2′-O-methyl nucleotides (represented herein as a lower case letter 'n' in a nucleotide sequence) , 2′-deoxy-2′-fluoro nucleotides (represented herein as Nf, also represented herein as 2′-fluoro nucleotide) , 2′-deoxy nucleotides (represented herein as dN) , 2′-methoxyethyl (2′-O-2-methoxylethyl) nucleotides (represented herein as NM or 2′-MOE) , 2′-amino  nucleotides, and 2′-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in the first or second RNAi agent or even in a single nucleotide thereof. The RNAi agent sense strands and antisense strands may be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide. Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine) , 5-methylcytosine (5-me-C) , 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil) , 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo) , 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.
In some embodiments, all or at least 90%of the nucleotides of the first or the second RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein at least 90%of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides. As used herein, a sense strand, wherein at least 90%of the nucleotides present are modified nucleotides, is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. As used herein, an antisense sense strand, wherein at least 90%of the nucleotides present are modified nucleotides, is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is a ribonucleotide.
Modified Internucleoside Linkages
In some embodiments, one or more nucleotides of the first or the second RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones) . In some embodiments, a modified internucleoside linkage is a non-phosphate-containing covalent internucleoside linkage. Modified internucleoside linkages or  backbones include, but are not limited to, 5’-phosphorothioate groups (represented herein as a lower case “s” ) , chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3′-alkylene phosphonates) , chiral phosphonates, phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates) , thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, a modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH 2 components.
In some embodiments, a sense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
In some embodiments, the first or the second RNAi agent sense strand contains at least two phosphorothioate internucleoside linkages. In some embodiments, the at least two phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6- 8 from the 5' end of the sense strand. In some embodiments, the first or the second RNAi agent antisense strand contains four phosphorothioate internucleoside linkages. In some embodiments, the four phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the sense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, the first or the second RNAi agent contains at least two phosphorothioate internucleoside linkages in the sense strand and three or four phosphorothioate internucleoside linkages in the antisense strand.
In some embodiments, the first or the second RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2′-modified nucleoside is combined with modified internucleoside linkage. In some embodiments, the first and the second RNAi agents disclosed herein comprise any of the modified sequences in Table 3.
Table 3. Exemplary modified sequences for first and second RNAi agents
Figure PCTCN2021123990-appb-000020
A            = adenosine-3′-phosphate;
C            = cytidine-3′-phosphate;
G            = guanosine-3′-phosphate;
U            = uridine-3′-phosphate
n            = any 2′-OMe modified nucleotide
a            = 2′-O-methyladenosine-3′-phosphate
as           = 2′-O-methyladenosine-3′-phosphorothioate
c            = 2′-O-methylcytidine-3′-phosphate
cs           = 2′-O-methylcytidine-3′-phosphorothioate
g            = 2′-O-methylguanosine-3′-phosphate
gs           = 2′-O-methylguanosine-3′-phosphorothioate
t            = 2′-O-methyl-5-methyluridine-3′-phosphate
ts           = 2′-O-methyl-5-methyluridine-3′-phosphorothioate
u            = 2′-O-methyluridine-3′-phosphate
us           = 2′-O-methyluridine-3′-phosphorothioate
Nf           = any 2′-fluoro modified nucleotide
Af           = 2′-fluoroadenosine-3′-phosphate
Afs          = 2′-fluoroadenosine-3′-phosporothioate
Cf           = 2′-fluorocytidine-3′-phosphate
Cfs          = 2′-fluorocytidine-3′-phosphorothioate
Gf           = 2′-fluoroguanosine-3′-phosphate
Gfs          = 2′-fluoroguanosine-3′-phosphorothioate
Tf           = 2′-fluoro-5′-methyluridine-3′-phosphate
Tfs          = 2′-fluoro-5′-methyluridine-3′-phosphorothioate
Uf           = 2′-fluorouridine-3′-phosphate
Ufs          = 2′-fluorouridine-3′-phosphorothioate
dN           = any 2′-deoxyribonucleotide
dT           = 2′-deoxythymidine-3′-phosphate
N UNA         = 2′, 3′-seco nucleotide mimics (unlocked nucleobase analogs)
N LNA         = locked nucleotide
Nf ANA        = 2'-F-Arabino nucleotide
NM           = 2′-methoxyethyl nucleotide
AM           = 2′-methoxyethyladenosine-3′-phosphate
AMs          = 2′-methoxyethyladenosine-3′-phosphorothioate
TM           = 2′-methoxyethylthymidine-3′-phosphate
TMs          = 2′-methoxyethylthymidine-3′-phosphorothioate
R            = ribitol
(invdN)      = any inverted deoxyribonucleotide (3′-3′ linked nucleotide)
(invAb)      = inverted (3′-3′ linked) abasic deoxyribonucleotide,
(invAb) s    = inverted (3′-3′ linked) abasic deoxyribonucleotide-5′-
             phosphorothioate,
(invn)       = any inverted 2′-OMe nucleotide (3′-3′ linked nucleotide)
s            = phosphorothioate linkage
In some embodiments, the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO: 1 and a sense strand comprising SEQ ID NO: 10; an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11; an antisense strand comprising SEQ ID NO: 3 and a sense strand comprising SEQ ID NO: 11; an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12; an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16; an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 17; an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 13; and an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 18.
In some embodiments, the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11; an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12; an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16; an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 13; and an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 18. In still another embodiment, the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1: 2 to about 5: 1.
In some embodiments, the ratio of the first RNAi agent to the second RNAi agent by weight is about 2: 1.
In some embodiments, the first and the second RNAi agents are each independently conjugated to (NAG37) s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11, and the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16.
In some embodiments, the first RNAi agent comprises SEQ ID NO: 5 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 6 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 7 and SEQ ID NO: 15. In some embodiments, the first RNAi agent comprises SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 3 and SEQ ID NO: 10, 11, or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 4 and SEQ ID NO: 12. In some embodiments, the second RNAi agent comprises SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the second RNAi agent comprises SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 5 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ  ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 6 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 7 and SEQ ID NO: 15 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19.
In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 3 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 4 and SEQ ID NO: 12 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 11 and the second RNAi agent comprising SEQ ID NO: 16 and SEQ ID NO: 8.
In some embodiments, the RNAi component comprises a first and a second RNAi agent in a ratio of about 1: 1, 2: 1, 3: 1, 4: 1 or 5: 1. In some embodiments, the two HBV RNAi agents are administered in a ratio of about 2: 1.
In some embodiments,
(1) the first or the second RNAi agent comprises at least one modified nucleotide and/or at least one modified internucleoside linkage,
(2) at least 90%of the nucleotides in the first and the second RNAi agents are modified nucleotides,
(3) the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and /or
(4) the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1: 2 to about 5: 1.
In some embodiments,
the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
(a) an antisense strand comprising SEQ ID NO: 1 and a sense strand comprising SEQ ID NO: 10;
(b) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11;
(c) an antisense strand comprising SEQ ID NO: 3 and a sense strand comprising SEQ ID NO: 11;
(d) an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12;
(e) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16;
(f) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 17;
(g) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 12; and
(h) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 18.
In some embodiments,
the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and
the targeting ligand comprises N-acetyl-galactosamine,
preferably selected from the group consisting of (NAG13) , (NAG13) s, (NAG18) , (NAG18) s, (NAG24) , (NAG24) s, (NAG25) , (NAG25) s, (NAG26) , (NAG26) s, (NAG27) , (NAG27) s, (NAG28) , (NAG28) s, (NAG29) , (NAG29) s, (NAG30) , (NAG30) s, (NAG31) , (NAG31) s, (NAG32) , (NAG32) s, (NAG33) , (NAG33) s, (NAG34) , (NAG34) s, (NAG35) , (NAG35) s, (NAG36) , (NAG36) s, (NAG37) , (NAG37) s, (NAG38) , (NAG38) s, (NAG39) , and (NAG39) s,
particularly preferably selected from the group consisting of (NAG25) , (NAG25) s, (NAG31) , (NAG31) s, (NAG37) , and (NAG37) s.
In some embodiments,
the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and
the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent, preferably the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent.
In some embodiments,
the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
(a) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11;
(b) an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12;
(c) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16;
(d) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 13; and
(e) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 18.
Heteroaryldihydropyrimidine (HAP) compound
In some embodiments, the heteroaryldihydropyrimidine (HAP) compound is a compound of Formula (Ia) or a stereoisomer or a tautomeric form thereof or a pharmaceutically acceptable salt or a solvate thereof:
Figure PCTCN2021123990-appb-000021
wherein in Formula (Ia) :
R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4alkyl, halogen and CN;
R 2 is C 1-4alkyl;
R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally substituted with one or more substituents selected from fluorine and C 1-6alkyl;
n is an integer of 0 or 1;
R 4 and R 5 are independently selected from H and -COOH;
Figure PCTCN2021123990-appb-000022
is a single bond or a double bond;
when X and Y are linked by a single bond, X is selected from the group consisting of C (=S) , C (=NR 6) , C (=CHR 7) and CHR 8, and Y is NR 9;
when X and Y are linked by a double bond, X is C-SR 9 or C-OR 9, and Y is N atom;
Z is selected from the group consisting of CH 2, and C (=O) ;
R 6 is selected from the group consisting of CN, C (=O) CH 3 and SO 2CH 3;
R 7 is CN;
R 8 is CF 3;
R 9 is selected from the group consisting of H, -C 1-6alkyl, -C 1-6alkyl-R 10, -C 1-6alkoxy-C 1- 6alkyl-R 10, - (CH 2)  p-C (R 11R 12) -R 10 and - (CH 2)  p-Q-R 10;
p is an integer of 0, 1, 2, or 3;
R 11 and R 12 together with carbon atom to which they are attached form a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with one or more substituents selected from the group consisting of with H, -C 1-6alkyl, -C 1-6alkoxy-C 1-6alkyl and -C 1-6alkylcarbonyl;
Q is selected from the group consisting of aryl, heteroaryl, and a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6alkyl, -C 1-6alkoxy-C 1-6alkyl and -C 1-6alkylcarbonyl; R 10 is selected from -COOH, -C (=O) NHS (=O)  2-C 1-6alkyl, tetrazolyl, and carboxylic acid bioisosteres.
In a particular embodiment of the compounds of Formula (Ia) , the carboxylic acid bioisosters of Formula (Ia) are -S (=O)  2 (OH) , -P (=O) (OH)  2, -C (=O) NHOH, -C (=O) NHCN, 1, 2, 4-oxadiazol-5 (4H) -one, and 3-hydroxy-4-methylcyclobut-3-ene-1, 2-dione.
In a particular embodiment of the compounds of Formula (Ia) , R 1 of Formula (Ia) is phenyl substituted with one or more substituents selected from halogens and C 1-6alkyl.
In a particular embodiment of the compounds of Formula (Ia) , R 2 of Formula (Ia) is methyl or ethyl.
In a particular embodiment of the compounds of Formula (Ia) , R 3 of Formula (Ia) is thiazolyl.
In a particular embodiment of the compounds of Formula (Ia) , R 4 and R 5 of Formula (Ia) are H.
In a particular embodiment of the compounds of Formula (Ia) , X of Formula (Ia) is C (=S) .
In a particular embodiment of the compounds of Formula (Ia) , Z of Formula (Ia) is CH 2
In a particular embodiment of the compounds of Formula (Ia) , R 9 of Formula (Ia) is -C 1- 6alkyl-CO 2H, - (CH 2)  p-C (R 11R 12) -R 10 or - (CH 2)  p-Q-R 10.
In a particular embodiment of the compounds of Formula (Ia) , Q of Formula (Ia) is phenyl.
In a particular embodiment of the compounds of Formula (Ia) , Q of Formula (Ia) is a C 3- 6cycloalkyl, or R 11 and R 12 of Formula (Ia) together with carbon atom to which they are attached form a C 3-6cycloalkyl.
In a further particular embodiment of the compounds of Formula (Ia) , Q of Formula (Ia) is a 3-to 6-saturated membered ring containing an oxygen, or R 11 and R 12 of Formula (Ia) together with carbon atom to which they are attached form a 3-to 6-saturated membered ring containing an oxygen.
In some embodiments, the compound of Formula (Ia) is selected from the group consisting of the compounds having the following formulae:
Figure PCTCN2021123990-appb-000023
Figure PCTCN2021123990-appb-000024
Figure PCTCN2021123990-appb-000025
Figure PCTCN2021123990-appb-000026
In some embodiments, the heteroaryldihydropyrimidine (HAP) compound is a compound of Formula (Ib) or a stereoisomer or a tautomeric form thereof or a pharmaceutically acceptable salt or a solvate thereof:
Figure PCTCN2021123990-appb-000027
wherein in Formula (Ib) :
R 1 is hydrogen, halogen or C 1-6alkyl;
R 2 is hydrogen or halogen;
R 3 is hydrogen or halogen;
R 4 is C 1-6alkyl;
R 5 is hydrogen, hydroxy C 1-6alkyl, aminocarbonyl, C 1-6alkoxycarbonyl or carboxy;
R 6 is hydrogen, C 1-6alkoxycarbonyl or carboxy-C mH 2m-;
X is carbonyl or sulfonyl;
Y is -CH 2-, -O-or -N (R 7) -,
wherein R 7 is hydrogen, C 1-6alkyl, haloC 1-6alkyl, C 3-7cycloalkyl-C mH 2m-, C 1-6alkoxycarbonyl-C mH 2m-, -C tH 2t-COOH, -haloC 1-6alkyl-COOH, - (C 1-6alkoxy) C 1-6alkyl-COOH, -C 1-6alkyl-O-C 1-6alkyl-COOH, -C 3- 7cycloalkyl-C mH 2m-COOH, -C mH 2m-C 3-7cycloalkyl-COOH, hydroxy-C tH 2t-, carboxyspiro [3.3] heptyl or carboxyphenyl-C mH 2m-, carboxypyridinyl-C mH 2m-;
W is -CH 2-, -C (C 1-6alkyl)  2-, -O-or carbonyl;
n is 0 or 1;
m is 0-7; and
t is 1-7.
In some embodiments, the compound of formula (Ib) is selected from the group consisting of the compounds:
Methyl (4R) -4- (2-chloro-4-fluoro-phenyl) -6- [ (6-oxo-1, 3, 4, 8, 9, 9a-hexahydropyrazino [1, 2-c] [1, 3] oxazin-2-yl) methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -4- (2-chloro-4-fluoro-phenyl) -6- [ (4-oxo-6, 7, 9, 9a-tetrahydro-1H-pyrazino [2, 1-c] [1, 4] oxazin-8-yl) methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aR) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aS) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aS) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Ethyl (4R) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Ethyl (4S) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (3, 4-difluorophenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ (6-oxo-3, 4, 7, 8, 9, 9a-hexahydro-1H-pyrido [1, 2-a] pyrazin-2-yl) methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aR) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aS) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Ethyl (4R) -6- [ [ (8aS) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Ethyl (4R) -6- [ [ (8aR) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aR) -1, 3-dioxo-5, 6, 8, 8a-tetrahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8aS) -1, 3-dioxo-5, 6, 8, 8a-tetrahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (3aS) -1, 1-dioxo-2, 3, 3a, 4, 6, 7-hexahydro- [1, 2, 5] thiadiazolo [2, 3-a] pyrazin-5-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (3aR) -1, 1-dioxo-2, 3, 3a, 4, 6, 7-hexahydro- [1, 2, 5] thiadiazolo [2, 3-a] pyrazin-5-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] - 2, 2-dimethyl-propanoic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chlorophenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2-methyl-propanoic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2-methyl-propanoic acid;
3- [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
Ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ [2- (2-methoxy-1, 1-dimethyl-2-oxo-ethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Methyl 7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-2, 5, 6, 8-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8a-carboxylate;
(R) -6- [ (S) -2- (4-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
(R) -6- [ (S) -2- (4-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [ (S) -2- (3-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [ (S) -2- (2-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [ (S) -2- (3-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-1, 5, 6, 8-tetrahydroimidazo [1, 5-a] pyrazin-8a-yl] acetic acid;
2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-1, 5, 6, 8-tetrahydroimidazo [1, 5-a] pyrazin-8a-yl] acetic acid;
(R) -6- [ (S) -2- (1-Carboxy-l-methyl-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3-methyl-butanoic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3-methyl-butanoic acid;
l- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] cyclopropane carboxylic acid;
l- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] cyclopropane carboxylic acid;
3- [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3-methyl-butanoic acid;
[ [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] cyclopropane carboxylic acid;
3- [ (2S, 8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclobutanecarboxylic acid;
3- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclobutanecarboxylic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
3- [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4- dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
3- [ (8aS) -7- [ [ (4S) -4- (3-fluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid.
2- [l- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopropyl] acetic acid;
2- [l- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopropyl] acetic acid;
2- [l- [ (8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopropyl] acetic acid;
(1R, 2R) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
(1S, 2R) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
(1R, 2S) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
(1S, 2S) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] butanoic acid;
4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] - 2, 2-dimethyl-butanoic acid;
4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3, 3-dimethyl-butanoic acid;
(R) -6- [ (S) -2- (2-Carboxy-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [ (S) -2- ( (R) -2-Carboxy-l-methyl-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylicacid ethyl ester;
(R) -6- [ (S) -2- ( (S) -2-Carboxy-l-methyl-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [ (S) -2- (1-Carboxy-cyclobutylmethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-acid methyl ester;
6- [ (S) -2- (1-Carboxy-cyclobutylmethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- ( (R) -2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [ (S) -2- ( (1R, 3S) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
(R) -6- [ (S) -2- ( (R) - (S) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [ (S) -2- ( (1R, 3R) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
(R) -6- [ (S) -2- ( (1R, 3R) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
(R) -6- [2- (4-Carboxy-benzyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
(R) -6- [2- (4-Carboxy-benzyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
2- [2- [7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] ethoxy] acetic acid;
2- [3- [7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] propoxy] acetic acid;
methyl (4R) -4- (2-chloro-4-fluoro-phenyl) -6- [ [2- (5-hydroxy-4, 4-dimethyl-pentyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ [2- (2-hydroxyethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ [2- (2-hydroxyethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclohexanecarboxylic acid;
4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclohexanecarboxylic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chlorophenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
2- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] butanoic acid;
3- [ (8aS) -7- [ [ (4S) -5-ethoxycarbonyl-4- (3-fluoro-2-methyl-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
3- [ (8aS) -7- [ [4- (4-chlorophenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2-methoxy-propanoic acid;
2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] spiro [3.3] heptane-6-carboxylic acid;
5- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] pentanoic acid;
3- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] cyclobutene carboxylic acid;
(8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aS) -2-cyclopropyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -2-cyclopropyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aS) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2- yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aS) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aS) -2-tert-butyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidaza] pyrazine-8-carboxylic acid;
(8S, 8aR) -2-tert-butyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
methyl (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-2, 5, 6, 8-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8a-carboxylate;
2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -1, 1-dimethyl-3-oxo-6, 8-dihydro-5H-oxazolo [3, 4-a] pyrazin-8a-yl] acetic acid;
2- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -1, 1-dimethyl-3-oxo-6, 8-dihydro-5H-oxazolo [3, 4-a] pyrazin-8a-yl] acetic acid;
(8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-methyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-methyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
Methyl (4R) -6- [ [ (8R, 8aS) -2-tert-butyl-8-carbamoyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4- dihydropyrimidine-5-carboxylate;
Methyl (4R) -6- [ [ (8S, 8aR) -2-tert-butyl-8-carbamoyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-propoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-butanoic acid;
5- [7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] pyridine-2-carboxylic acid;
(S) -6- [ (S) -2- (2-Carboxy-2, 2-difluoro-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (3, 4-difluoro-2-methyl-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carbo acid ethyl ester;
(8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (cyclopropylmethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (cyclopropylmethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2- (4-methylthiazol-2-yl) -1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
2- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazine-1-carboxylic acid;
(8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isobutyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isobutyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8R, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazine-8-carboxylic acid;
(8S, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazine-8-carboxylic acid;
Ethyl (4R) -6- [ [ (8R, 8aS) -2-tert-butyl-8- (hydroxymethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Ethyl (4R) -6- [ [ (8S, 8aR) -2-tert-butyl-8- (hydroxymethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
Ethyl (4R) -6- [ [ (8aR) -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate; and
Ethyl (4R) -6- [ [ (8aS) -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
(8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (2, 2-difluroethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
(8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (2, 2-difluoroethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
Ethyl (4R) -6- [ [ (8aR) -2- (2-hydroxy-2-methyl-propyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate ;
and Ethyl (4R) -6- [ [ (8aS) -2- (2-hydroxy-2-methyl-propyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate.
In one embodiment, the heteroaryldihydropyrimidine (HAP) compound is selected from the group consisting of:
Figure PCTCN2021123990-appb-000028
Compounds of Formula (Ia) or (Ib) are capable of capsid assembly modulation.
Compounds of Formula (Ia) or (Ib) may modulate (e.g., accelerate, delay, inhibit, disrupt or reduce) normal viral capsid assembly or disassembly, bind to HBV core protein and capsid or alter metabolism of cellular polyproteins and precursors. The modulation may occur during the assembly process when the capsid protein is mature, or during viral infectivity. Compounds of Formula (Ia) or (Ib) may be used in methods of modulating the activity or properties of HBV cccDNA, or the generation or release of HBV RNA particles from within an infected cell.
Compounds of Formula (Ia) or (Ib) may accelerate the kinetics of HBV capsid assembly, thereby preventing or competing with the encapsidation of the Pol-pgRNA complex and thus blocking the reverse transcription of the pgRNA.
The compounds of Formula (Ia) or (Ib) may possess one or more stereocenters, and each stereocenter may exist independently in either R or S configuration. The stereochemical configuration may be assigned at indicated centers as (*R) , (*S) , (R*) or (S*) when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure. Compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. When the absolute R or S stereochemistry of a compound cannot be determined, it can be identified by the retention time after chromatography under  particular chromatographic conditions as determined by chromatographic column, eluent, etc. Additionally, the notation *S, and *R has been used herein to denote different atropoisomers, in the case where the atropoisomer (s) have been separated but the orientation has not been determined.
A stereoisomeric form of a compound refers to all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which are not interchangeable.
Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. A mixture of one or more isomers can be utilized as the disclosed compound described herein. Compounds described herein may contain one or more chiral centers. These compounds can be prepared by any means, including stereoselective synthesis, enantioselective synthesis or separation of a mixture of enantiomers or diastereomers. Resolution of compounds and isomers thereof can be achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
Compounds of Formula (Ia) and (Ib) are disclosed in WO2015/132276A1 and WO2020/125730A1. The references WO2015/132276A1 and WO2020/125730A1 are incorporated herein in their entirety, particularly the descriptions of the heteroaryldihydropyrimidine (HAP) compounds as capsid assembly modulators and the method of preparing them.
RNAi Agent Pharmaceutical Compositions and Formulations
In another aspect, described herein are methods for therapeutic and/or prophylactic treatment of diseases/disorders which are associated with HBV infection or inhibition of expression of one or more HBV genes comprising administering a pharmaceutical composition comprising one or more HBV RNAi agents that can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted  device) , and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.
In another aspect, methods described herein comprise one or more HBV RNAi agents, wherein the one or more HBV agents are prepared as pharmaceutical compositions or formulations. In some embodiments, pharmaceutical compositions include at least one HBV RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism. The pharmaceutical compositions can be used to treat a subject having a disease or disorder that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering an HBV RNAi agent linked to a targeting ligand as described herein, to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions including an HBV RNAi agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a human.
The pharmaceutical compositions that include an HBV RNAi agent and methods disclosed herein may decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, or subject, including: administering to the subject a therapeutically effective amount of a herein described HBV RNAi agent, thereby inhibiting the expression of a target mRNA in the subject.
In some embodiments, the described pharmaceutical compositions including an HBV RNAi agent are used for treating or managing clinical presentations associated with HBV infection. In some embodiments, a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment, prevention or management. In some embodiments, administration of any of the disclosed HBV RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
The described pharmaceutical compositions including an HBV RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of HBV mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions including an HBV RNAi agent thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more HBV RNAi agents, thereby preventing the at least one symptom.
The route of administration is the path by which an HBV RNAi agent is brought into contact with the body. In general, methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The HBV RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some embodiments, there herein described pharmaceutical compositions via subcutaneous injection.
The pharmaceutical compositions including an HBV RNAi agent described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a herein described compositions. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering) , systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal) , intramuscular, transdermal, airway (aerosol) , nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.
Accordingly, in some embodiments, the herein described pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.
As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., HBV RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.
Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol) , and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
The HBV RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc. ) . It is also envisioned that cells, tissues or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions. ” As used herein, “pharmacologically effective amount, ” “therapeutically effective amount, ” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic or preventive result.
Generally, an effective amount of an active compound will be in the range of from about 0.1 to about 100 mg/kg of body weight/day, e.g., from about 1.0 to about 50 mg/kg of body weight/day. In some embodiments, an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg/kg of body weight per dose. In some embodiments, an effective amount of an active compound will be in the range of 25-400 mg per 1-18 weeks or 1-6 months. In some embodiments, an effective amount of an active compound will be in the range of 50-125 mg per 4 weeks or per one month. In some embodiments, an effective amount of an active ingredient will be in the range of from about 0.5 to about 3 mg/kg of body weight per dose. In some embodiments, an effective amount of an active ingredient will be in the range of from about 25-400 mg per dose. In some embodiments, an effective amount of an active ingredient will be in the range of from about 50-125 mg per dose. The amount administered will also likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.
In some embodiments, an effective amount of the RNAi component is in the range of about 25-600 mg per dose. In some embodiments, an effective amount of the RNAi component is in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose. In some embodiments, an effective amount of the RNAi component is about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50  mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, an effective amount of the RNAi component is about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
The one or more (e.g., at least two) HBV RNAi agents described herein can be formulated into one single composition or separate individual compositions. In some embodiments, the HBV RNAi agents in separate individual compositions can be formulated with the same or different excipients and carriers. In some embodiments, the HBV RNAi agents in separate individual compositions agents can be administered through same or different administration routes. In some embodiments, the HBV RNAi agents are administered subcutaneously.
For treatment of disease or for formation of a medicament or composition for treatment of a disease, the pharmaceutical compositions described herein including an HBV RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and/or a vaccine.
The described HBV RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
In some embodiments, the composition comprises an effective amount of an RNAi component in the range of about 25-600 mg and an effective amount of a heteroaryldihydropyrimidine (HAP) compound in the range of about 75-600 mg per dose. In some embodiments, the composition comprises an effective amount of an RNAi component in the range of about 25-300 mg and an effective amount of a heteroaryldihydropyrimidine (HAP) compound in the range of about 75-300 mg per dose. In some embodiments, the composition comprises an effective amount of an RNAi of about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg and an effective amount of a heteroaryldihydropyrimidine (HAP) compound of about 100 mg, about 150 mg or about 250 mg per dose.
Heteroaryldihydropyrimidine (HAP) compound Pharmaceutical Compositions and  Formulations
In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is in the range of about 75-600 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is in the range of about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 150 mg or about 250 mg per dose.
The heteroaryldihydropyrimidine (HAP) compound, or a pharmaceutically acceptable salt thereof, or any subgroup thereof may be formulated into various pharmaceutical forms for administration purposes. As appropriate compositions there may be cited all compositions usually employed for systemically administering drugs. To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration orally, rectally, percutaneously, or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the  carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations intended to be converted, shortly before use, to liquid form preparations. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. The compounds of the present invention may also be administered via oral inhalation or insufflation in the form of a solution, a suspension or a dry powder using any art-known delivery system.
It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets) , capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions and the like, and segregated multiples thereof.
In some embodiments, the first and the second RNAi agents are each independently conjugated to (NAG25) or (NAG37) s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11, the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16 and the heteroaryldihydropyrimidine (HAP) compound is
Figure PCTCN2021123990-appb-000029
or a pharmaceutically acceptable salt thereof.
In some embodiments, the pharmaceutical combination further comprises another agent for treating infection caused by hepatitis B virus HBV. The other agent can be a nucleoside analog. In some embodiments, the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
The combinations described herein can be used in any methods or kits described.
In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof included in the combinations described herein are provided in separate containers. In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof included in the combinations described herein are provided in the same container. In some embodiments, the RNAi component includes a first RNAi agent in a first container and a second RNAi agent in a second container. For example, in an exemplary embodiment, the first RNAi agent is in a first container, the second RNAi agent is in a second container, and the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof is in a third container. In another embodiment, the RNAi component includes the first RNAi agent and the second RNAi agent in the same container. For example, in some embodiments, the first RNAi agent and the second RNAi agent are in a first container, and the heteroaryldihydropyrimidine (HAP) compound or pharmaceutically acceptable salt thereof is in a second container. Exemplary containers include vials, bags, tubes, or other suitable containers. In some embodiments, the contents of the container are sterile.
METHOD
Also provided herein is a method for treating a Hepatitis B viral (HBV) infection in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided herein is a method for enhancing an immune response in a subject with an HBV infection, wherein the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided herein is a method for decreasing viral replication in a subject with a HBV infection, wherein the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided  is a method for decreasing the expression of one or more HBV polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided herein is a method for increasing the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA in a subject with an HBV infection, the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided is a method for modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject in need thereof, the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound. Also provided is a method for inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof, the method comprises administering to the subject an effective amount of the RNAi component and the heteroaryldihydropyrimidine (HAP) compound.
In some embodiments, the HBV infection is a chronic HBV infection. In some embodiments, administering the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound decreases the immune tolerogenicity of the subject to HBV, more particularly the immune tolerogenicity of the liver of the subject to HBV. Decreasing the immune tolerogenicity of the subject to HBV or the immune tolerogenicity of the liver of the subject to HBV infection is characterized by the reactivation of the immune system in the subject with the chronic HBV infection. Reactivation of the immune system can occur after administration of the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound, which serves to decrease or reduce the level of HBsAg in the subject, which allows for the reactivation of the immune system.
In some embodiments, administering the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound at least until the subject meets at least one of, at least two of, at least three of, at least four of, or the five following features: (i) a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 IU/mL, more particularly is lower than 15 IU/mL, more particularly is lower than 10 IU/mL; (ii) a serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108  U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; (iii) a HBeAg-negative serum, (iv) a serum HBsAg level of 100 IU/mL or lower, more particularly of 10 IU/mL or lower; and (v) HBs seroconversion. In some embodiments, the at least one of, at least two of, at least three of, at least four of, or five of features (i) , (ii) , (iii) , (iv) , and (v) are still met six (6) months after the end of treatment.
In some embodiments, administering the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound at least until the subject meets at least one of, at least two of, at least three of, or four of the following features: (i) a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 IU/mL, more particularly is lower than 15 IU/mL, more particularly is lower than 10 IU/mL; (ii) a serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; (iii) a HBeAg-negative serum; and (iv) a serum HBsAg level of 100 IU/mL or lower, more particularly of 10 IU/mL or lower. In some embodiments, the at least one of, at least two of, at least three of, or four of features (i) , (ii) , (iii) , and (iv) are still met six (6) months after the end of treatment.
In some embodiments, the serum HBsAg level in the subject is reduced at a faster rate, is reduced at a greater level, and/or is reduced for a greater time period than in a subject in which only an effective amount of the RNAi component is administered or an effective amount of the heteroaryldihydropyrimidine (HAP) compound is administered.
In some embodiments, enhancing an immune response in the subject results in an increase in an innate immune system and/or an adaptive immune system. The increase in the innate immune system results in an increase in the expression of one or several from among an interferon stimulated gene (ISG) , an interferon gamma-induced protein 10 (IP10) , an interferon alpha (IFNα) , an interleukin 12 (IL12) , or an interleukin-6 (IL-6) .
In some embodiments, the increase in the adaptive immune system results in an increase in the level of HBV-specific T cell responses and/or an increased activation of the HBV-specific T cells. Activation of HBV-specific T cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., an interferon gamma ELISPOT) .
The increase in expression of the at least one of ISG, IP10, IFNα, IL12, or IL-6; the increase in the level of HBV-specific T cell responses; and/or the increased activation of HBV-specific T cells can be compared to a control. The control can be an expression level of ISG, IP10, IFNα, IL12, or IL-6; the level of HBV-specific T cell responses; the level of activation of HBV-specific T cells from a sample from the subject prior to administration of the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound. By way of another example, the control can be the expression level of ISG, IP10, IFNα, IL12, or IL-6; the level of HBV-specific T cell responses; and/or the level of activation of HBV-specific T cells from a subject administered the effective amount of the RNAi component, but not the effective amount of the heteroaryldihydropyrimidine (HAP) compound; or from a subject administered the effective amount of the heteroaryldihydropyrimidine (HAP) compound, but not the effective amount of the RNAi component. A person skilled in the art will understand the proper control for determining an increase in the expression level of ISG, IP10, IFNα, IL12, or IL-6; an increase in the level of HBV-specific T cell responses; and/or an increased activation level of HBV-specific T cells in the subject.
In some embodiments, enhancing an immune response in the subject results in an increase in the immunocompetence of the subject. An increase in the immunocompetence of the subject can, for example, result in an increase in the level of HBV-specific T cells, B cells, or NK cells in the liver and/or an increased activation of HBV-specific T cells, B cells, or NK cells in the liver. An increase in the immunocompetence of the subject can, for example, result in an increase in NK cells, T cells, or B cells in a peripheral immune cell compartment and/or an increased activation of NK cells, T cells, or B cells in the peripheral immune cell compartment. An increase in the immunocompetence of the subject can, for example, result in a decrease of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or in a  peripheral immune cell compartment; and/or a decreased immunosuppressive activity of MDSCs in the lever and/or in a peripheral immune cell compartment.
The increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the increase in the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or the decrease in the level of or decreased immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment can, for example, be determined by comparing to a control. The control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment from a sample from the subject prior to administration of the effective amount of the RNAi component and the effective amount of the heteroaryldihydropyrimidine (HAP) compound. By way of another example, the control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment from a subject administered the effective amount of the RNAi component, but not the effective amount of the heteroaryldihydropyrimidine (HAP) compound; or from a subject administered the effective amount of the heteroaryldihydropyrimidine (HAP) compound, but not the effective amount of the RNAi component. A person skilled in the art will understand the proper control for determining an increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; and/or a decrease in the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment in the subject.
Activation of HBV-specific NK cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., interferon gamma ELISPOT) . Activation of HBV-specific B cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by anti-HBs antibody production (e.g., anti-HBs ELISPOT) . The immunosuppressive activity of  MDSCs can be measured by any means that the person of average skill in the art may find appropriate, for example, by arginase expression.
In some embodiments, decreasing the viral replication in the subject results in a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 IU/mL, more particularly is lower than 15 IU/mL, more particularly is lower than 10 IU/mL.
In some embodiments, the targeted hepatocytes comprise cccDNA. In some embodiments, the targeted hepatocytes comprise integrated HBV DNA.
In some embodiments, the RNAi component comprises: (i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; and (ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
In some embodiments, the first RNAi agent comprises SEQ ID NO: 5 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 6 and SEQ ID NO: 14. In some embodiments, the first RNAi agent comprises SEQ ID NO: 7 and SEQ ID NO: 15. In some embodiments, the first RNAi agent comprises SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 3 and SEQ ID NO: 10, 11, or 13. In some embodiments, the first RNAi agent comprises SEQ ID NO: 4 and SEQ ID NO: 12. In some embodiments, the second RNAi agent comprises SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the second RNAi agent comprises SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 5 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent  comprising SEQ ID NO: 6 and SEQ ID NO: 14 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 7 and SEQ ID NO: 15 and a second RNAi agent comprising SEQ ID NO: 9 and SEQ ID NO: 19.
In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 1 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 3 and SEQ ID NO: 10, 11 or 13 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 4 and SEQ ID NO: 12 and a second RNAi agent comprising SEQ ID NO: 8 and SEQ ID NO: 16, 17 or 18.
In some embodiments, the two HBV RNAi agents are administered in a ratio of about 1: 1, 2: 1, 3: 1, 4: 1 or 5: 1. In some embodiments, the two HBV RNAi agents are administered in a ratio of about 2: 1.
In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the  ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-100 mg per dose administration and in the ratio of about 2: 1, about 3: 1, about 1: 1, about 4: 1, about 5: 1 or about 1: 2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-400 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 125-225 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 40 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi  agents are administered in a combined amount of about 50 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75 mg per dose administration and in the ratio of about 2: 1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200 mg per dose administration and in the ratio of about 2: 1.
In some embodiments, the first RNAi agent is administered in an amount of about 3-650 mg per dose administration, and the second RNAi agent is administered in an amount of about 2-325 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-150 mg per dose administration, and the second RNAi agent is administered in an amount of about 5-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 35-265 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 50-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-75 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 20-125 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 25-50 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 5-40 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 17 mg per dose administration, and the second RNAi agent is administered in an amount of about 8 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 23 mg per dose administration, and the second RNAi agent is administered in an amount of about 12 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 27 mg per dose administration, and the second RNAi agent is administered in an amount of about 13 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 33 mg per dose administration, and the second RNAi agent is administered in an amount of about 17 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 67 mg per dose administration, and the second RNAi agent is administered in an amount of about 33 mg per dose administration.
In some embodiments, two RNAi agents are administered at a combined dose of 25-400 mg per dose administration. In some embodiments, two RNAi agents are administered at a  combined dose of 25-400 mg, and the first RNAi agent is administered with the second RNAi agent at a ratio of 1: 1. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 12 mg for a combined dose of about 25 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 17 mg for a combined dose of about 35 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 20 mg for a combined dose of about 40 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 25 mg for a combined dose of about 50 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 50 mg for a combined dose of about 100 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 100 mg for a combined dose of about 200 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 150 mg for a combined dose of about 300 mg. In some embodiments, the dose of each of the first and second RNAi agents is in an amount of about 200 mg for a combined dose of about 400 mg.
In some embodiments, two RNAi agents are administered at a combined dose of 25-400 mg per dose, and the first RNAi agent is administered with the second RNAi agent at a ratio of 2: 1. In some embodiments, the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 24 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 35 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 65 mg, and the dose of the second RNAi agent is in an amount of about 35 mg for a combined dose of about 100 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 133 mg, and the dose of the second RNAi agent is in an amount of about 67 mg for a combined dose of about 200 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 200 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 300 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 270 mg,  and the dose of the second RNAi agent is in an amount of about 135 mg for a combined dose of about 400 mg.
In some embodiments, two RNAi agents are administered at a combined dose of 25-400 mg per dose, the first RNAi agent is administered with the second RNAi agent at a ratio of 3: 1. In some embodiments, the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 9 mg for a combined dose of about 35 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 75 mg, and the dose of the second RNAi agent is in an amount of about 25 mg for a combined dose of about 100 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 150 mg, and the dose of the second RNAi agent is in an amount of about 50 mg for a combined dose of about 200 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 225 mg, and the dose of the second RNAi agent is in an amount of about 75 mg for a combined dose of about 300 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 300 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 400 mg.
In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275-300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 25-75 mg, 50-100 mg, 100-150 mg, 150-200 mg, 200-250 mg, 250-300 mg, 300-350 mg, 350-400 mg, 25-100 mg, 50-150 mg, 100-200 mg, 150-250 mg, 200-300 mg, 300-400 mg, 25-200 mg, or 200-400 mg per dose administration. In some embodiments, the first RNAi agent to the second RNAi agent are administered in a combined amount of about 25 mg, about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg,  about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, or about 400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 50 mg, about 75 mg, about 100 mg, or about 125 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25 mg, about 35 mg, about 40 mg, or about 200 mg per dose administration.
In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-10 mg/kg per dose administration. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-5 mg/kg per dose administration. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-1.5 mg/kg, about 1.5-2.0 mg/kg, about 2.0-2.5 mg/kg, about 2.5-3.0 mg/kg, about 3.0-3.5 mg/kg, about 3.5-4.0 mg/kg, about 4.0-4.5 mg/kg, about 4.5-5.0 mg/kg, about 5.0-5.5 mg/kg, about 5.5-6.0 mg/kg, about 6.0-6.5 mg/kg, about 6.5-7.0 mg/kg, about 7.0-7.5 mg/kg, about 7.5-8.0 mg/kg, about 8.0-8.5 mg/kg, about 8.5-9.0 mg/kg, about 9.0-9.5 mg/kg, about 9.5-10 mg/kg, about 1-2.5 mg/kg, about 2.5-5.0 mg/kg, about 5.0-7.5 mg/kg, about 7.5-10 mg/kg, about 1-5.0 mg/kg, or about 5.0-10 mg/kg per dose administration.
In some embodiments, the first RNAi agent is administered in an amount of about 0.6-7 mg/kg per dose administration, and the second RNAi agent is administered in an amount of about 0.3-5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.5-2.5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.3-1.5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 0.6-5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 1-2.5 mg/kg per dose administration.
In some embodiments, the two RNAi agents are administered in about 1-18 week intervals. In some embodiments, the two RNAi agents are administered in about 1-week intervals, about 2-week intervals, about 3-week intervals, about 4-week intervals, about 5-week intervals, about 6-week intervals, about 7-week intervals, about 8-week intervals, about 9-week intervals, about 10-week intervals, about 11-week intervals, about 12-week intervals, about 13-week intervals, about 14-week intervals, about 15-week intervals, about 16-week  intervals, about 17-week intervals, or about 18-week intervals. In some embodiments, the two RNAi agents are administered in about 1-6 month intervals. In some embodiments, the two RNAi agents are administered in about 1-month intervals, about 2-month intervals, about 3-month intervals, about 4-month intervals, about 5-month intervals, or about 6-month intervals. In some embodiments, the two RNAi agents are administered in about 4-week intervals or 1-month intervals. In some embodiments, the two RNAi agents are administered once per month.
In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of about 1-12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of about 1-18 weeks. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, at least about 80 weeks, at least about 90 weeks, or at least 96 weeks.
In some embodiments, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg, and the first RNAi agent is administered with the second RNAi agent at a ratio of 1: 1. In some embodiments, the dose of the first RNAi agent is administered with the second RNAi agent is in an amount of about 12 mg for a combined dose of about 25 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 17 mg for a combined dose of about 35 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 20 mg for a combined dose of about 40 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 25 mg for a combined dose of about 50 mg. In  some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 50 mg for a combined dose of about 100 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 100 mg for a combined dose of about 200 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 150 mg for a combined dose of about 300 mg. In some embodiments, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 200 mg for a combined dose of about 400 mg.
In some embodiments, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, and the second RNAi agent is administered with the first RNAi agent at a ratio of 1: 2. In some embodiments, the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 12 mg, and the dose of the first RNAi agent is in an amount of about 24 mg for a combined dose of about 35 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 35 mg, and the dose of the first RNAi agent is in an amount of about 65 mg for a combined dose of about 100 mg. In some embodiments, the dose of v is in an amount of about 67 mg, and the dose of the first RNAi agent is in an amount of about 133 mg for a combined dose of about 200 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 200 mg for a combined dose of about 300 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 135 mg, and the dose of the first RNAi agent is in an amount of about 270 mg for a combined dose of about 400 mg.
In some embodiments, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, the second RNAi agent is administered with the first RNAi agent at a ratio of 1: 3. In some embodiments, the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg. In some embodiments, the dose of the second RNAi  agent is in an amount of about 9 mg, and the dose of the first RNAi agent is in an amount of about 27 mg for a combined dose of about 35 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In some embodiments, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 25 mg, and the dose of the first RNAi agent is in an amount of about 75 mg for a combined dose of about 100 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 50 mg, and the dose of the first RNAi agent is in an amount of about 150 mg for a combined dose of about 200 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 75 mg, and the dose of the first RNAi agent is in an amount of about 225 mg for a combined dose of about 300 mg. In some embodiments, the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 300 mg for a combined dose of about 400 mg.
In some embodiments, about 1 mg/kg (mpk) of the first RNAi agent and about 1 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of the first RNAi agent and about 1.5 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.2 mg/kg of the first RNAi agent and about 0.8 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.7 mg/kg of the first RNAi agent and about 1.3 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 4.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.3 mg/kg of the first RNAi agent and about 1.7 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, between about 0.05 and about 5 mg/kg of the first RNAi agent and between about 0.05 and about 5 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about the first RNAi agent and about the second RNAi agent are administered separately (e.g., in separate injections) . In some embodiments, the respective  dose of the first RNAi agent and the respective dose of the second RNAi agent are administered together (e.g., in the same injection) . In some embodiments, the respective dose of the first RNAi agent and the respective dose of the second RNAi agent are prepared in a single pharmaceutical composition.
In some embodiments, the RNAi component is administered to the subject once monthly in a dose of about 40-350 mg, such as about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg.
In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt is administered in the amount of about 100-500 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is in the range of about 100-500 mg, about 100-450 mg, about 100-400 mg, about 100-350 mg, about 100-300 mg, about 100-250 mg, about 100-200 mg, about 100-150 mg, about 150-500 mg, about 150-450 mg, about 150-400 mg, about 150-350 mg, about 150-300 mg, about 150-250 mg, about 150-200 mg, about 200-500 mg, about 200-450 mg, about 200-400 mg, about 200-350 mg, about 200-300 mg, about 200-250 mg, about 250-500 mg, about 250-450 mg, about 250-400 mg, about 250-350 mg, about 250-300 mg, about 300-500 mg, about 300-450 mg, about 300-400 mg, about 300-350 mg, about 350-500 mg, about 350-450 mg, about 350-400 mg, about 400-500 mg, about 400-450 mg, or about 450-500 mg. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg per dose. In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at  least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, at least about 80 weeks, at least about 90 weeks, or at least about 96 weeks. In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is administered for a duration of about 24 weeks or 48 weeks. In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is administered daily, every other day, once a week, twice a week, once every 2 weeks, once every 3 weeks, or once every month.
In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt is formulated in a solid form, such as a tablet or capsule. In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized. In some embodiments, the RNAi component is formulated in a solid form, such as a tablet or capsule. In some embodiments, the RNAi component is formulated for subcutaneous injection. In some embodiments, the RNAi component is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized.
In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are administered simultaneously or intermittently. In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are administered and formulated separately and administered with different dosing frequencies. In some embodiments, the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are formulated as one or separate compositions. In some embodiments, the RNAi component is formulated as a solution and administered once per month or once every four weeks via subcutaneous injection. In some embodiments, the heteroaryldihydropyrimidine (HAP) compound is formulated as an oral tablet or oral liquid solution and administered daily.
In some embodiments, the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, such as any of those described herein, is administered to the subject in a daily dose of about 100-500 mg, about 100-400 mg, about 100-300 mg, about 100-200 mg, about 200-500 mg, about 200-400 mg, about 200-300 mg, more particularly of about 200-300 mg, more particularly of about 250 mg.
In some embodiments, the first and the second RNAi agents are each independently conjugated to (NAG25) or (NAG37) s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11, the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16 and the heteroaryldihydropyrimidine (HAP) compound is
Figure PCTCN2021123990-appb-000030
or a pharmaceutically acceptable salt thereof.
In some embodiments, the method further comprises administering a nucleoside analog. In some embodiments, the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof. In some embodiments, the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.01-5 mg, about 0.01-0.05 mg, about 0.05-0.1 mg, about 0.1-0.5 mg, about 0.5-1 mg, about 1-2 mg, about 2-3 mg, about 3-4 mg or about 4-5 mg. In some embodiments, the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.5 mg. In some embodiments, the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 100-500 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, 300-400 mg, about 400-500 mg. In some embodiments, the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 300 mg. In some embodiments, the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 5-100 mg, about 5-25 mg, about 25-50 mg, about 50-75 or about 75-100 mg. In some embodiments, the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 25 mg. In some embodiments, the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of about 50-600 mg, about 50-300 mg, about 100-300 mg, about 100-500 mg, about 150-400 mg, about 200-350, or about  250-300 mg. In some embodiments, the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of 100 mg, 150 mg, or 300 mg. In some embodiments, the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of about 300-800 mg, about 400-700 mg, about 300-600 mg, about 300-400 mg, about 400-500 mg, or about 500-600 mg. In some embodiments, the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of 600 mg. In some embodiments, the patients have been exposed to the nucleoside analog prior to the combination therapy. In some embodiments, the patients have been administered the nucleoside analog for at least 1 month, at least 3 months, at least 6 months, or at least 1 year prior to receiving the combination therapy.
In some embodiments, the patients are screened for HBeAg status prior to administration of the combination therapy. In some embodiments, the patients are HBeAg positive. In some embodiments, the patients are HBeAg negative. In some embodiment, the patients are screened for immune tolerance prior to administration of the combination therapy.
In some embodiments, the HBsAg level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1. In some embodiments, the HBeAg level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1. In some embodiments, the HBcrAg level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1. In some embodiments, the HBV DNA level in the patient is reduced by at least about log 10 0.5, about log 10 1, about log 10 1.5, about log 10 2, about log 10 3, about log 10 4, about log 10 5 or about log 10 7.5 from base line on Day 1. In some embodiments, the HBV RNA level in the patient is reduced by at least about log 10 0.5, about log 10 0.75, about log 10 1, about log 10 1.25, about log 10 1.5, about log 10 1.75, about log 10 2 or about log 10 2.5 from base line on Day 1.
KIT
Provided herein is a kit comprising an effective amount of the RNAi component and an effective amount of the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof.
The described HBV RNAi agents and the heteroaryldihydropyrimidine (HAP) compounds may be presented in the form of a kit such as when added to pharmaceutically acceptable excipients or adjuvants, and packaged into kits, containers, packs, or dispensers. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
The kit can comprise any combinations or compositions described herein.
In another aspect, the kit further comprises a package insert including, without limitation, appropriate instructions for preparation and administration of the formulation, side effects of the formulation, and any other relevant information. The instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, optical disc or directions to internet-based instructions.
In another aspect, kits for treating an individual who suffers from or is susceptible to the conditions described herein are provided, comprising a first container comprising a dosage amount of a composition or formulation as disclosed herein, and a package insert for use. The container can be any of those known in the art and appropriate for storage and delivery of intravenous formulation. In certain embodiments, the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for preparation of the formulation to be administered to the individual.
In some embodiments, the kit comprises one or more doses of the heteroaryldihydropyrimidine (HAP) compound in the range of about 100-500 mg per dose. In some embodiments, the kit comprises one or more doses of the heteroaryldihydropyrimidine (HAP) compound in the range of about 100-500 mg, about 100-450 mg, about 100-400 mg, about 100-350 mg, about 100-300 mg, about 100-250 mg, about 100-200 mg, about 100-150 mg, about 150-500 mg, about 150-450 mg, about 150-400 mg, about 150-350 mg, about 150-300 mg, about 150-250 mg, about 150-200 mg, about 200-500 mg, about 200-450 mg, about 200-400 mg, about 200-350 mg, about 200-300 mg, about 200-250 mg, about 250-500 mg, about 250-450 mg, about 250-400 mg, about 250-350 mg, about 250-300 mg, about 300-500 mg, about 300-450 mg, about 300-400 mg, about 300-350 mg, about 350-500 mg, about 350-450 mg, about 350-400 mg, about 400-500 mg, about 400-450 mg, or about 450-500 mg. In  some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg per dose. In some embodiments, an effective amount of the heteroaryldihydropyrimidine (HAP) compound is about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg per dose.
In some embodiments, the kit comprises one or more doses of the RNAi component in the range of about 25-600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component of about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
In some embodiments, the kit contains an RNAi component useful for the invention, such as those described herein, for once monthly (or every four weeks) administration to a subject in a dose of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg; more particularly 200 mg; and a heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, such as those described herein, for administration to a subject in a dose of about 0.6-3.0 mg, about 0.6-2.5 mg, about 0.6-2.0 mg, more particularly of about 100-500 mg, about 100-400 mg, about 100-300 mg, about 100-200 mg, about 200- 500 mg, about 200-400 mg, about 200-300 mg, or about 250 mg, wherein said dose is administered daily.
In some embodiments, the kit further comprises instructions for using the RNAi component and the heteroaryldihydropyrimidine (HAP) compound contained therein for administration to treat a subject with an HBV infection, in particular, a subject having a chronic HBV infection. In some embodiments, the kit further comprises instructions for using the RNAi component and the heteroaryldihydropyrimidine (HAP) compound contained therein for administration to a subject with an HBV infection to enhance the immune response, to decrease viral replication, to decrease expression of one or more HBV polypeptide (s) , to modulate Hepatitis B viral (HBV) capsid assembly or disassembly, and/or to increase the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA.
In another aspect, kits may also be provided that contain sufficient dosages of the compositions described herein (including pharmaceutical compositions thereof) to provide effective treatment for an individual for an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles or more. In some embodiments, one cycle of treatment is about 1-24 months, about 1-3 months, about 3-6 months, about 6-9 months, about 9-12 months, about 12-18 months, about 18-21 months or about 21-24 months. In some embodiments, one cycle of treatment is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months or about 24 months.
In some embodiments, the kits can also include multiple doses and may be packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies. In certain embodiments the kits may include a dosage amount of at least one composition as disclosed herein.
The application also relates to an effective amount of an RNAi component and a heteroaryldihydropyrimidine (HAP) compound, optionally a nucleoside analog, each of which as that described herein, in the manufacture of a medicament for treating a Hepatitis B  viral (HBV) infection in a subject; enhancing an immune response in a subject with an HBV infection; decreasing viral replication in a subject with HBV; decreasing the expression of one or more HBV polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof; modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject with a HBV infection; and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA in a subject with a HBV infection. In some embodiments, the RNAi component is for once monthly or once every four weeks administration to a subject in a dose of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg; the heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof is for administration to a subject in a dose of about 100-500 mg, particularly of 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg, more particularly of 250 mg, wherein said dose is administered daily. In some embodiment, the medicament is for administering to a subject infected by HBV, in particular, a subject having a chronic HBV infection.
BRIEF DESCRIPTION OF THE FIGURE
Fig. 1 indicates the treatment schedule and the dosage of each animal group of the examples.
EXAMPLES
Combination
The combination comprised Compound A having the following structure and an RNAi component comprising a first RNAi agent comprising SEQ ID NO: 2 and SEQ ID NO: 11 and a second RNAi agent comprising SEQ ID NO: 16 and SEQ ID NO: 8 in a ratio of 2: 1. Compound A can be prepared according to WO2020125730A1 and the RNAi component can be prepared according to WO2018027106A1.
Figure PCTCN2021123990-appb-000031
Compound A
Method
C57BL/6 mice (male, approximately 4-5 weeks old, commercially available from Shanghai Lingchang Bio Tech co., Ltd, Shanghai, China) received a single tail vein injection with 200μL PBS containing 1x10 11vg rAAV-HBV virus (this recombinant virus carries 1.3 copies of the HBV genome (genotype D; serotype ayw) and was packaged in AAV serotype 8 (AAV8) capsids, commercially available from Beijing FivePlus Molecular Medicine Institute, China) . Plasma samples were obtained from mice by submandibular vein bleeding at various time point after injection, to monitor HBsAg, HBeAg and HBV genomic DNA. This model can mimic chronic HBV infection. Stable HBV replication and continuous expression of HBV antigens are usually established after 4 weeks of the injection with rAAV-HBV, at which time the model is ready for compound dosing.
Based on HBV-DNA, HBsAg and HBeAg levels and bodyweight, rAAV-HBV-infected mice were selected, and group randomized into four matched groups as described in Table 4. Compound A was formulated with 2-hydroxypropyl-β-cyclodextrin in PBS to form the Compound A formulation. The RNAi component was formulated in PBS to form the RNAi component formulation.
Vehicle A (Group 1, vehicle of the Compound A formulation) and the Compound A formulation (Group 2 and Group 4) were dosed once daily by oral gavage with a volume of 10 mL/kg during Dosing Day 0 ～ 83. The dosage of Compound A is 12 mg/kg.
Vehicle B (Group 1, vehicle of the RNAi component formulation) and the RNAi component formulation (Group 3 and Group 4) were dosed subcutaneously with a volume of 3 mL/kg on  Dosing Day  0, 21, 42 and 63. The dosage of the RNAi component is 3 mg/kg.
The treatment schedule and the dosage of each animal group are listed in Table 4 and Fig 1.
Table 4. Group and dose information
Figure PCTCN2021123990-appb-000032
Abbreviations: Tx Grp = Treatment group, Vol = Volume, No. An = Number of animals, QD = Once daily, Q3W = Once every three weeks; SC = Subcutaneous (injection) , PO = Oral Gavage
Mice in all groups were bled for plasma preparation every week to quantify HBsAg, HBeAg, HBcrAg and HBV DNA. Blood was collected by submandibular vein bleeding without anesthesia and transferred into pre-labeled tubes containing EDTA-K2, followed by centrifuging (3500 g for 5 minutes at 4℃) within half an hour of collection to isolate plasma. Plasma was diluted 60-fold in DPBS for HBsAg and HBeAg quantification, and diluted 200 fold in PBS for HBcrAg. HBsAg levels were determined with AlphaLISA as describe in Antimicrob Agents Chemother, 2017, Jul 25; 61 (8) : e00560-17 (doi: 10.1128/AAC. 00560-17) . HBeAg levels were determined with Autobio ELISA kit (05150312) as described by the manufacturer. HBcrAg levels were determined with Lumipulse CLEIA (Fujirebio) . HBV DNA was isolated from plasma using QIAamp 96 Virus QIAcube HT Kit (Qiagen, 57731) following the manufacturer’s instructions. The number of copies of HBV DNA was determined by LightCycler 480 Real-Time PCR as described in Antiviral Res, 2017 Jun 21; 144: 205 (doi: 10.1016/j. antiviral. 2017.06.016) .
Results
Table 5. Average HBeAg levels (Mean, Log PEIU/ml) and standard deviation (SD) of mice in each group following the administration
Figure PCTCN2021123990-appb-000033
HBeAg reduction was observed in  mouse Group  2, 3 and 4 (Table 5) among which Group 4 induced the fastest HBeAg reduction.
Table 6. Average HBcrAg levels (Mean, Log U/ml) and standard deviation (SD) of mice in each group following the administration
Figure PCTCN2021123990-appb-000034
HBcrAg reduction was observed in  mouse Group  2, 3 and 4 (Table 6) among which Group 4 induced the fastest HBcrAg reduction.
Table 7. Average HBsAg levels (Mean, Log ng/ml) and standard deviation (SD) of mice in each group following the administration
Figure PCTCN2021123990-appb-000035
HBsAg reduction was observed in  mouse Group  2, 3 and 4 (Table 7) . Group 4 induced faster HBsAg redction compared with Group 2, and smaller individual variability compared with Group 3.
Table 8. Average HBV DNA levels (Mean, Log copies/μl) and standard deviation (SD) of mice in each group following the administration
Figure PCTCN2021123990-appb-000036
2.120 [Log copies/μl] is the lower limit of quantification (LLOQ) here.
HBV DNA was significantly reduced in  mouse Group  2, 3 and 4 (Table 8) . HBV DNA was efficiently reduced under LLOQ in Group 2 and Group 4.

Claims (17)

  1. A pharmaceutical combination comprising an RNAi component and a heteroaryldihydropyrimidine (HAP) compound,
    wherein:
    (a) the RNAi component comprises
    (i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; and
    (ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 8 and SEQ ID NO: 9, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; and
    (b) the heteroaryldihydropyrimidine (HAP) compound is a compound of Formula (Ia) or a compound of Formula (Ib) or a stereoisomer or a tautomeric form thereof or a pharmaceutically acceptable salt or a solvate thereof:
    Figure PCTCN2021123990-appb-100001
    wherein in Formula (Ia) :
    R 1 is selected from the group consisting of phenyl, thiophenyl, pyridyl, and pyridonyl, optionally substituted with one or more substituents selected from the group consisting of C 1- 4alkyl, halogen and CN;
    R 2 is C 1-4alkyl;
    R 3 is selected from the group consisting of thiazolyl, pyridyl, and oxazolyl, optionally  substituted with one or more substituents selected from fluorine and C 1-6alkyl;
    n is an integer of 0 or 1;
    R 4 and R 5 are independently selected from H and -COOH;
    Figure PCTCN2021123990-appb-100002
    is a single bond or a double bond;
    when X and Y are linked by a single bond, X is selected from the group consisting of C (=S) , C (=NR 6) , C (=CHR 7) and CHR 8, and Y is NR 9;
    when X and Y are linked by a double bond, X is C-SR 9 or C-OR 9, and Y is N atom;
    Z is selected from the group consisting of CH 2, and C (=O) ;
    R 6 is selected from the group consisting of CN, C (=O) CH 3 and SO 2CH 3;
    R 7 is CN;
    R 8 is CF 3;
    R 9 is selected from the group consisting of H, -C 1-6alkyl, -C 1-6alkyl-R 10, -C 1-6alkoxy-C 1- 6alkyl-R 10, - (CH 2)  p-C (R 11R 12) -R 10 and - (CH 2)  p-Q-R 10;
    p is an integer of 0, 1, 2, or 3;
    R 11 and R 12 together with carbon atom to which they are attached form a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with one or more substituents selected from the group consisting of with H, -C 1-6alkyl, -C 1-6alkoxy-C 1-6alkyl and -C 1-6alkylcarbonyl;
    Q is selected from the group consisting of aryl, heteroaryl, and a 3-to 7-saturated membered ring, optionally containing a heteroatom, the heteroatom being an oxygen or a nitrogen, the nitrogen being substituted with H, -C 1-6alkyl, -C 1-6alkoxy-C 1-6alkyl and -C 1-6alkylcarbonyl;
    R 10 is selected from -COOH, -C (=O) NHS (=O)  2-C 1-6alkyl, tetrazolyl, and carboxylic acid bioisosteres;
    Figure PCTCN2021123990-appb-100003
    wherein in Formula (Ib) :
    R 1 is hydrogen, halogen or C 1-6alkyl;
    R 2 is hydrogen or halogen;
    R 3 is hydrogen or halogen;
    R 4 is C 1-6alkyl;
    R 5 is hydrogen, hydroxy C 1-6alkyl, aminocarbonyl, C 1-6alkoxycarbonyl or carboxy;
    R 6 is hydrogen, C 1-6alkoxycarbonyl or carboxy-C mH 2m-;
    X is carbonyl or sulfonyl;
    Y is -CH 2-, -O-or -N (R 7) -,
    wherein R 7 is hydrogen, C 1-6alkyl, haloC 1-6alkyl, C 3-7cycloalkyl-C mH 2m-, C 1-6alkoxycarbonyl-C mH 2m-, -C tH 2t-COOH, -haloC 1-6alkyl-COOH, - (C 1-6alkoxy) C 1-6alkyl-COOH, -C 1-6alkyl-O-C 1-6alkyl-COOH, -C 3-7cycloalkyl-C mH 2m-COOH, -C mH 2m-C 3-7cycloalkyl-COOH, hydroxy-C tH 2t-, carboxyspiro [3.3] heptyl or carboxyphenyl-C mH 2m-, carboxypyridinyl-C mH 2m-;
    W is -CH 2-, -C (C 1-6alkyl)  2-, -O-or carbonyl;
    n is 0 or 1;
    m is 0-7; and
    t is 1-7.
  2. The pharmaceutical combination of claim 1, wherein
    (1) the first or the second RNAi agent comprises at least one modified nucleotide and/or at least one modified internucleoside linkage,
    (2) at least 90%of the nucleotides in the first and the second RNAi agents are modified nucleotides,
    (3) the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and /or
    (4) the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1: 2 to about 5: 1.
  3. The pharmaceutical combination of any one of the preceding claims, wherein the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
    (a) an antisense strand comprising SEQ ID NO: 1 and a sense strand comprising SEQ ID NO: 10;
    (b) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO:  11;
    (c) an antisense strand comprising SEQ ID NO: 3 and a sense strand comprising SEQ ID NO: 11;
    (d) an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12;
    (e) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16;
    (f) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 17;
    (g) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 12; and
    (h) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 18.
  4. The pharmaceutical combination of any one of the preceding claims, wherein
    the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and
    the targeting ligand comprises N-acetyl-galactosamine,
    preferably selected from the group consisting of (NAG13) , (NAG13) s, (NAG18) , (NAG18) s, (NAG24) , (NAG24) s, (NAG25) , (NAG25) s, (NAG26) , (NAG26) s, (NAG27) , (NAG27) s, (NAG28) , (NAG28) s, (NAG29) , (NAG29) s, (NAG30) , (NAG30) s, (NAG31) , (NAG31) s, (NAG32) , (NAG32) s, (NAG33) , (NAG33) s, (NAG34) , (NAG34) s, (NAG35) , (NAG35) s, (NAG36) , (NAG36) s, (NAG37) , (NAG37) s, (NAG38) , (NAG38) s, (NAG39) , and (NAG39) s,
    particularly preferably selected from the group consisting of (NAG25) , (NAG25) s, (NAG31) , (NAG31) s, (NAG37) , and (NAG37) s.
  5. The pharmaceutical combination of any one of the preceding claims, wherein
    the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent, and
    the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent, preferably the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent.
  6. The pharmaceutical combination of any one of the preceding claims, wherein
    the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
    (a) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 11;
    (b) an antisense strand comprising SEQ ID NO: 4 and a sense strand comprising SEQ ID NO: 12;
    (c) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 16;
    (d) an antisense strand comprising SEQ ID NO: 2 and a sense strand comprising SEQ ID NO: 13; and
    (e) an antisense strand comprising SEQ ID NO: 8 and a sense strand comprising SEQ ID NO: 18.
  7. The pharmaceutical combination of any one of the preceding claims, wherein the heteroaryldihydropyrimidine (HAP) compound is selected from the group consisting of:
    Figure PCTCN2021123990-appb-100004
    Figure PCTCN2021123990-appb-100005
    Figure PCTCN2021123990-appb-100006
    Figure PCTCN2021123990-appb-100007
    Figure PCTCN2021123990-appb-100008
  8. The pharmaceutical combination of any one of the preceding claims 1-6, wherein the heteroaryldihydropyrimidine (HAP) compound is selected from the group consisting of:
    Methyl (4R) -4- (2-chloro-4-fluoro-phenyl) -6- [ (6-oxo-1, 3, 4, 8, 9, 9a-hexahydropyrazino [1, 2-c] [1, 3] oxazin-2-yl) methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -4- (2-chloro-4-fluoro-phenyl) -6- [ (4-oxo-6, 7, 9, 9a-tetrahydro-1H-pyrazino [2, 1-c] [1, 4] oxazin-8-yl) methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aR) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aS) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aS) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Ethyl (4R) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Ethyl (4S) -6- [ [ (8aR) -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazin-2-yl] methyl] -4- (3, 4-difluorophenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ (6-oxo-3, 4, 7, 8, 9, 9a-hexahydro-1H-pyrido [1, 2-a] pyrazin-2-yl) methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aR) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aS) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Ethyl (4R) -6- [ [ (8aS) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Ethyl (4R) -6- [ [ (8aR) -3-oxo-1, 2, 5, 6, 8, 8a-hexahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-bromo-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aR) -1, 3-dioxo-5, 6, 8, 8a-tetrahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8aS) -1, 3-dioxo-5, 6, 8, 8a-tetrahydroimidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (3aS) -1, 1-dioxo-2, 3, 3a, 4, 6, 7-hexahydro- [1, 2, 5] thiadiazolo [2, 3-a] pyrazin-5-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (3aR) -1, 1-dioxo-2, 3, 3a, 4, 6, 7-hexahydro- [1, 2, 5] thiadiazolo [2, 3-a] pyrazin-5-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chlorophenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2-methyl-propanoic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2-methyl-propanoic acid;
    3- [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4- dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    Ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ [2- (2-methoxy-1, 1-dimethyl-2-oxo-ethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl 7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-2, 5, 6, 8-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8a-carboxylate;
    (R) -6- [ (S) -2- (4-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
    (R) -6- [ (S) -2- (4-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [ (S) -2- (3-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [ (S) -2- (2-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [ (S) -2- (3-Carboxy-phenyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
    2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-1, 5, 6, 8-tetrahydroimidazo [1, 5-a] pyrazin-8a-yl] acetic acid;
    2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-1, 5, 6, 8-tetrahydroimidazo [1, 5-a] pyrazin-8a-yl] acetic acid;
    (R) -6- [ (S) -2- (1-Carboxy-l-methyl-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3-methyl-butanoic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3- methyl-butanoic acid;
    l- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] cyclopropane carboxylic acid;
    l- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] cyclopropane carboxylic acid;
    3- [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3-methyl-butanoic acid;
    [ [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] cyclopropane carboxylic acid;
    3- [ (2S, 8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclobutanecarboxylic acid;
    3- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclobutanecarboxylic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    3- [ (8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    3- [ (8aS) -7- [ [ (4S) -4- (3-fluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid.
    2- [l- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopropyl] acetic acid;
    2- [l- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopropyl] acetic acid;
    2- [l- [ (8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopropyl] acetic acid;
    (1R, 2R) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
    (1S, 2R) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
    (1R, 2S) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
    (1S, 2S) -2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclopentanecarboxylic acid;
    4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] butanoic acid;
    4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-butanoic acid;
    4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -3, 3-dimethyl-butanoic acid;
    (R) -6- [ (S) -2- (2-Carboxy-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [ (S) -2- ( (R) -2-Carboxy-l-methyl-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5- carboxylicacid ethyl ester;
    (R) -6- [ (S) -2- ( (S) -2-Carboxy-l-methyl-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [ (S) -2- (1-Carboxy-cyclobutylmethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-acid methyl ester;
    6- [ (S) -2- (1-Carboxy-cyclobutylmethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- ( (R) -2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [ (S) -2- ( (1R, 3S) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
    (R) -6- [ (S) -2- ( (R) - (S) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [ (S) -2- ( (1R, 3R) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
    (R) -6- [ (S) -2- ( (1R, 3R) -3-Carboxy-cyclopentyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    (R) -6- [2- (4-Carboxy-benzyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid methyl ester;
    (R) -6- [2- (4-Carboxy-benzyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carboxylic acid ethyl ester;
    2- [2- [7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] ethoxy] acetic acid;
    2- [3- [7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] propoxy] acetic acid;
    methyl (4R) -4- (2-chloro-4-fluoro-phenyl) -6- [ [2- (5-hydroxy-4, 4-dimethyl-pentyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4- dihydropyrimidine-5-carboxylate;
    ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ [2- (2-hydroxyethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    ethyl (4R) -4- (2-chloro-3-fluoro-phenyl) -6- [ [2- (2-hydroxyethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclohexanecarboxylic acid;
    4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] cyclohexanecarboxylic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chlorophenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    2- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] methyl] butanoic acid;
    3- [ (8aS) -7- [ [ (4S) -5-ethoxycarbonyl-4- (3-fluoro-2-methyl-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    3- [ (8aS) -7- [ [4- (4-chlorophenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2-methoxy-propanoic acid;
    2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] spiro [3.3] heptane-6-carboxylic acid;
    5- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] pentanoic acid;
    3- [ [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2- yl] methyl] cyclobutene carboxylic acid;
    (8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-cyclopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aS) -2-cyclopropyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -2-cyclopropyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aS) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aS) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aS) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -2-tert-butyl-7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aS) -2-tert-butyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidaza] pyrazine-8-carboxylic acid;
    (8S, 8aR) -2-tert-butyl-7- [ [ (4S) -4- (3, 4-difluoro-2-methyl-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    methyl (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-2, 5, 6, 8-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8a-carboxylate;
    2- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -1, 1-dimethyl-3-oxo-6, 8-dihydro-5H-oxazolo [3, 4-a] pyrazin-8a-yl] acetic acid;
    2- [ (8aR) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -1, 1-dimethyl-3-oxo-6, 8-dihydro-5H-oxazolo [3, 4-a] pyrazin-8a-yl] acetic acid;
    (8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-methyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-methyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    Methyl (4R) -6- [ [ (8R, 8aS) -2-tert-butyl-8-carbamoyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Methyl (4R) -6- [ [ (8S, 8aR) -2-tert-butyl-8-carbamoyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-4-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-propoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    4- [ (8aS) -7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4- dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-butanoic acid;
    5- [7- [ [ (4R) -4- (2-chloro-4-fluoro-phenyl) -5-methoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] pyridine-2-carboxylic acid;
    (S) -6- [ (S) -2- (2-Carboxy-2, 2-difluoro-ethyl) -3-oxo-hexahydro-imidazo [1, 5-a] pyrazin-7-ylmethyl] -4- (3, 4-difluoro-2-methyl-phenyl) -2-thiazol-2-yl-1, 4-dihydro-pyrimidine-5-carbo acid ethyl ester;
    (8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (cyclopropylmethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (cyclopropylmethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    3- [ (8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2- (4-methylthiazol-2-yl) -1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-2-yl] -2, 2-dimethyl-propanoic acid;
    2- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -6-oxo-1, 3, 4, 7, 8, 8a-hexahydropyrrolo [1, 2-a] pyrazine-1-carboxylic acid;
    (8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isobutyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2-isobutyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8R, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazine-8-carboxylic acid;
    (8S, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-diydropyrimidin-6-yl] methyl] -3-oxo-5, 6, 8, 8a-tetrahydro-1H-oxazolo [3, 4-a] pyrazine-8-carboxylic acid;
    Ethyl (4R) -6- [ [ (8R, 8aS) -2-tert-butyl-8- (hydroxymethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4- dihydropyrimidine-5-carboxylate;
    Ethyl (4R) -6- [ [ (8S, 8aR) -2-tert-butyl-8- (hydroxymethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    Ethyl (4R) -6- [ [ (8aR) -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate; and
    Ethyl (4R) -6- [ [ (8aS) -2-isopropyl-3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    (8R, 8aS) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (2, 2-difluroethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    (8S, 8aR) -7- [ [ (4R) -4- (2-chloro-3-fluoro-phenyl) -5-ethoxycarbonyl-2-thiazol-2-yl-1, 4-dihydropyrimidin-6-yl] methyl] -2- (2, 2-difluoroethyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazine-8-carboxylic acid;
    Ethyl (4R) -6- [ [ (8aR) -2- (2-hydroxy-2-methyl-propyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate;
    and Ethyl (4R) -6- [ [ (8aS) -2- (2-hydroxy-2-methyl-propyl) -3-oxo-5, 6, 8, 8a-tetrahydro-1H-imidazo [1, 5-a] pyrazin-7-yl] methyl] -4- (2-chloro-3-fluoro-phenyl) -2-thiazol-2-yl-1, 4-dihydropyrimidine-5-carboxylate.
  9. The pharmaceutical combination of any one of the preceding claims 1-6, wherein the heteroaryldihydropyrimidine (HAP) compound is selected from the group consisting of:
    Figure PCTCN2021123990-appb-100009
  10. The pharmaceutical combination of any one of the preceding claims, wherein the combination further comprises a nucleoside analog, preferably the nucleoside is selected from the group consisting of entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, and telbivudine, or a combination thereof.
  11. The pharmaceutical combination of any one of the preceding claims, for a use selected from the group consisting of:
    (1) treating a Hepatitis B viral (HBV) infection in a subject;
    (2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
    (3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
    (4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
    (5) increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
    (6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
    (7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof.
  12. Use of an RNAi component and a heteroaryldihydropyrimidine (HAP) compound in the manufacture of a pharmaceutical combination for a use selected from the group consisting of:
    (1) treating a Hepatitis B viral (HBV) infection in a subject;
    (2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
    (3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
    (4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
    (5) increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
    (6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
    (7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof;
    wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound and the pharmaceutical combination are as defined in any one of the preceding claims 1-11.
  13. A method for a use selected from the group consisting of:
    (1) treating a Hepatitis B viral (HBV) infection in a subject;
    (2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
    (3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
    (4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
    (5) for increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
    (6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
    (7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof;
    wherein the method comprises administering to the subject an RNAi component and a heteroaryldihydropyrimidine (HAP) compound;
    wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as above defined in any one of the preceding claims 1-11.
  14. An RNAi component and a heteroaryldihydropyrimidine (HAP) compound for a use selected from the group consisting of:
    (1) treating a Hepatitis B viral (HBV) infection in a subject;
    (2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
    (3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
    (4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
    (5) for increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
    (6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
    (7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof;
    wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as defined in any one of the preceding claims 1-11.
  15. An RNAi component for a use selected from the group consisting of:
    (1) treating a Hepatitis B viral (HBV) infection in a subject;
    (2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
    (3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
    (4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
    (5) for increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
    (6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
    (7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof;
    wherein the RNAi component is to be administered in combination therapy with a heteroaryldihydropyrimidine (HAP) compound,
    wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as defined in any one of the preceding claims 1-11.
  16. A heteroaryldihydropyrimidine (HAP) compound for a use selected from the group consisting of:
    (1) treating a Hepatitis B viral (HBV) infection in a subject;
    (2) enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection;
    (3) decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection;
    (4) decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide (s) , more particularly of one or more polypeptide (s) selected from HBsAg and HBeAg, in a subject in need thereof;
    (5) for increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a Hepatitis B viral (HBV) infection;
    (6) modulating Hepatitis B Virus (HBV) viral capsid assembly or disassembly in a subject with an HBV infection;
    (7) inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof;
    wherein the heteroaryldihydropyrimidine (HAP) compound is to be administered in combination therapy with a RNAi component,
    wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as defined in any one of the preceding claims 1-11.
  17. A kit comprising an effective amount of an RNAi component and a heteroaryldihydropyrimidine (HAP) compound or a pharmaceutically acceptable salt thereof, wherein the RNAi component and the heteroaryldihydropyrimidine (HAP) compound are as defined in any one of the preceding claims 1-11.
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