A pharmaceutical composition and uses thereof

Abstract

A pharmaceutical composition comprising a pharmaceutically active component, wherein the pharmaceutically active component consists of an RNAi agent and an immune response modulator, the RNAi agent and the immune response modulator being present independently; the RNAi agent refers to one or more of an siRNA composition, an siRNA conjugate, and pharmaceutically acceptable salts thereof; the siRNA composition contains siRNA and a pharmaceutically acceptable carrier; the siRNA conjugate contains an siRNA group and a conjugate group conjugated to the siRNA group; the siRNA group refers to a group formed by removing one or more atoms or groups from siRNA, the siRNA being siRNA capable of inhibiting HBV mRNA; the weight ratio of the RNAi agent to the immune response modulator is (0.5-5000):1, based on siRNA. And a method for treating HBV-related diseases using the RNAi agent and the immune response modulator.

Description

A pharmaceutical composition and its use Technical Field

[0001] The present disclosure relates to a pharmaceutical composition and uses thereof, and in particular, to a pharmaceutical composition for treating diseases related to hepatitis B virus infection and uses thereof. Background Art

[0002] Hepatitis B (also known as HBV or HBV), caused by infection with the HBV virus, is a serious infectious disease posing a global threat, particularly in China. Chronic HBV infection (CHB) increases the risk of developing chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC).

[0003] Currently, the two main types of drugs for treating hepatitis B are interferon and nucleoside analogues. However, these two types of drugs are prone to drug resistance after use. Interferon is prone to adverse reactions after use, and nucleoside drugs are prone to drug resistance and recurrence after discontinuation of the drug.

[0004] In recent years, emerging small nucleic acid drugs have also shown high activity and durability in inhibiting hepatitis B virus.

[0005] However, in the prior art, there is still no drug or treatment method that can functionally cure hepatitis B.

[0006] Summary of the Invention

[0007] The inventors of the present invention have discovered that a composition comprising an RNAi agent and an immune response modifier unexpectedly exhibits excellent therapeutic effects when used to eliminate HBsAg in a subject and / or to treat HBV infection. Therefore, the inventors have made the following invention.

[0008] In one aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically active component, wherein the pharmaceutically active component consists of an RNAi agent and an immune response regulator, and the RNAi agent and the immune response regulator exist independently; the RNAi agent refers to one or more of an siRNA composition, an siRNA conjugate, and a pharmaceutically acceptable salt thereof; the siRNA composition contains siRNA and a pharmaceutically acceptable carrier; the siRNA conjugate contains an siRNA group and a conjugated group conjugated to the siRNA group; the siRNA group refers to a group formed after removing one or more atoms or groups from the siRNA, and the siRNA is an siRNA that can inhibit HBV mRNA; based on the siRNA, the weight ratio of the RNAi agent to the immune response regulator is (0.5-5000):1.

[0009] In another aspect, the present disclosure provides use of the pharmaceutical composition of the present disclosure in the preparation of a medicament for treating diseases associated with hepatitis B virus infection.

[0010] In yet another aspect, the present disclosure provides a method for treating diseases associated with hepatitis B virus infection, comprising administering an effective amount of the pharmaceutical composition of the present disclosure to a subject.

[0011] In yet another aspect, the present disclosure further provides a kit comprising the pharmaceutical composition described in the present disclosure.

[0012] Incorporated by reference

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Beneficial effects

[0014] Compared to administering RNAi agents or immune response modifiers alone, the pharmaceutical compositions and treatment methods disclosed herein exhibit superior anti-HBV effects, have the potential to achieve a functional cure for HBV, and demonstrate significant synergistic effects and significantly better therapeutic efficacy. They can achieve higher reduction rates in serum HBsAg, HBeAg, and HBV DNA levels while administering lower doses of the drug to subjects, and can also promote the production of anti-HBV antibodies in subjects.

[0015] For example, compared with the use of siRNA conjugates or immune response regulators alone, the use of the pharmaceutical composition of the present invention can further reduce HBV DNA levels within an experimental period of up to 78 days, with the maximum HBV DNA inhibition rate reaching 99.9992%. Moreover, the magnitude of HBV DNA reduction unexpectedly far exceeds the sum of the inhibitory effects of siRNA conjugates or immune response regulators alone. Compared with the use of RNAi agents alone, the HBV DNA level can be further significantly reduced by 99.9% on the basis of the significant reduction in HBV DNA levels in the RNAi agent group alone. Further results showed that among the 6 experimental animals given the pharmaceutical composition of the present invention, the HBV DNA level of one experimental animal was reduced to the detection limit (10 3.18 IU / mL). For another example, the present disclosure further verified the inhibitory effect of a lower dose of RNAi agent and immune response modifier pharmaceutical composition on HBV DNA in mice. The results showed that HBV DNA remained at a low level for up to 85 days after the first administration, with the maximum reduction of 4.48 log 10IU / mL, that is, the maximum HBV DNA inhibition rate reached 99.9967%. Moreover, compared with the inhibition level of the RNAi agent at a single dose of 9mpk, the HBV DNA level in mice given a single dose of 3mpk of RNAi agent and the pharmaceutical combination of immune response modifiers was further greatly reduced, with a maximum reduction of 2.76log 10 IU / mL, that is, the HBV DNA level was further reduced by 99.83%.

[0016] For another example, compared with the use of siRNA conjugates or immune response regulators alone, the use of the pharmaceutical composition of the present invention can further reduce the maximum HBsAg inhibition rate to 99.9930% within an experimental period of up to 78 days. And the reduction rate unexpectedly greatly exceeds the sum of the inhibitory effects of siRNA conjugates or immune response regulators alone. Compared with the use of RNAi agents alone, it can further reduce the HBsAg level by 97.76% on the basis of the significant reduction in the HBsAg level in the conjugate group. Furthermore, on the 78th day, in the experimental animals of the test group given the composition of the present invention, the HBsAg in some animals was already below the detection limit, showing a very excellent inhibitory effect. For another example, the present disclosure further verified the inhibitory effect of HBsAg after administering a lower dose of RNAi agents and immune response regulators to mice. The results showed that HBsAg was maintained at a low level for up to 85 days after the first administration, with a maximum reduction of 3.32 log 10 IU / mL, that is, the maximum HBsAg inhibition rate reached 99.9521%. Moreover, compared with the inhibition level of the RNAi agent at a single dose of 9 mpk, the HBsAg level in mice given a single dose of 3 mpk of RNAi agent and the pharmaceutical composition of the immune response modifier was further greatly reduced, with the maximum reduction of 1.31 log 10 IU / mL, that is, the HBsAg level was further reduced by 95.1022%.

[0017] For another example, compared with the use of siRNA conjugates or immune response modifiers alone, the use of the pharmaceutical composition of the present disclosure can further reduce HBeAg levels within an experimental period of up to 78 days, and the reduction exceeds the inhibitory effect of siRNA conjugates or immune response modifiers alone. Compared with the use of RNAi agents alone, the pharmaceutical composition of the present disclosure shows an approximately 0.3 log 10 For another example, the present disclosure further verified that after administering a lower dose of the RNAi agent and the immune response modifier pharmaceutical composition to mice, the HBeAg level in the mice also showed an increase of about 0.325 log compared to the administration of a higher dose of the RNAi agent alone. 10IU / mL further decreased.

[0018] For another example, the pharmaceutical composition disclosed herein can induce significant production of HBsAb in the serum of mice, indicating that while effectively inhibiting HBV antigens and DNA, it can also stimulate the immune response of mice, showing excellent prospects for achieving functional cure of hepatitis B. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 is a line graph showing changes in HBV DNA levels in the serum of HBV transgenic mice over time after administration of a control group or the pharmaceutical composition of the present disclosure in vivo.

[0020] FIG2 is a line graph showing changes in HBsAg levels in the serum of HBV transgenic mice over time after administration of a control group or the pharmaceutical composition of the present disclosure in vivo.

[0021] FIG3 is a line graph showing changes in serum HBeAg levels over time in HBV transgenic mice after in vivo administration of a control group or the pharmaceutical composition of the present disclosure.

[0022] FIG4 is a line graph showing changes in HBV DNA levels in the serum of HBV transgenic mice over time after administration of a control group or the pharmaceutical composition of the present disclosure in vivo.

[0023] FIG5 is a line graph showing changes in HBsAg levels in the serum of HBV transgenic mice over time after administration of a control group or the pharmaceutical composition of the present disclosure in vivo.

[0024] FIG6 is a line graph showing changes in serum HBeAg levels over time in HBV transgenic mice after in vivo administration of a control group or the pharmaceutical composition of the present disclosure. DETAILED DESCRIPTION

[0025] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0026] definition

[0027] Unless otherwise specified, the nouns or terms used in this disclosure have the following meanings.

[0028] HBV DNA refers to a DNA sequence such as that shown in Genbank Accession No. NC_003977.1. Further, unless otherwise specified, "HBV mRNA" used in this disclosure refers to the mRNA transcribed from the above-mentioned HBV DNA. The complete coding sequence of the reference sequence of the HBV genome can be found, for example, in GenBank gene accession Nos. GI:21326584 and GI:3582357. It is well known to those skilled in the art that, based on comparison of the whole gene nucleotide sequence, HBV can be divided into 9 subtypes: A, B, C, D, E, F, G, H and I. These 9 HBV subtypes are all within the scope of the HBV DNA described in this disclosure.

[0029] The capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group; P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide. In some embodiments, P1 is VP, Ps, or P representing a specific modification, wherein the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide, the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate-modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0030] A "fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group is replaced by a fluorine group. A "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose group is replaced by a non-fluorinated group. A "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), and acyclic nucleotides. A "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0031] "Complementary" or "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are each paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair consists of a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of that strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" is used in the art to mean that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary manner.

[0032] "Substantially reverse complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than one base mismatch between the two nucleotide sequences; and "perfectly reverse complementary" means that there are no base mismatches between the two nucleotide sequences.

[0033] A "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleotide sequences are considered to have a nucleotide difference at that position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleotide difference at that position can also be considered.

[0034] In the context of the present disclosure, particularly when describing the preparation method of siRNA or siRNA conjugate in the pharmaceutical composition and / or RNAi agent of the present disclosure, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to, according to the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared, the modified or unmodified nucleoside phosphoramidite monomer (modified or unmodified RNA / nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis, which is a method used in RNA synthesis well known to those skilled in the art. Unless otherwise specified, the nucleoside monomers used in the present disclosure are all commercially available or can be prepared by methods well known to those skilled in the art.

[0035] "Conjugation" refers to the connection between two or more chemical moieties, each of which has a specific function, in a covalently linked manner; accordingly, "conjugate" refers to a compound formed by covalently linking the chemical moieties. Furthermore, "siRNA conjugate" means a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA. siRNA conjugates should be understood as a general term for multiple siRNA conjugates or an siRNA conjugate shown by a certain chemical formula, depending on the context. In the context of the present disclosure, "conjugated molecule" should be understood as a specific compound that can be conjugated to siRNA through a reaction to ultimately form a siRNA conjugate. As used in the present disclosure, "siRNA conjugate" comprises an siRNA group and a conjugated group portion, wherein the siRNA group refers to a chemical portion formed by removing one or more atoms from the siRNA molecule. It will be understood by those skilled in the art that the removal of one or more of the above atoms will not destroy the inhibitory activity or stability of the siRNA against the target mRNA. For example, the siRNA group can be a chemical part formed by removing the hydrogen atom in the phosphate bond of the siRNA, or the chemical part formed by removing the hydrogen atom in the 5' hydroxyl group of the 5' terminal nucleotide of the sense strand or antisense strand of the siRNA, or the chemical part formed by removing the hydrogen atom in the 3' hydroxyl group of the 3' terminal nucleotide of the sense strand or antisense strand of the siRNA.

[0036] It will be understood by those skilled in the art that, for any group comprising one or more substituents, these groups are not intended to introduce any substituent groups or substitution patterns that are sterically impractical, synthetically infeasible, and / or inherently unstable.

[0037] "Alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbons is mentioned, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to residues that are the same as alkyl, but with two points of attachment.

[0038] "Alkenyl" refers to an unsaturated branched or straight chain hydrocarbon radical having at least one carbon-carbon double bond obtained by removing one hydrogen molecule from adjacent carbon atoms of the parent alkyl group. The group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues identical to alkenyl but with two points of attachment.

[0039] "Alkynyl" refers to an unsaturated, branched or straight-chain hydrocarbon radical having at least one carbon-carbon triple bond derived by removing two hydrogen atoms from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like. In certain embodiments, alkynyl groups have from 2 to 20 carbon atoms, and in other embodiments, from 2 to 10, from 2 to 8, or from 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues identical to alkynyl, but with two points of attachment.

[0040] "Alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. The alkoxy group typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through the oxygen bridge.

[0041] "Aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2) π-electron system according to Hückel's theory. Aryl includes, but is not limited to, radicals such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to residues identical to aryl, but with two points of attachment.

[0042] "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, comprising 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprising a cyclic delocalized (4n+2)π-electron system according to Hückel theory. Heteroaryl groups include fused or bridged ring systems. In some embodiments, the heteroatom in the heteroaryl group is an oxidized heteroatom. In some embodiments, the heteroaryl group comprises one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in the heteroaryl group is a quaternized nitrogen atom. The heteroaryl group is attached to the remainder of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to, azacycloheptatrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzo[b][1,4] naphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5 ]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, furano[3,2-c]pyridinyl, 5,6,7,8 ,9,10-hexahydrocyclooctanol[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctanol[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctanol[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, dihydroindolinyl, isoindolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl),8-tetrahydroquinazolinyl), naphthyl, 1,6-naphthyl-onyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, ]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl / thienyl. ,

[0043] Various hydroxy protecting groups can be used in the present disclosure. In general, protecting groups make chemical functionality insensitive to specific reaction conditions and can be added and removed on the functionality in the molecule without substantially damaging the rest of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2nd ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0044] The term "subject," as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any type of poultry.

[0045] "Treatment" refers to an approach for obtaining beneficial or desired results, including, but not limited to, a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Moreover, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0046] "Hepatitis B surface antigen (HBsAg)" refers to the main surface antigen protein of hepatitis B virus (HBV), and its meaning is well known to those skilled in the art, for example, the protein with NCBI GENBANK data accession number AAF24729.1. As used herein, "hepatitis B core antigen protein (HBcAg)" refers to the core antigen protein of HBV, and its meaning is well known to those skilled in the art, for example, the protein with NCBI GENBANK data accession number AAO63517.1. As used herein. "Hepatitis B e antigen (HBeAg)" refers to the hepatitis B virus protein located between the envelope and capsid of HBV, and its meaning is well known to those skilled in the art, for example, the protein with NCBI GENBANK data accession number BAJ51621.1. Those skilled in the art will understand that mutations or variations may occur naturally or be artificially introduced into the amino acid sequences of the above-mentioned viral proteins, including but not limited to substitutions, deletions and / or additions, such as HBsAg, HBcAg and / or HBeAg of different genotypes or subtypes. These mutations or compilations will not affect their biological functions, and all such natural or artificial variants are also included within the scope of the present disclosure.

[0047] Pharmaceutical composition

[0048] In one aspect, the present disclosure provides a pharmaceutical composition, which comprises a pharmaceutically active component, wherein the pharmaceutically active component consists of an RNAi agent and an immune response regulator, and the RNAi agent and the immune response regulator exist independently; the RNAi agent refers to one or more of an siRNA composition, an siRNA conjugate, and a pharmaceutically acceptable salt thereof; the siRNA composition contains siRNA and a pharmaceutically acceptable carrier; the siRNA conjugate contains an siRNA group and a conjugated group conjugated to the siRNA group; the siRNA group refers to a group formed after removing one or more atoms or groups from the siRNA, and the siRNA is an siRNA that can inhibit HBV mRNA; based on the siRNA, the weight ratio of the RNAi agent to the immune response regulator is (0.5-5000):1.

[0049] In the context of the present disclosure, the term "based on siRNA in the RNAi agent" in the siRNA composition refers to the siRNA contained in the composition; in the siRNA conjugate, it refers to the siRNA group. Since the siRNA group contained in the siRNA conjugate has substantially the same molecular weight as the siRNA that forms the siRNA group, for convenience, the dosage of the siRNA group in the siRNA conjugate is also described as "based on siRNA."

[0050] In the pharmaceutical composition of the present disclosure, the pharmaceutical active ingredient is composed of an RNAi agent and an immune response regulator. The inventors of the present disclosure have surprisingly found that, unlike in the prior art, by comprising an RNAi agent and an immune response regulator in a specific weight ratio, the pharmaceutical composition of the present disclosure does not need to be further used in conjunction with an HBV vaccine to obtain an enhanced HBV inhibitory effect, and shows excellent synergistic effects of HBsAg and HBV DNA inhibition. Therefore, in some embodiments, in the pharmaceutical composition of the present disclosure, based on the siRNA in the RNAi agent, the weight ratio of the RNAi agent to the immune response regulator is (0.5-2000): 1 or (1-2000): 1. In some embodiments, based on the siRNA in the RNAi agent, the weight ratio of the RNAi agent to the immune response regulator is (1-400): 1 or (2-400): 1. In some embodiments, based on the siRNA in the RNAi agent, the weight ratio of the RNAi agent to the immune response regulator is (2-150): 1 or (3-150): 1. In some embodiments, the weight ratio of the RNAi agent to the immune response modifier, calculated based on the siRNA in the RNAi agent, is (2.4-50):1 or (4-50):1. The pharmaceutical composition of the present disclosure having the above weight ratio can better exert the synergistic effect of the RNAi agent and the immune response modifier, resulting in a higher inhibitory effect on HBV-related diseases or symptoms.

[0051] In some embodiments, the dosage ratio of the RNAi agent to the immune response modifier in the pharmaceutical composition is calculated according to the dosage. The dosage of the RNAi agent in the pharmaceutical composition is calculated according to mg / kg subject body weight, and the dosage of the immune response modifier is calculated by weight. In some embodiments, based on the siRNA in the RNAi agent, the dosage ratio of the RNAi agent to the immune response modifier is (0.02-540) mg / kg subject body weight: 1 mg. In some embodiments, based on the siRNA in the RNAi agent, the dosage ratio of the RNAi agent to the immune response modifier is (0.03-400) mg / kg subject body weight: 1 mg. In some embodiments, based on the siRNA in the RNAi agent, the dosage ratio of the RNAi agent to the immune response modifier is (0.06-380) mg / kg subject body weight: 1 mg. In some embodiments, based on the siRNA in the RNAi agent, the dosage ratio of the RNAi agent to the immune response modifier is (0.03-180) mg / kg subject body weight: 1 mg. In some embodiments, the dosage ratio of the RNAi agent to the immune response regulator is (0.06-180) mg / kg body weight of the subject: 1 mg based on the siRNA in the RNAi agent. In some embodiments, the dosage ratio of the RNAi agent to the immune response regulator is (0.1-80) mg / kg body weight of the subject: 1 mg based on the siRNA in the RNAi agent. In some embodiments, the dosage ratio of the RNAi agent to the immune response regulator is (0.2-30) mg / kg body weight of the subject: 1 mg based on the siRNA in the RNAi agent. Based on the dosage of the RNAi agent and the immune response regulator in the animal model for verification of the effect in the present disclosure, those skilled in the art can infer the dosage range suitable for use in human subjects.

[0052] In some embodiments, the siRNA composition contains siRNA and a pharmaceutically acceptable carrier, wherein the siRNA is in an siRNA conjugate.

[0053] In some embodiments, the siRNA conjugate in the pharmaceutical composition of the present disclosure contains a siRNA group and a conjugated group conjugated to the siRNA group.

[0054] In the pharmaceutical compositions disclosed herein, the "siRNA group" contained in the siRNA conjugate refers to a group formed by removing one or more atoms or groups from the siRNA molecule to form a covalent bond with the conjugated group. In some embodiments, the siRNA group is a group formed by removing one atom or one group from one or more nucleotide residues in the siRNA molecule. In some embodiments, the siRNA group is a group formed by removing one atom or one group from the ribose ring, the base, or the phosphate group of one or more nucleotide residues in the siRNA molecule. In some embodiments, the siRNA group is a group formed by removing one hydrogen atom or one hydroxyl group from the 3' and / or 5' terminal hydroxyl group in the sense strand or antisense strand of the siRNA molecule. In some embodiments, the siRNA group is a group formed by removing one hydrogen atom from the 3' or 5' terminal hydroxyl group in the sense strand of the siRNA molecule. In some embodiments, the siRNA group is a group formed by removing one hydrogen atom from the 3' terminal hydroxyl group and / or the 5' terminal hydroxyl group in the sense strand of the siRNA molecule.

[0055] In some embodiments, the RNAi agent refers to an siRNA conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, wherein the siRNA group, the linker and the targeting group are connected in sequence, and each of the targeting groups is selected from a ligand capable of binding to a surface receptor of a hepatocyte.

[0056] In general, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA group, the linker and the targeting group are connected in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA group can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of the siRNA group and the conjugated group can be at the 3' end or 5' end of the sense strand in the siRNA group, or at the 5' end of the antisense strand, or inside the sequence of the siRNA group. In this context, "sequence inside" refers to the nucleotides or internucleotide linkage groups other than the 3' end and 5' end nucleotides of the siRNA sense strand and antisense strand. In some embodiments, the conjugation site of the siRNA group and the conjugated group is at the 3' end of the sense strand.

[0057] In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base in the siRNA group. In some embodiments, the conjugate group can be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA group, the conjugate group is usually connected to the phosphate group or 2'- or 5'-hydroxyl group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA group, the conjugate group is usually connected to the ribose sugar ring or the base. For various connection methods, please refer to: Muthiah Manoharanet.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.

[0058] In some embodiments, the siRNA group and the conjugate group can be connected by acid-labile or reducible chemical bonds. In the acidic environment of endosomes, these chemical bonds can degrade, thereby freeing the siRNA group. For example, the siRNA group may reform into the siRNA molecule to fully exert the RNA interference effect. For non-degradable conjugation methods, the conjugate group can be attached to the sense strand of the siRNA group to minimize the effect of the conjugation on the inhibitory activity of the siRNA group.

[0059] The targeting group can be connected to the siRNA group via a suitable linker, and those skilled in the art can select a suitable linker based on the specific type of the targeting group. These linkers, the type of targeting group, and the mode of connection to the siRNA group can be found, for example, in WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0060] In some embodiments, the targeting group can be a ligand commonly used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

[0061] In some embodiments, at least one or each of the targeting groups is selected from ligands that can bind to a surface receptor on a cell expressing the target gene.

[0062] In some embodiments, at least one or each of the targeting groups is selected from a ligand that can bind to a receptor on the surface of a mammalian hepatocyte. In some embodiments, each of the targeting groups is independently a ligand that is affinity-bound to an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian hepatocyte. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or sugar. In some embodiments, each of the targeting groups is independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose , α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2 -amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4 - one of tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose. In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

[0063] In some embodiments, the linker in the siRNA conjugate has a structure as shown in formula (301):

[0064] Wherein, k is an integer from 1 to 3;

[0065] L A Having a structure including an amide bond as shown in formula (302), L B It has a structure containing N-acylpyrrolidine as shown in formula (303), containing a carbonyl group and an oxygen atom, L Cis a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trishydroxymethylaminomethane;

[0066] Among them, n 302 ,q 302 and p 302 Each independently represents an integer from 2 to 6, optionally, n 302 ,q 302 and p 302 Each independently is 2 or 3; n 303 An integer between 4 and 16. Optionally, n 303 is an integer between 8 and 12, Indicates the site of covalent attachment of a group.

[0067] In the joint, each L A Each of the targeting groups is connected via an ether bond and connected via L C The oxygen atom of the hydroxyl group in the part is connected with L C Partially form ether bonds and connect; L B Through the carbonyl group in formula (303) and L C The nitrogen atom of the amino group in the moiety forms an amide bond to connect, and is connected to the siRNA group through the oxygen atom in formula (303) to form a phosphate bond or a phosphorothioate bond.

[0068] In some embodiments, the RNAi agent is a conjugate having a structure as shown in Formula (305) or a pharmaceutically acceptable salt thereof:

[0069] Wherein, Nu represents the siRNA group.

[0070] In some embodiments, the linker in the siRNA conjugate has a structure represented by formula (306):

[0071] Among them, n 306 is an integer from 0 to 3, each p 306 are independently an integer from 1 to 6, Indicates the site of covalent attachment of a group; the linking group is connected to the targeting group through an ether bond formed by the oxygen atom marked by *; the linking group is connected to the siRNA group by forming a phosphate bond or a phosphorothioate bond by at least one of the oxygen atoms marked by #, and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form hydroxyl groups, or are connected to C1-C3 alkyl groups to form C1-C3 alkoxy groups.

[0072] In some embodiments, the siRNA conjugate has a structure as shown in formula (307):

[0073] Wherein, Nu represents the siRNA group.

[0074] In some embodiments, the RNAi agent is a conjugate having a structure as shown in Formula (308) or a pharmaceutically acceptable salt thereof:

[0075] in,

[0076] n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

[0077] Each m1, m2 or m3 is independently an integer selected from 2-10;

[0078] R 10 、R 11 、R 12 、R 13 、R 14 or R 15 are each independently H, or selected from the group consisting of: C1-C 10 Alkyl, C1-C 10 Halogenated alkyl and C1-C 10 alkoxy;

[0079] R3 has the structure shown in formula (A59):

[0080] Wherein, E1 is OH, SH or BH2, and Nu represents the siRNA group;

[0081] R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C5-C 10 Cycloalkylene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein R2 may optionally have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0082] Each L1 is independently a linear alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein L1 may optionally have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0083] represents the site of covalent attachment of a group;

[0084] M1 represents a targeting group, and its definition and selectable range are the same as above. In some embodiments, each M1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

[0085] The skilled artisan will appreciate that although L1 is defined as a linear alkyl group for convenience, it may not be a linear group or may be named differently, such as an amine or alkenyl group resulting from the above-mentioned substitutions and / or replacements. For the purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two points of attachment. For this purpose, rings resulting from the replacement of carbon atoms of the linear alkylene group (e.g., heterocyclylene or heteroarylene) are counted as one atom.

[0086] When M1 is a ligand with affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer from 1 to 3, and n3 can be an integer from 0 to 4, ensuring that the number of M1 ligands in the conjugate is at least 2. In some embodiments, n1 + n3 ≥ 2, which can result in the number of M1 ligands being at least 3, making it easier for the M1 ligand to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the conjugate to enter the cell via endocytosis. Experiments have shown that when the number of M1 ligands is greater than 3, the increased ease of binding of the M1 ligand to the asialoglycoprotein receptor on the liver surface is not significantly increased. Therefore, considering multiple factors such as ease of synthesis, structural / processing costs, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2-3.

[0087] In some embodiments, when m1, m2 and m3 are independently selected from integers of 2-10, the spatial positions between multiple M1 ligands can be made suitable for the binding of M1 ligands to the asialoglycoprotein receptor on the liver surface. In order to make the conjugate provided by the present disclosure simpler, easier to synthesize and / or reduce costs, in some embodiments, m1, m2 and m3 are each independently an integer of 2-5. In some embodiments, m1=m2=m3.

[0088] Those skilled in the art will understand that when R 10 、R 11 、R 12 、R 13、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, and C1-C 10 In some embodiments, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each is independently selected from H, methyl and ethyl. 10 、R 11 、R 12 、R 13 、R 14 and R 15 Both are H.

[0089] In some embodiments, R3 is a group represented by the structure of formula A59, wherein E1 is OH, SH or BH2. Based on the consideration of the availability of raw materials for preparation, in some embodiments, E1 is OH or SH.

[0090] In some embodiments, R2 is selected to achieve connection with N and A59 on the nitrogen-containing skeleton. In the context of this disclosure, "nitrogen-containing skeleton" refers to a skeleton having R 10 、R 11 、R 12 、R 13 、R 14 and R 15 The carbon atoms of R2 are connected to each other in a chain structure. Therefore, R2 can be any connecting group that can connect the A59 group to the N on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, when the siRNA conjugate is prepared by a solid phase synthesis process, the R2 group needs to contain both a connection site connected to the N on the nitrogen-containing skeleton and a connection site connected to the P in R3. In some embodiments, the site in R2 connected to the N on the nitrogen-containing skeleton forms an amide bond with N, and the site connected to the P on R3 forms a phosphate bond with P. In some embodiments, the length of R2 is 3-25 atoms, 3-20 atoms, 4-15 atoms or 5-12 atoms. In some embodiments, R2 includes a first connection point and an optional second functional group, wherein the first connection point is a connection point that forms a phosphate bond or a phosphorothioate bond with the oligonucleotide or nucleotide, and the second functional group is a functional group formed after the covalent connection between the solid phase support and the solid phase support is cleaved. In some embodiments, R2 is B5, B6, B5' or B6':

[0091] in, Indicates the site of covalent attachment of a group.

[0092] The value range of q2 can be an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.

[0093] L1 functions to connect the M1 ligand to the nitrogen-containing backbone, providing targeting functionality to the siRNA conjugate. In some embodiments, L1 is selected from a combination of one or more of the groups in formulae A1-A26. In some embodiments, L1 is selected from a combination of one or more of A1, A4, A5, A6, A8, A10, A11, and A13; in some embodiments, L1 is selected from a combination of at least two of A1, A4, A8, A10, and A11; in some embodiments, L1 is selected from a combination of at least two of A1, A8, and A10.

[0094] In some embodiments, L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 atoms in length.

[0095] In some embodiments, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, and in some embodiments, j2 is an integer of 3-5. R' is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, and Rb are each selected in formulas A1-A26 to achieve connection between the M1 ligand and the N on the nitrogen-containing skeleton, and to make the spatial position between the M1 ligands more suitable for binding of the M1 ligand to the liver surface asialoglycoprotein receptor.

[0096] In some embodiments, the siRNA conjugate has a structure represented by formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

[0097] In some embodiments, P in formula (A59) can be connected to any possible position in the siRNA group, for example, P in formula (A59) can be connected to any nucleotide of the sense strand or antisense strand in the siRNA group; in some embodiments, P in formula (A59) is connected to any nucleotide of the sense strand. In some embodiments, P in formula (A59) is connected to the end of the sense strand or antisense strand; in some embodiments, P in formula (A59) is connected to the end of the sense strand. The end refers to the first 4 nucleotides of the sense strand or antisense strand from one end. In some embodiments, P in formula (A59) is connected to the end of the sense strand or antisense strand in the siRNA group; in some embodiments, P in formula (A59) is connected to the 3' end of the sense strand. When connected to the above-mentioned position of the sense strand, after the siRNA conjugate enters the cell, when unwinding, the separate siRNA antisense strand can be released to regulate target gene expression.

[0098] The P in formula (A59) can be connected to any possible position on the nucleotide in the siRNA group, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or the base of the nucleotide. In some embodiments, the P in formula (A59) can be connected to the 2' position, 3' position or 5' position of the nucleotide in the siRNA group by forming a phosphodiester bond. In some embodiments, the P in formula (A59) is connected to the oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the sense strand, or the P in formula (A59) is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl group of a nucleotide in the sense strand, or the P in formula (A59) is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the sense strand.

[0099] In some embodiments, the siRNA group contained in the siRNA conjugate can be formed by removing one or more atoms from the above-mentioned siRNA molecule. siRNA and / or siRNA conjugates containing the sequences shown in SEQ ID NOs: 11-24 exhibit low off-target effects and high HBV mRNA inhibitory activity. The siRNA conjugates also exhibit higher efficiency in entering cells. In some embodiments, the siRNA conjugates have the structure shown in formula (403) and the siRNA sequences shown in SEQ ID NOs: 11 and 12.

[0100] siRNA composition capable of inhibiting HBV mRNA

[0101] The RNAi agent disclosed herein may also be an siRNA composition capable of inhibiting HBV mRNA, wherein the siRNA composition comprises the above-mentioned siRNA and a pharmaceutically acceptable carrier.

[0102] In some embodiments, the siRNA composition can be in the form of a liposome. In some embodiments, the pharmaceutically acceptable carrier used in the liposome comprises an amine-containing transfection compound (hereinafter also referred to as a key lipid), an auxiliary lipid and / or a pegylated lipid. Wherein, the key lipid, auxiliary lipid and pegylated lipid can be selected from one or more of the amine-containing transfection compound or its pharmaceutically acceptable salt or derivative, auxiliary lipid and pegylated lipid described in Chinese patent application CN103380113A (incorporated herein by reference in its entirety).

[0103] In some embodiments, the key lipid may be a compound described in Chinese patent application CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:

[0104] in:

[0105] X 101 and X 102 are each independently O, S, NA or CA, wherein A is hydrogen or C1-C 20 hydrocarbon chain;

[0106] Y 101 and Z 101 Each independently is C=O, C=S, S=O, CH-OH or SO2;

[0107] R 101 、R 102 、R 103 、R 104 、R 105 、R106 and R 107 are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;

[0108] x is an integer from 1 to 10;

[0109] n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; wherein, if m=p=0, then R 102 It is hydrogen;

[0110] And, if at least one of n or m is 2, then R 103 and the nitrogen in formula (201) to form a structure as shown in formula (202) or formula (203):

[0111] wherein g, e, and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).

[0112] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 In some embodiments, R in formula (201) 101 and R 102 Each of is independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0113] In some embodiments, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following formulas (204) to (213):

[0114] Wherein, in formula (204) to formula (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R 103 Possible points of attachment to the nitrogen atom in formula (201), wherein each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).

[0115] Those skilled in the art can obtain the compound represented by formula (201) by any reasonable method. In some embodiments, the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.

[0116] In some embodiments, the key lipid is a key lipid as shown in formula (214) and / or a key lipid as shown in formula (215):

[0117] The helper lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative;

[0118] The PEGylated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0119] In some embodiments, in the siRNA composition, the molar ratio of the key lipid, the auxiliary lipid and the PEGylated lipid is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).

[0120] In some embodiments, the siRNA composition particles formed by the conjugates provided herein and the above-mentioned amine-containing transfection reagents have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.

[0121] In some embodiments, in the siRNA composition formed by the conjugate provided by the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio of the conjugate to all lipids (e.g., key lipids, helper lipids and / or PEGylated lipids) (weight / weight ratio) is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the weight ratio of the conjugate provided by the present disclosure to all lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.

[0122] In some embodiments, the components of the siRNA composition can be sold separately and can be in the form of a liquid when used. In some embodiments, the siRNA composition disclosed herein can be prepared according to various known methods, simply by replacing the existing nucleic acid components with the above-mentioned siRNA; in some embodiments, the siRNA composition can be prepared according to the following methods:

[0123] The key lipid, helper lipid, and PEGylated lipid are suspended in alcohol in the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the obtained lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at around room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, for example, ethanol.

[0124] The siRNA is dissolved in a buffered saline solution to obtain an siRNA aqueous solution. The concentration of the buffered saline solution is 0.05-0.5 M, for example, 0.1-0.2 M. The pH of the buffered saline solution is adjusted to 4.0-5.5, for example, 5.0-5.2. The amount of buffered saline solution used is such that the concentration of the siRNA does not exceed 0.6 mg / mL, for example, 0.2-0.4 mg / mL. The buffered salt is selected from one or more soluble acetates and soluble citrates, for example, sodium acetate and / or potassium acetate.

[0125] The lipid solution and the siRNA aqueous solution are mixed, and the resulting mixture is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome formulation. The volume ratio of the lipid solution to the conjugate aqueous solution is 1:(2-5), for example, 1:4.

[0126] The incubated liposome agent is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the siRNA composition provided by the present disclosure, wherein the physicochemical parameters thereof are as follows: pH 6.5-8, encapsulation efficiency not less than 80%, particle size 40-200 nm, polydispersity index not higher than 0.30, and osmotic pressure 250-400 mOsm / kg; for example, the physicochemical parameters may be as follows: pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100 nm, polydispersity index not higher than 0.20, and osmotic pressure 300-400 mOsm / kg.

[0127] Concentration or dilution can be performed before, after, or simultaneously with impurity removal. Impurity removal can be performed using various existing methods, such as ultrafiltration at 100 kDa using a tangential flow system or a hollow fiber column, with the ultrafiltration exchange solution being phosphate buffered saline (PBS) at pH 7.4. Sterilization can be performed using various existing methods, such as filtration sterilization on a 0.22 μm filter.

[0128] It will be appreciated by those skilled in the art that, in the RNAi agent, any suitable ratio of siRNA and siRNA conjugate can achieve the purpose of the present disclosure. For example, in some embodiments, the molar ratio of siRNA and siRNA conjugate can be 0:1000-1000:0. In some embodiments, in the RNAi agent, the molar ratio of siRNA and siRNA conjugate is 1:100-100:1, 1:50-50:1 or 1:10-10:1. In some embodiments, in the RNAi agent, the molar ratio of siRNA and siRNA conjugate is 1:5-5:1. In some embodiments, the RNAi agent is a siRNA conjugate.

[0129] siRNA

[0130] In the pharmaceutical composition disclosed herein, the RNAi agent comprises an siRNA composition or an siRNA conjugate. In some embodiments, the siRNA group in the siRNA or siRNA conjugate in the pharmaceutical composition comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are both composed of 19 nucleotides, each of the nucleotides in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complemented to form a double-stranded region, the nucleotide sequence II is at least partially reverse-complemented with the first section of nucleotide sequence, and the first section of nucleotide sequence is a nucleotide sequence of 19 nucleotides in length in HBV mRNA.

[0131] In some embodiments, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2 and differs by no more than 3 nucleotides:

[0132] 5'-CCUUGAGGCAUACUUCAAZ1-3' (SEQ ID NO: 1);

[0133] 5'-Z2UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO: 2);

[0134] Wherein, Z1 is A, Z2 is U, the nucleotide sequence I contains the nucleotide Z3 corresponding to the position of Z1, the nucleotide sequence II contains the nucleotide Z4 corresponding to the position of Z2, and Z4 is the first nucleotide at the 5' end of the antisense strand;

[0135] Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 3 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 4 and differs by no more than 3 nucleotides:

[0136] 5'-GUGUGCACUUCGCUUCACZ5-3' (SEQ ID NO: 3);

[0137] 5'-Z6GUGAAGCGAAGUGCACAC-3' (SEQ ID NO: 4);

[0138] Wherein, Z5 is A, Z6 is U, the nucleotide sequence I contains the nucleotide Z7 corresponding to the position of Z5, the nucleotide sequence II contains the nucleotide Z8 corresponding to the position of Z6, and Z8 is the first nucleotide at the 5' end of the antisense strand;

[0139] Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 5 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 6 and differs in no more than 3 nucleotides:

[0140] 5'-GGACUUCUCUCAAUUUUCZ9-3' (SEQ ID NO: 5);

[0141] 5'-Z 10 GAAAAUUGAGAGAAGUCC-3' (SEQ ID NO: 6);

[0142] Among them, Z9 is U, Z 10 A, the nucleotide sequence I contains the nucleotide Z corresponding to position Z9 11 The nucleotide sequence II contains a position corresponding to Z 10 The nucleotide Z 12 , the Z 12 is the first nucleotide at the 5' end of the antisense strand;

[0143] Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 7 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 8 and differs in no more than 3 nucleotides:

[0144] 5'-CUGUAGGCAUAAAUUGGUZ 13 -3' (SEQ ID NO: 7);

[0145] 5'-Z 14 ACCAAUUUAUGCCUACAG-3' (SEQ ID NO:8);

[0146] Among them, Z 13 A, Z 14 is U, the nucleotide sequence I contains a position corresponding to Z 13 The nucleotide Z 15 The nucleotide sequence II contains a position corresponding to Z 14 The nucleotide Z 16 , the Z 16 is the first nucleotide at the 5' end of the antisense strand;

[0147] Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 9 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 10 and differs in no more than 3 nucleotides:

[0148] 5'-GUGCACUUCGCUUCACZ 17 -3' (SEQ ID NO: 9);

[0149] 5'-Z 18 ACCAAUUUAUGCCUACAG-3' (SEQ ID NO: 10);

[0150] Among them, Z 17 A, Z 18 is U, the nucleotide sequence I contains a position corresponding to Z 17 The nucleotide Z 19 The nucleotide sequence II contains a position corresponding to Z 18 The nucleotide Z 20 , the Z 20 is the first nucleotide at the 5' end of the antisense strand.

[0151] In some embodiments, the sense strand and antisense strand are the same or different in length, with the sense strand being 19-23 nucleotides long and the antisense strand being 20-26 nucleotides long. Thus, the length ratio of the sense strand to the antisense strand in the siRNA or siRNA group can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0152] In some embodiments, the nucleotide sequence I further comprises a nucleotide sequence III, and the nucleotide sequence II further comprises a nucleotide sequence IV, wherein the nucleotide sequence III and nucleotide sequence IV are identical in length, ranging from 1 to 4 nucleotides; the nucleotide sequence III is linked to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II. In some embodiments, the base sequence of the nucleotide sequence IV is the reverse complement of a second nucleotide sequence, which is a nucleotide sequence in HBV mRNA that is adjacent to the 5' end of the first nucleotide sequence and has the same length as the nucleotide sequence IV.

[0153] In some embodiments, nucleotide sequence III and nucleotide sequence IV are the same length and are completely reverse complementary, so that when the base of nucleotide sequence III is given, the base of nucleotide sequence IV is also determined.

[0154] In some embodiments, the sense strand and the antisense strand are of different lengths, and the nucleotide sequence II further contains a nucleotide sequence V, which is 1 to 3 nucleotides long and is connected to the 3' end of the antisense strand to form the 3' overhang end of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand can be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length of the nucleotide sequence V is 2 nucleotides, and thus, the length ratio of the sense strand and the antisense strand can be 19 / 21, 21 / 23 or 23 / 25.

[0155] Each nucleotide in the nucleotide sequence V can be any nucleotide. To facilitate synthesis and save costs, in some embodiments, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU). Alternatively, to improve the affinity of the antisense strand for the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding position in the target mRNA. Therefore, in some embodiments, the ratio of the sense strand to the antisense strand length is 19 / 21 or 21 / 23. In this case, the siRNA or siRNA conjugate has better mRNA silencing activity.

[0156] In some embodiments, each nucleotide in the siRNA group in the siRNA or siRNA conjugate in the pharmaceutical composition is a modified nucleotide. In some embodiments, each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a fluorinated or non-fluorinated modified nucleotide; in the sense strand, from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides; in the antisense strand, from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides; each fluorinated modified nucleotide is independently selected from a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted with fluorine, and each non-fluorinated modified nucleotide is independently selected from a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is substituted with a non-fluorinated group. In some embodiments, in the sense strand, from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 or positions 5, 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides; in the antisense strand, from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 or positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides; and the other nucleotides in the sense strand and the antisense strand are all non-fluorinated modified nucleotides.

[0157] In the context of the present disclosure, a fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure as shown in the following formula (7). A non-fluoro-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluoro group. In some embodiments, each non-fluoro-modified nucleotide is independently selected from one of a 2'-alkoxy-modified nucleotide, a 2'-substituted alkoxy-modified nucleotide, a 2'-alkyl-modified nucleotide, a 2'-substituted alkyl-modified nucleotide, a 2'-amino-modified nucleotide, a 2'-substituted amino-modified nucleotide, and a 2'-deoxynucleotide. In some embodiments, a 2'-substituted alkoxy-modified nucleotide, in some embodiments, a 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, a 2'-substituted alkoxy-modified nucleotide, for example, can be a 2'-O-methoxyethyl-modified nucleotide (2'-MOE), as shown in formula (9). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is represented by formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is represented by formula (11):

[0158] BNA refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):

[0159] Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):

[0160] In the above formulae (15) and (16), R is selected from H, OH or alkoxy (O-alkyl).

[0161] An isonucleotide is a compound formed by a change in the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide can be a compound formed by a base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18).

[0162] In the compounds of formula (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; and R is selected from H, OH, F or the non-fluorinated groups described above.

[0163] In some embodiments, the nucleotide analog is selected from one of an isonucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0164] In the above and below, “fluorinated nucleotides”, “2’-fluorinated nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by fluorine” and “nucleotides having a 2’-fluorinated ribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (7) formed by replacing the 2’-hydroxyl group of the nucleotide with fluorine; “methoxy-modified nucleotides”, “2’-methoxy-modified nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by a methoxy group” and “nucleotides having a 2’-methoxyribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (8) formed by replacing the 2’-hydroxyl group of the ribose group of the nucleotide with a methoxy group.

[0165] In some embodiments, in the pharmaceutical composition, at least one phosphate group in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a thiophosphate group having a structure as shown in formula (1):

[0166] In some embodiments, the phosphorothioate linkage is present at at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:

[0167] between the first and second nucleotides at the 5' end of the sense strand;

[0168] between the second and third nucleotides at the 5' end of the sense strand;

[0169] between the first and second nucleotides at the 3' end of the sense strand;

[0170] between the second and third nucleotides at the 3' end of the sense strand;

[0171] between the first and second nucleotides at the 5' end of the antisense strand;

[0172] between the second and third nucleotides at the 5' end of the antisense strand;

[0173] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0174] between the second and third nucleotides at the 3' end of the antisense strand.

[0175] In some embodiments, in the pharmaceutical composition of the present disclosure, the 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. Commonly used 5'-phosphate nucleotides or 5'-phosphate analog-modified nucleotides are well known to those skilled in the art. For example, a 5'-phosphate nucleotide may have a structure as shown in formula (2):

[0176] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four 5'-phosphate analogue-modified nucleotides:

[0177] Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a base, which is selected from A, U, C, G, or T.

[0178] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a thiophosphate modified nucleotide as shown in formula (5).

[0179] In some embodiments, the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 11, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 12:

[0180] 5'-CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 11);

[0181] 5'-VP-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm-3' (SEQ ID NO: 12);

[0182] Alternatively, the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 13, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 14:

[0183] 5'-GmsUmsGmUmGfCmAfCfUfUmCmGmCmUmUmCmAmCmAm-3' (SEQ ID NO: 13);

[0184] 5'-UmsGfsUmGmAm(Agn)GmCfGfAmAmGmUmGfCmAfCmAmCmsUmsUm-3' (SEQ ID NO: 14);

[0185] Alternatively, the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 15, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 16:

[0186] 5'-(invAb)sGmUmGmGmAmCmUmUmCfUfCfUmCmAmAmUmUmUmUmCmUms(invAb)-3' (SEQ ID NO: 15);

[0187] 5'-AmsGfsAmsAfAmAfUmUfGmAfGmAfGmAfAmGfUmCfCmAmsCm-3' (SEQ ID NO: 16);

[0188] Alternatively, the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 17, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 18:

[0189] 5'-(invAb)sCmGmCmUmGmUmAmGmGfCfAfUmAmAmAmUmUmGmGmUmAms(invAb)-3' (SEQ ID NO: 17);

[0190] 5'-UmsAfsCmsCfAmAfUmUfUmAfUmGfCmCfUmAfCmAfGmCmsGm-3' (SEQ ID NO: 18);

[0191] Alternatively, the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 19, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 20:

[0192] 5'-GmsUmsGmCmAfCfUfUmCmGmCmUmUmCmAmCmAm-3' (SEQ ID NO: 19);

[0193] 5'-UmsGfsUmGmAmAmGmCmGmAmAmGmUmGfCmAfCmAmCmsGmsGmUf-3' (SEQ ID NO: 20);

[0194] Alternatively, the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 21, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 22:

[0195] 5'-GmsUmsGmCmAfCfUfUmCmGmCmUmUmCmAmCmAm-3' (SEQ ID NO: 21);

[0196] 5'-UmsGfsUmGm(Agn)AmGmCmGmAmAmGmUmGfCmAfCmAmCmsGmsGmUf-3' (SEQ ID NO: 22);

[0197] Alternatively, the sense strand of the siRNA is the nucleotide sequence shown in SEQ ID NO: 23, and the antisense strand is the nucleotide sequence shown in SEQ ID NO: 24:

[0198] 5'-GmsUmsGmCmAfCfUfUmCmGmCmUmUmCmAmCmAm-3' (SEQ ID NO: 23);

[0199] 5'-UmsGfsUmGmAm(Agn)GmCmGmAmAmGmUmGfCmAfCmAmCmsGmsGmUf-3' (SEQ ID NO: 24).

[0200] The capital letters C, G, U, and A represent the base composition of the nucleotide. The lowercase letter m indicates that the nucleotide to the left of the letter m is a methoxy-modified nucleotide. The lowercase letter f indicates that the nucleotide to the left of the letter f is a fluorinated nucleotide. The lowercase letter s indicates that the nucleotides to the left of the letter are linked by a phosphorothioate group. The letter combination VP indicates that the nucleotide to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide. invAb represents an inverted abasic deoxyribonucleotide. Agn represents adenosine glycerol nucleic acid (GNA).

[0201] immune response modifiers

[0202] "Immune response modifier" refers to an agent that modulates an immune response and participates in immune response regulation by promoting or inhibiting the mechanisms and effects of cellular and humoral immunity. In some embodiments, immune response modifiers include, but are not limited to, adjuvants or immune response stimulants. In some embodiments, the adjuvant is selected from one or more agents that can promote an immune response. In some embodiments, the immunostimulant is selected from one or more independently administered agents that can stimulate an immune response.

[0203] As used herein, the term "adjuvant" or "vaccine adjuvant" is understood to be an agent that promotes (e.g., enhances, accelerates, or prolongs) the immune response to the antigen administered with it to induce long-term protective immunity. There is no substantial immune response to the adjuvant itself. Adjuvants include, but are not limited to, pathogen components, particulate adjuvants, and combination adjuvants (see, e.g., www.niaid.nih.gov / research / vaccine-adjuvants-types). Pathogen components (e.g., monophosphatidyl lipid A (MPL), poly (I: C), polyICLC adjuvants, CpG DNA, c-di-AMP, c-di-GMP, c-di-CMP; short, blunt-ended 5'-triphosphate dsRNA (3pRNA) RIG-1 ligands, and emulsions such as poly [sodium di(carboxylate ethylphenoxy) phosphazene] (PCEP)) can help trigger early nonspecific or innate immune responses to vaccines by targeting various receptors inside or on the surface of innate immune cells. The innate immune system influences the acquired immune response, which provides long-term protection against the pathogens targeted by the vaccine. Particulate adjuvants (e.g., alum, virosomes, cytokines, such as IL-12) form very small particles that can stimulate the immune system and also enhance the delivery of antigens to immune cells. Combination adjuvants (e.g., AS02, AS03, and AS04 (all GSK); MF59 (Novartis); (CSL Limited); and (Altimmune) elicit a variety of protective immune responses. Adjuvants that have a modest effect when used alone can induce a more effective immune response when used together.

[0204] In some embodiments, the adjuvants used in the present disclosure promote humoral and cellular immune responses. To this end, a balanced Th1 / Th2 helper T cell response is required to support neutralizing antibody responses and effector cytotoxic T cell responses. In some embodiments, the adjuvant provides a balanced Th1 / Th2 response. In certain embodiments, the adjuvant is a poly I:C adjuvant, a polyICLC adjuvant, a C pG adjuvant, a STING agonist (c-bis-AMP adjuvant, c-bis-GM P adjuvant or c-bis-CM P adjuvant), In some embodiments, the adjuvant is a poly I:C adjuvant, a CpG adjuvant, a STING agonist, or a PCEP adjuvant. In some embodiments, the adjuvant is a CpG adjuvant.

[0205] As used herein, "immunostimulant" is a reagent that can be administered or not administered independently of an antigen to stimulate an immune response. Immunostimulants include, but are not limited to, PEGylated interferon α2a (PEG-IFN-α-2a), interferon α-2b, PEG-IFN α-2b, interferon λ, recombinant human interleukin 7 and Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonists, virus entry inhibitors (such as Myrcludex), oligonucleotides (such as REP 9AC) that inhibit HBsAg secretion or release, capsid inhibitors (such as Bay41-4109 and NVR-1221), cccDNA inhibitors (such as IHVR-25). In some embodiments, immunostimulants can include viral capsids, optionally empty viral capsids, for example, MVA capsids. In some embodiments, immunostimulants can also include immune checkpoint regulators. Immune checkpoint regulators can be stimulating or inhibitory. As used herein, immune checkpoint regulators enhance immune responses. Immune checkpoint regulators include, but are not limited to, CTLA-4 inhibitors (e.g., ipilimumab), PD-1 inhibitors (e.g., nivolumab, pembrolizumab, and BGB-A317 antibodies). In addition to affimer biotherapeutics, PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab.

[0206] In some embodiments, the immune response modifier in the pharmaceutical composition of the present invention is selected from one or more TLR agonists. In some embodiments, the immune response modifier is selected from one or more TLR9 agonists. In some embodiments, the immune response modifier is selected from one or more of CpG DNA or a pharmaceutically acceptable salt thereof and alum adjuvant. In some embodiments, the CpG DNA contains the nucleotide sequence shown in SEQ ID NO 25 or SEQ ID NO 26:

[0207] 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′(SEQ ID NO 25)

[0208] 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO 26);

[0209] Wherein, each nucleotide in the CpG DNA is a deoxynucleotide, and each nucleotide in the CpG DNA is connected by a phosphorothioate bond. In some embodiments, the CpG DNA is the commercial adjuvant CpG 7909 or CpG 1018.

[0210] In some embodiments, the compositions of the present disclosure comprise an RNAi agent and one or more immune response modifiers; or comprise an RNAi agent and one or more adjuvants; or comprise an RNAi agent and one or more of a particulate adjuvant and a pathogen component; or comprise an RNAi agent and CpG DNA and / or an alum adjuvant. In some embodiments, the pharmaceutically active components of the compositions of the present disclosure comprise only an RNAi agent and CpG DNA. In some embodiments, the pharmaceutically active components of the compositions of the present disclosure comprise an RNAi agent and CpG DNA and / or an alum adjuvant. In some embodiments, the CpG DNA and alum adjuvant can be administered simultaneously or at different times, or simultaneously.

[0211] In some embodiments, in the composition of the present disclosure, the RNAi agent is a conjugate of the structure shown in formula (403), its sodium salt or partial sodium salt, wherein Nu represents an siRNA group, the siRNA group has a sense chain shown in SEQ ID NO: 11 and an antisense chain shown in SEQ ID NO: 12, and the siRNA group is formed by removing a hydrogen atom from the 3' hydroxyl group of the 3' terminal nucleotide of the sense chain; and the immune response regulator is CpG DNA shown in SEQ ID NO: 25, its sodium salt or partial sodium salt thereof.

[0212] For the above-mentioned siRNA, siRNA conjugates and immune response modifiers, those skilled in the art can easily prepare or commercially obtain them by appropriate prior art methods. For example, WO2015006740A2 describes in detail the preparation methods of various siRNA conjugates. WO2014025805A1 describes the preparation method of the structure shown in formula (305). Rajeev et al. described the preparation method of the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908. Chinese patent application CN110959011A also discloses in detail the method for preparing the siRNA conjugate shown in formula (308). The contents of the above-mentioned documents are incorporated herein by reference in their entirety. For another example, siRNA and CpG DNA can be obtained by nucleic acid solid phase synthesis methods well known in the art by connecting nucleoside phosphoramidite monomers one by one according to the base sequence of the siRNA nucleic acid sequence.

[0213] In some embodiments, the pharmaceutically acceptable salt of one or more of the siRNA, siRNA conjugate, and CpG DNA is a water-soluble salt or a partial salt. In some embodiments, the pharmaceutically acceptable salt is an alkali metal salt or a partial alkali metal salt. In some embodiments, the pharmaceutically acceptable salt is a sodium salt or a partial sodium salt. In some embodiments, the RNAi agent is a sodium salt of the siRNA conjugate, and the immune response modifier is a sodium salt of the CpG DNA.

[0214] In some embodiments, in order to facilitate transportation and / or storage, the siRNA and siRNA conjugates in the RNAi agent and the immune response regulator are independently present in the form of powders, for example, in the form of freeze-dried powder injections. When the administration is implemented, the freeze-dried powder injection is mixed with a liquid excipient to be formulated into a liquid agent. In some embodiments, for ease of use, the siRNA and siRNA conjugates in the RNAi agent and the immune response regulator are present in the form of various RNAi preparations and immune response regulator preparations commonly used in the art. For example, the RNAi agent and the immune response regulator preparation can each independently be a liquid agent, such as an injection. The liquid agent can be an injection for subcutaneous injection, an injection for intraperitoneal injection, an injection for intramuscular injection, or an injection for intravenous injection, or a spray administered to the lungs or to other organ tissues (such as the liver) via the lungs by spraying, or an inhaler inhaled by the oropharynx, or a pharmaceutical agent for nasal administration. Therefore, the RNAi agent contains at least one of siRNA and siRNA conjugate and a pharmaceutically acceptable carrier and / or excipient. The types and contents of the carrier and / or excipient in the injection for subcutaneous injection, the injection for intramuscular injection, the injection for intravenous injection, the spray for administration to the lungs or to other organs and tissues (such as the liver) through the lungs, the inhaler for inhalation through the oropharynx, or the pharmaceutical agent for nasal administration are well known to those skilled in the art. In some embodiments, the RNAi agent is an injection for subcutaneous injection.

[0215] additives

[0216] The pharmaceutical composition further contains an auxiliary agent, which is selected from one or more of a solvent and a pharmaceutically acceptable carrier.

[0217] When the pharmaceutical composition is an injection, the adjuvant comprises at least a solvent. The solvent may be, for example, deionized water, water for injection, ethanol, or a pH buffer. The pH buffer may be a tris-hydroxymethylaminomethane hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.

[0218] The amount of the solvent is adjusted according to the required solution concentration. Based on the nucleotide sequence groups in the conjugate, the concentration of the conjugate in the injection can be 0.01 mg / mL-20 mg / mL, 0.1 mg / mL-10 mg / mL, or 0.5 mg / mL-5 mg / mL.

[0219] The pharmaceutically acceptable carrier is one or more of various components conventionally used in the art, such as a protective agent, an osmotic pressure regulator, and one or more other pharmaceutically acceptable carriers.

[0220] The other pharmaceutically acceptable carriers may be carriers conventionally used in the art, such as, but not limited to, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine, PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate), ... phosphate), PPEEA) and poly (methacrylate-N, N-dimethylaminoethyl ester) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and one or more of their derivatives.

[0221] In some embodiments, the pharmaceutically acceptable carrier contains a physiologically acceptable compound that acts, for example, to stabilize the pharmaceutical composition or to increase or decrease the absorption of the conjugate and / or pharmaceutical composition. The physiologically acceptable compound is selected from one or more of the following compounds: carbohydrates, such as glucose, sucrose, and / or dextran; antioxidants, such as ascorbic acid and / or glutathione; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of any co-administered substances; excipients; stabilizers, and buffers. Detergents may also be used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition. The physiologically acceptable compound may also include one or more wetting agents, emulsifiers, dispersants, or preservatives specifically for preventing microbial growth or action. The physiologically acceptable compounds are known to those skilled in the art and will not be described in detail in this disclosure. It will be readily understood by those skilled in the art that the selection of a pharmaceutically acceptable carrier and a physiologically acceptable compound depends, for example, on the route of administration and the specific physiochemical properties of any co-administered substances.

[0222] In some embodiments, the pharmaceutically acceptable carrier is sterile and generally free of undesirable substances. The pharmaceutical compositions provided herein may further comprise pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the drug conjugate provided herein in the pharmaceutical composition may vary over a wide range and is primarily selected based on fluid volume, viscosity, body weight, and the like according to a specific administration route.

[0223] In some embodiments, there are no particular requirements for the content of the active pharmaceutical ingredient and the pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of the RNAi agent to the auxiliary agent, calculated as the siRNA in the RNAi agent, can be 1:(1-600). In some embodiments, the weight ratio is 1:(1-50). In some embodiments, the weight ratio of the immune response modifier to the auxiliary agent can be 1:(1-5000). In some embodiments, the weight ratio of the immune response modifier to the auxiliary agent can be 1:(1-5000).

[0224] In some embodiments, the carrier can be selected from an osmotic pressure regulator, which can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmoles / kilogram (mOsm / kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage of the agent made from the pharmaceutical composition during administration can be adjusted due to different modes of administration.

[0225] In some embodiments, the carrier may be selected from a protective agent, which may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be present in an amount of 0.01-30% by weight based on the total weight of the pharmaceutical composition.

[0226] In some embodiments, the pharmaceutical composition can be a liquid, such as an injection; or it can be a lyophilized powder injection, which is mixed with a liquid excipient during administration to form a liquid. The liquid can be, but is not limited to, for subcutaneous, intramuscular, or intravenous administration, and can also be delivered by, but is not limited to, puncture injection, or oropharyngeal inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is for subcutaneous, intramuscular, intravenous, or intrathecal administration. In some embodiments, the RNAi agent is in the form of a formulation for subcutaneous injection, and the immune response modifier is in the form of a formulation for intraperitoneal or subcutaneous injection.

[0227] Uses and treatment methods of the pharmaceutical composition disclosed herein

[0228] In another aspect, the present disclosure provides use of the pharmaceutical composition of the present disclosure in the preparation of a medicament for treating a disease associated with hepatitis B virus infection. In some embodiments, the disease associated with hepatitis B virus infection is one or more of inflammation, liver fibrosis, hepatic proliferative disease, liver failure, and hepatocellular carcinoma caused by hepatitis B virus infection. In some embodiments, inflammation caused by hepatitis B virus infection refers to hepatitis B and / or hepatitis D.

[0229] In another aspect, the present disclosure further provides a method for treating a disease associated with hepatitis B virus infection, the method comprising administering an effective amount of a pharmaceutical composition of the present disclosure to a subject in need thereof. In some embodiments, the method comprises administering an effective amount of an RNAi agent and an effective amount of an immune response modifier to a subject in need thereof, wherein the RNAi agent is capable of inhibiting mRNA expressed by HBV. In some embodiments, the disease associated with hepatitis B virus infection is selected from one or more of the following groups: hepatitis, liver fibrosis, and proliferative liver disease.

[0230] The pharmaceutical composition of the present disclosure can significantly reduce the levels of HBV antigens such as HBsAg and HBeAg, and HBV DNA in the subject. Furthermore, the pharmaceutical composition of the present disclosure can also induce the production of HBV antibodies in the subject, showing excellent potential for functional cure. In the context of the present disclosure, when HBV antigen, DNA content and HBV antibody content are calculated using logarithmic coordinates, the unit of measurement is log10 (IU / ml or S / CO), which means the logarithmic content value obtained by calculating the common logarithm of the concentration content (IU / ml or S / CO). For example, for example, the initial content is 5log 10 (IU / mL), reduced to 4 log 10 (IU / mL) concentration, the concentration decreased by 1log 10 (IU / mL), refers to the concentration from 10 5 IU / ml reduced to 10 4 The IU / mL decreases, that is, it decreases to 1 / 10 of the initial concentration.

[0231] In some embodiments, the treatment method of the present invention comprises administering the RNAi agent and the anti-hepatitis B virus antibody to a subject in need thereof over one or more cycles. In some embodiments, the treatment method of the present invention comprises 1-4 of the cycles. In some embodiments, the length of the cycle is 5 weeks to 120 weeks. In some embodiments, the length of the cycle is 5 weeks to 96 weeks. In some embodiments, the length of the cycle is 7 weeks, 50 days, 60 days, 70 days, 80 days, 12 weeks, 20 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or 60 weeks. In some embodiments, one of the cycles is referred to as one complete "course of treatment."

[0232] It will be understood by those skilled in the art that, after achieving functional cure of HBV in a subject, further treatment is generally not required. Therefore, in some embodiments, the treatment method disclosed herein does not require the completion of a final full course of treatment, and the administration is stopped when the HBsAg concentration in the subject's serum is below the detection limit of the test kit, 0.05 IU / mL. In some embodiments, the administration can be stopped when the absolute value of HBsAg in the subject's serum is below 100 IU / mL.

[0233] In some embodiments, in order to obtain better therapeutic effects of the prepared medicine, the RNAi agent and the immune response regulator are administered separately. In some embodiments, the subject is first administered the RNAi agent and then the immune response regulator. In some embodiments, the subject is first administered the immune response regulator and then the RNAi agent. In some embodiments, the time interval between the first administration of the RNAi agent and the first administration of the immune response regulator is 0 days to one month, for example, it can be 0 days, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks or 1 month. In some embodiments, the first administration of the RNAi agent and the immune response regulator is 2 weeks apart.

[0234] In some embodiments, the methods of the present disclosure include one or more treatment courses, wherein the RNAi agent and the immune response modifier are each independently administered one or more times during a treatment course. In some embodiments, in a treatment course, the subject is administered an effective amount of the RNAi agent followed by administration of the immune response modifier. In some embodiments, in a treatment course, the subject is administered 0.01-27 mg / kg of the RNAi agent followed by an initial administration of 0.05-3 mg of the immune response modifier.

[0235] In some embodiments, within one course of treatment, the RNAi agent is administered more than once, and the immune response modifier is administered more than once. In some embodiments, the RNAi agent is administered 1-5 times, and the immune response modifier is administered 1-6 times. In some embodiments, to achieve better therapeutic effects, the RNAi agent is administered 1-3 times, and the immune response modifier is administered 2-4 times.

[0236] In some embodiments, to reduce the total dosage and frequency of administration, the interval between each administration of the RNAi agent is 5 days to 60 weeks, 7 days to 60 weeks, or 7 days to 1 year. In some embodiments, to achieve better therapeutic effects, specifically, in some embodiments, the interval between each administration of the RNAi agent is 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, half a year, or 1 year.

[0237] In some embodiments, the interval between each administration of the immune response modifier is 1 day to 2 months, 2 days to 1 month, 3 days to 1 month, 1 week to 1 month, or 2 weeks to 1 month. Specifically, in some embodiments, the interval between each administration of the immune response modifier is 3 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month.

[0238] In some embodiments, during the preparation of the medicament, the RNAi agent and the immune response modifier are in a form suitable for administration to a subject once or more within a course of treatment, wherein the length of the course of treatment is 25-840 days. In some embodiments, the length of the course of treatment is 25-672 days, 25-504 days, 25-420 days, 25-336 days, 25-252 days, 25-168 days, 70-504 days, 70-336 days, or 70-168 days.

[0239] In some embodiments, within one course of treatment, the RNAi agent is administered more than once, and the immune response modifier is administered more than once. In some embodiments, the RNAi agent is administered 1-5 times, and the immune response modifier is administered 2-6 times. In some embodiments, to achieve better therapeutic effects, the RNAi agent is administered 1-3 times, and the immune response modifier is administered 3-4 times.

[0240] In some embodiments, to achieve a more desired therapeutic effect within a certain period of time, the interval between each administration of the RNAi agent is 5 days to 60 weeks, 7 days to 60 weeks, 7 days to 1 year, or 10 days to 40 weeks. In some embodiments, to achieve a better therapeutic effect, specifically, in some embodiments, the interval between each administration of the RNAi agent is 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, half a year, or 1 year.

[0241] In some embodiments, the interval between each administration of the immune response modifier is 1 day to 2 months, 2 days to 1 month, 3 days to 1 month, 1 week to 1 month, or 2 weeks to 1 month. Specifically, in some embodiments, the interval between each administration of the immune response modifier is 3 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month.

[0242] In some embodiments, the dosage of the RNAi agent for a single administration is 0.1 mg / kg-10 mg / kg, 0.2 mg / kg-8 mg / kg, 0.3 mg / kg-9 mg / kg, 0.5 mg / kg-6 mg / kg of the subject's body weight, based on the total amount of siRNA and siRNA groups. Specifically, in some embodiments, the dosage of the RNAi agent for a single administration is 0.1 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.8 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg of the subject's body weight.

[0243] In some embodiments, the single dose of the immune response modifier is 0.03-3 mg. In some embodiments, the single dose of the immune response modifier is 0.05-2 mg, 0.1-2 mg, or 0.12-1 mg. Specifically, in some embodiments, the single dose of the immune response modifier is 0.05 mg, 0.075 mg, 0.1 mg, 0.125 mg, 0.15 mg, 0.2 mg, 0.5 mg, or 0.8 mg.

[0244] In some embodiments, the RNAi agent and the immune response modifier are both administered multiple times. In some embodiments, the interval between each administration of the RNAi agent is 5 days to 60 weeks, and the interval between each administration of the immune response modifier is 1 day to 60 days. In some embodiments, the interval between each administration of the RNAi agent is 10 days to 40 weeks, and the interval between each administration of the immune response modifier is 5 days to 45 days.

[0245] In some embodiments, the immune response modifier is administered after an effective amount of the RNAi agent is administered, and the interval between administration of the RNAi agent and the immune response modifier is 5 days to 2 months. In some embodiments, the interval between administration of the RNAi agent and the immune response modifier is 7 days to 45 days. In some embodiments, the first administration of the immune response modifier is 2 weeks after the first administration of the RNAi agent.

[0246] In some embodiments, in one course of treatment, the subject is administered the RNAi agent 1-5 times, each time at 0.1-9 mg / kg of the subject's body weight, and then administered the immune response modifier 1-5 times, each time at 0.05-1 mg.

[0247] In some embodiments, the RNAi agent and the immune response modifier are both in the form of a formulation for subcutaneous injection. In some embodiments, the RNAi agent is in the form of a formulation for subcutaneous injection and the immune response modifier is in the form of a formulation for intraperitoneal injection.

[0248] In some embodiments, the administration of the immune response regulator can be the administration of one or more immune response regulators; or the administration of one or more adjuvants; or the administration of aluminum hydroxide adjuvant and / or CpG DNA. In some embodiments, the single administration of the immune response regulator is a single administration of CpG DNA. In some embodiments, the single administration of the immune response regulator is a single administration of alum adjuvant and CpG DNA respectively. In the case of a single administration of alum adjuvant and CpG DNA respectively, the dosage, interval and number of single administrations of the immune response regulator are calculated based on the dosage, interval and number of administrations of one of the adjuvants. In some embodiments, the dosage, interval and number of administrations of alum adjuvant and CpG DNA may be the same or different, or the same.

[0249] In some embodiments, the RNAi agent and the immune response modifier are present in a form suitable for administration to the subject during the course of treatment, and the course of treatment includes one or more courses of treatment. The number of courses of treatment can be determined based on relevant indicators such as hepatitis B surface antigen and HBV DNA, for example, HBsAg can be continuously reduced to below 300 IU / mL. In some embodiments, the course of treatment includes 1-4 courses of treatment. In some embodiments, it will be understood by those skilled in the art that after achieving functional cure of HBV in the subject, there is generally no need to continue treatment. The functional cure of HBV refers to a state in which the hepatitis B surface antigen (HBsAg) is negative (with or without the appearance of hepatitis B surface antibodies), HBV DNA is undetectable, and liver function indicators are normal. Therefore, in some embodiments, it is not necessary to complete the last full course of treatment, and the administration is stopped when the HBsAg concentration in the subject's serum is lower than the detection limit of the kit of 0.05 IU / mL. In some embodiments, the administration can be stopped when the absolute value of HBsAg in the subject's serum is below 100 IU / mL.

[0250] The definitions and selection ranges of the RNAi agent and the immune response modifier are as described above in the description of the pharmaceutical composition. In some embodiments, the RNAi agent is a sodium salt of an siRNA conjugate, wherein the siRNA group contained in the siRNA conjugate contains the nucleotide sequences shown in SEQ ID NOs: 11 and 12, and the immune response modifier is a sodium salt of CpG7909 containing the sequence shown in SEQ ID NO: 25.

[0251] Reagent test kit

[0252] In yet another aspect, the present disclosure further provides a kit comprising the pharmaceutical composition provided by the present disclosure.

[0253] In some embodiments, the kit described in the present disclosure may provide a pharmaceutical composition in a container. In some embodiments, the kit described in the present disclosure may include a container for providing a pharmaceutically acceptable excipient. In some embodiments, the kit may also include other ingredients, such as stabilizers or preservatives. In some embodiments, the kit described in the present disclosure may include at least one other therapeutic agent in a container other than the container providing the pharmaceutical composition described in the present disclosure. In some embodiments, the kit may include instructions for mixing the active pharmaceutical ingredient in the pharmaceutical composition with a pharmaceutically acceptable carrier and / or excipient or other ingredients (if any).

[0254] In the kits of the present disclosure, the pharmaceutical composition, the pharmaceutically active ingredient in the pharmaceutical composition, and / or the pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dry form, or lyophilized form. In some embodiments, the pharmaceutical composition, the pharmaceutically active ingredient in the pharmaceutical composition, and the optional pharmaceutically acceptable excipients are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kits of the present disclosure.

[0255] The present disclosure will be further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0256] Example

[0257] Unless otherwise specified, all reagents and culture media used in the following examples are commercially available products, and nucleic acid electrophoresis, real-time PCR and other operations used are performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0258] For the conjugates and CpG DNA synthesized and used in the following examples, unless otherwise specified, the obtained conjugates were sodium salts in which the hydroxyl hydrogen ions in all phosphate groups in the conjugates were replaced by sodium ions, and the obtained CpG DNA were sodium salts in which the hydroxyl hydrogen ions in all phosphate groups in the CpG DNA were replaced by sodium ions.

[0259] Preparation Example 1 Preparation of the pharmaceutical composition provided by the present disclosure

[0260] (1-1) Preparation of RNAi Agents in the Pharmaceutical Compositions of the Present Disclosure

[0261] According to the preparation method described in Preparation Example 13 of CN110959011A, conjugate 1 was prepared. The sense chain and antisense chain contained in conjugate 1 were the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the sense chain and antisense chain were synthesized respectively. After conjugate 1 was diluted to a concentration of 0.2 mg / mL (based on the amount of siRNA groups) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), the molecular weight was detected using liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). Among them, the theoretical molecular weight of the sense chain of conjugate 1 was 8218.83, the measured molecular weight was 8218, the theoretical molecular weight of the antisense chain was 7061.57, and the measured molecular weight was 7061.5. The measured values ​​were consistent with the theoretical values, indicating that the synthesized conjugate 1 was the target designed double-stranded nucleic acid sequence.

[0262] Conjugate 1 has a structure shown in formula (403), and the siRNA group contained in Conjugate 1 has the sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12:

[0263] 5'-CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 11);

[0264] 5'-VPUmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm-3' (SEQ ID NO: 12);

[0265] Among them, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group, and VP indicates that the nucleotide on the right of the letter VP is a 5'-vinyl phosphate-modified nucleotide.

[0266] (1-1-1) Preparation of Component I using Conjugate 1: The prepared Conjugate 1 was stored in the dark at 2-8°C. Conjugate 1 was diluted with phosphate buffer to a 1.8 mg / mL solution (calculated as siRNA groups), designated as Component I. Component I was administered at a concentration of 1.8 mg / mL at a rate of 5 mL / kg of subject body weight, resulting in a single dose of 9 mg / kg. Based on a 25 g mouse weight, each single dose of Component I injected into the mouse contained 0.225 mg of Conjugate 1 (calculated as siRNA).

[0267] (1-1-2) Preparation of component III using conjugate 1: Another conjugate 1 was diluted with phosphate buffer to a 0.6 mg / mL solution (calculated as siRNA group), recorded as component III. Component III was administered at a concentration of 0.6 mg / mL at a dose of 5 mL / kg of subject body weight, i.e., a single dose of 3 mg / kg. Based on the weight of a mouse of 25 g, each single dose of component III preparation injected into the mouse contained 0.075 mg of conjugate 1 (calculated as siRNA). (1-2) Preparation of the immune response modifier in the pharmaceutical composition provided by the present disclosure

[0268] By solid phase phosphoramidite method, nucleoside monomers were linked one by one in the 3'-5' direction according to the sequence shown in SEQ ID NO: 25.

[0269] 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′(SEQ ID NO 25)

[0270] Each nucleotide is a deoxyribonucleotide, and each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and sulfurization. Two nucleotides are linked using phosphorothioate. The synthesis conditions are as follows:

[0271] The nucleoside monomer was provided as a 0.1 M acetonitrile solution. The deprotection reaction conditions for each step were the same, namely, a temperature of 25° C., a reaction time of 70 seconds, a deprotection reagent of 3% v / v dichloroacetic acid in dichloromethane, and a molar ratio of 5:1 between dichloroacetic acid and the 4,4'-dimethoxytrityl protecting group on the solid support.

[0272] The coupling reaction conditions were the same for each step, including a temperature of 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a coupling reagent of 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT).

[0273] The capping conditions were identical for each step, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution consisted of a 1:1 molar ratio of CapA and CapB. CapA consisted of a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile), while CapB consisted of a 20% by volume acetic anhydride solution in acetonitrile. The molar ratio of capping reagent to nucleic acid sequence attached to the solid support was 1:1:1 (acetic anhydride:N-methylimidazole:nucleic acid sequence attached to the solid support).

[0274] The sulfurization reaction conditions for each step were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the sulfurization reagent being hydroxanthin. The molar ratio of the sulfurization reagent to the nucleic acid sequence attached to the solid support during the coupling step was 120:1. The reactions were performed in a 1:1 acetonitrile:pyridine mixture.

[0275] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the carrier is added to 25 wt% ammonia water, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55° C. for 16 h, the liquid is removed, and the mixture is concentrated to dryness in vacuo.

[0276] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) using a NaCl gradient elution. Specifically, the following conditions were used: eluent A: 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9:1 (volume ratio); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9:1 (volume ratio); the elution gradient was: eluent A:eluent B = 100:0 to 50:50. The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler, and elution with deionized water.

[0277] Testing: Purity was determined using ion exchange chromatography (IEX-HPLC) and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS). The measured values ​​were consistent with the theoretical values, confirming that the obtained product was the CpG 7909 compound (Fraction II) represented by SEQ ID NO: 25. Store in the dark at 2-8°C. Dilute 50 μg of Fraction II to 200 μL of solution with phosphate buffer and store separately as a single Fraction II preparation.

[0278] (1-3) Preparation of the Pharmaceutical Composition 1 of the Present Disclosure

[0279] Two parts of component I and three parts of component II were combined to prepare pharmaceutical composition 1, wherein each part of component I and each part of component II were stored in different containers. At this time, based on the amount of siRNA groups and immune response modifiers in the siRNA conjugate, the total weight ratio of component I to component II in the prepared pharmaceutical composition 1 was 3:1, and the total dosage ratio was 120 mg / kg mouse body weight: 1 mg. (1-4) Preparation of pharmaceutical composition 2 of the present disclosure

[0280] Pharmaceutical Composition 2 was prepared by combining 2 parts of Component III with 3 parts of Component II, wherein each part of Component III and each part of Component II were stored in separate containers. The total weight ratio of Component III to Component II in Pharmaceutical Composition 2, based on the amount of siRNA groups and immune response modifier in the siRNA conjugate, was 1:1, and the total dosage ratio was 40 mg / kg mouse body weight:1 mg.

[0281] (1-5) Preparation of the pharmaceutical composition 3 disclosed herein

[0282] 50 μg of alum adjuvant (AH, purchased from Beijing Puxitang Biotechnology Co., Ltd., product number A10853) was diluted to 200 μL of solution with phosphate buffer and stored separately as one AH preparation.

[0283] Pharmaceutical Composition 4 was prepared by combining 2 parts of Component I, 3 parts of Component II, and 3 parts of the AH formulation. Each part of Component I, each part of Component II, and each part of the AH formulation was stored in separate containers. The total weight ratio of Component I, Component II, and the AH formulation in Pharmaceutical Composition 4, based on the amount of siRNA moieties and immune response modifier in the siRNA conjugate, was 3:1:1, resulting in a total dosage ratio of 180 mg / kg mouse body weight:1 mg:1 mg.

[0284] Experimental Example 1 Anti-HBV Effect of the Pharmaceutical Composition 1 of the Present Disclosure in a Recombinant Adenovirus-Hepatitis B Virus (AAV-HBV) Transfected Mouse Model

[0285] The mice used in this experiment were 3-4 week old male C57BL / 6 mice, weighing approximately 25 g on average. They were purchased from Shanghai Lingchang Biotechnology Co., Ltd. and transferred from Lebotech Laboratory Unit 8326405S529. The animal production license number for this batch is SCXK(Shanghai)2018-0003, and the animal health certificate numbers are 20180003016137 and 20180003016138. Lebotech's experimental animal use license number is SYXK(Shanghai)2021-0001.

[0286] The ARCHITECT i2000 (Abbott Laboratories, Lake Bluff, IL, USA) and its supporting reagents used in this experiment were used to detect serum hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B surface antibody (HBsAb). The hepatitis B virus DNA (HBV DNA) detection kit was purchased from Shengxiang Biotechnology Co., Ltd. (Changsha, Hunan, China). The PCR instrument used was QuantStudio TM 3) Purchased from Applied Biosystems (Foster City, CA, USA).

[0287] The conjugate 1 used in this experiment is the sodium salt of conjugate 1 prepared in step (1-1) of Preparation Example 1. During the experiment, it was diluted with phosphate buffer solution to the required concentration and then administered by subcutaneous injection. The immune response regulator 1 used in this experiment is the CpG 7909 sodium salt compound prepared in step (1-2) of Preparation Example 1. During the experiment, it was diluted with phosphate buffer solution to the required concentration and then administered by intraperitoneal injection.

[0288] The pharmaceutical composition used in this experiment is Pharmaceutical Composition 1 prepared in Preparation Example 1.

[0289] The specific experimental methods of this experiment are as follows:

[0290] [1] Establishment of AAV-HBV mouse model

[0291] To simulate the HBV infection environment, 24 C57BL / 6 male mice were acclimated for 14 days and each mouse was injected with 200 μL (1×10 11 vg) AAV-HBV (purchased from Wuhan Shumi Brain Technology Co., Ltd., model: rAAV8-1.3HBV (ayw)), the injection day was recorded as the first day of the modeling period. The mice were weighed on the 1st, 22nd and 29th day of the modeling period, and serum samples of the mice were taken on the 22nd and 29th day of the modeling period. The amount of serum collected from each mouse was 15 μL, and the collected serum was used for quantitative detection of hepatitis B virus indicators HBsAg, HBeAg and HBV DNA content. Based on the measured values ​​on the 29th day, mice with qualified blood virology index levels after infection were selected for subsequent treatment experiments. The qualified level of blood virology indexes means that the HBsAg level in the mouse serum is 4.28log 10 IU / mL~4.90log 10 IU / mL, HBeAg level was 3.73log 10 S / CO~3.88log 10S / CO and HBV DNA levels were 6.98 log 10 IU / mL~8.64log 10 IU / mL, these levels are slightly higher than those typically observed in human patients with HBV infection and are suitable for evaluating long-term drug efficacy.

[0292] The 24 selected mice were randomly divided into 4 groups, each with 6 mice. From the first day to the end of the experiment, the health status of the animals was observed twice a day, and the body weight of the animals was measured once or twice a week.

[0293] The specific dosing regimen for each group is as follows:

[0294] Group 1: blank control group. On day 1 and day 15, mice were subcutaneously injected with 0.9% PBS buffer at a dose of 5 mL / kg of mouse body weight.

[0295] Group 2: Conjugate control group, on day 1 and day 15, mice were subcutaneously injected with 1.8 mg / mL solution of Conjugate 1 at a dose of 5 mL / kg mouse body weight, respectively. The dose was a single dose of 9 mpk (mg / kg) per mouse.

[0296] Group 3: immunomodulator control group, on days 29, 43, and 57, 50 μg of immunomodulator solution was injected into the peritoneal cavity of mice at a dose of 200 μL per mouse.

[0297] Group 4: Pharmaceutical composition group 1, on day 1 and day 15, the mice were subcutaneously injected with a 1.8 mg / mL concentration of component I solution at a dose of 5 mL / kg mouse body weight, with a single dose of 9 mpk (mg / kg) per mouse; and on days 29, 43 and 57, the mice were intraperitoneally injected with 50 μg of component II solution at a dose of 200 μL per mouse.

[0298] [2] Blood sample collection and quantitative detection of HBV virus-related indicators during the medication period:

[0299] Whole blood samples were collected from each mouse before dosing on day 1 and on days 8, 15, 22, 29, 36, 43, 57, 64, 71, and 78 to prepare serum for HBV DNA, HBsAg, HBeAg, and HBsAb testing. The experimental results are shown in Figures 1-3.

[0300] Figure 1 is a line graph showing the HBV DNA levels in the serum of mice in Groups 1 to 4 during the dosing period. The blank control group (Group 1) showed almost no decrease in HBV DNA levels. The immune response modifier control group (Group 3) showed only a slight decrease in HBV DNA levels, with the maximum decrease not exceeding 1 log. 10 (IU / mL). On the other hand, the conjugate control group (Group 2) achieved a maximum decrease of 2.57 log on day 43 after administration. 10 IU / mL, that is, the maximum HBV DNA inhibition rate reached 99.73%.

[0301] Compared with the control group, the pharmaceutical composition group 1 (group 4) of the present disclosure showed an exceptionally high HBV DNA inhibitory effect. HBV DNA levels rapidly decreased further after administration of component II and remained at a low level for up to 78 days after the first dose. The pharmaceutical composition group 1 (group 4) achieved the maximum reduction on day 36, with a 5.09 log reduction in HBV DNA. 10 IU / mL, that is, the maximum HBV DNA inhibition rate reached 99.9992%. Moreover, compared with the control group 2, the pharmaceutical composition of the present disclosure unexpectedly further significantly reduced the HBV DNA level, with a maximum reduction of 3.01log 10 IU / mL, that is, compared with the use of RNAi agents alone, the conjugate group has significantly reduced HBV DNA levels, and the HBV DNA level has been further significantly reduced by 99.9%. The reduction in HBV DNA levels far exceeds the sum of the inhibitory effects of using siRNA conjugates or immune response modifiers alone. Further results showed that among the 6 experimental animals in Group 4, the HBV DNA level of one experimental animal was reduced to the detection limit (10 3.18 IU / mL) or less.

[0302] Figure 2 is a line graph showing the changes in HBsAg levels in the serum of mice in groups 1 to 4 over time during the administration period, wherein the blank control group (group 1) showed almost no decrease in HBsAg levels. The immune response modifier control group (group 3) showed only a slight decrease in HBV DNA levels, with the maximum decrease not exceeding 0.3 log 10 On the other hand, the conjugate control group (Group 2) reached the maximum reduction on the 43rd day after administration, with a maximum reduction of 2.71 log (IU / mL) compared with the blank control group. 10 IU / mL, that is, the maximum HBsAg inhibition rate reached 99.80%.

[0303] Compared with the above control group, similar to the results of HBV DNA, the pharmaceutical composition group 1 of the present disclosure also unexpectedly showed an abnormally high HBsAg inhibition effect. The HBsAg level decreased rapidly after the first administration of component II and remained stable at a low level during the experimental period of up to 78 days. The pharmaceutical composition group 1 achieved the maximum reduction on the 64th day, with HBsAg reduced by 4.17 log 10 IU / mL, that is, the maximum HBsAg inhibition rate reached 99.9930%; and at the 78th day after the end of the experiment, there was still 4.14log 10 IU / mL of HBsAg levels were reduced, that is, the inhibition rate was still 99.9927%. Further, compared with the control group 2, the pharmaceutical composition 1 of the present disclosure was unexpectedly able to further significantly reduce the HBsAg level, with a maximum reduction of 1.65log 10 IU / mL, that is, compared with the use of RNAi agents alone, the conjugate group further reduced the HBsAg level by 97.76%, and the reduction was far greater than the sum of the inhibitory effects of the siRNA conjugate or immune response modifier alone. Furthermore, among the 6 experimental animals in Group 4, the HBsAg levels of 2 experimental animals decreased to the detection limit (10 0.18 IU / mL), and the HBsAg level of another experimental animal decreased below the detection limit on day 78.

[0304] Figure 3 is a line graph showing the changes in HBeAg levels in the serum of mice in Groups 1 to 4 over time during the administration period. The blank control group (Group 1) showed almost no decrease in HBeAg levels. The immune response modifier control group (Group 3) showed only a slight decrease in HBeAg levels, with the maximum decrease not exceeding 0.1 log. 10 On the other hand, the conjugate control group (Group 2) reached the maximum reduction on the 43rd day after administration, with a maximum reduction of about 1 log (IU / mL) relative to the blank control group. 10 IU / mL, that is, the maximum HbeAg inhibition rate reaches 90%.

[0305] Compared with the control group, the pharmaceutical composition group 1 (Group 4) of the present disclosure showed a higher HBeAg inhibition effect, and the HBeAg level after the first administration of component II showed an increase of about 0.3 log 10 It can be seen that compared with the use of RNAi agents alone, the pharmaceutical composition of the present disclosure can further reduce HBeAg levels, and the degree of reduction also exceeds the sum of the effects of using RNAi agents or immune response modifiers alone.

[0306] In addition, the test results showed that three mice in group 1 of the drug composition showed a significant increase in the content of HBsAb in the serum after administration, with the highest increase in HBsAb (mIU / mL) being 7.76×10 1 , 2.98×10 2 and 5.72×10 2 (mIU / mL).

[0307] Experimental Example 2 Anti-HBV Effect of Pharmaceutical Composition 2-3 of the Present Disclosure in a Recombinant Adenovirus-Hepatitis B Virus (AAV-HBV) Transfected Mouse Model

[0308] The mice used in this experiment were 3-4 week old male C57BL / 6 mice with an average weight of approximately 25 g, purchased from Shanghai Lingchang Biotechnology Co., Ltd. The ARCHITECT i2000 (Abbott Laboratories, Lake Bluff, IL, USA) and its supporting reagents used in this experiment were used to detect serum hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B surface antibody (HBsAb). The hepatitis B virus DNA (HBV DNA) detection kit was purchased from Shengxiang Biotechnology Co., Ltd. (Changsha, Hunan, China), and the PCR instrument used was QuantStudio TM 3) Purchased from Applied Biosystems (Foster City, CA, USA).

[0309] The conjugate 1 used in this experiment is the sodium salt of conjugate 1 prepared in step (1-1) of Preparation Example 1. During the experiment, it was diluted with phosphate buffer solution to the required concentration and then administered by subcutaneous injection; the immune response regulator 1 used in this experiment is the CpG 7909 sodium salt compound prepared in step (1-2) of Preparation Example 1. During the experiment, it was diluted with phosphate buffer solution to the required concentration and then administered by intraperitoneal injection; the AH preparation used in this experiment was diluted with phosphate buffer solution to the required concentration and then administered by intraperitoneal injection.

[0310] The pharmaceutical composition 2-3 used in this experiment is the pharmaceutical composition 2-3 prepared in Preparation Example 1.

[0311] The specific experimental methods of this experiment are as follows:

[0312] [1] Establishment of AAV-HBV mouse model

[0313] Following the procedures described in Experimental Example 1, 42 C57BL / 6 male mice were modeled. The 42 mice were randomly divided into seven groups of six mice each. From Day 1 to the endpoint of the study, the animals were observed twice daily for their health and weighed once or twice weekly.

[0314] The specific dosing regimen for each group is as follows:

[0315] Group 1: blank control group. On day 1 and day 15, mice were subcutaneously injected with 0.9% PBS buffer at a dose of 5 mL / kg of mouse body weight.

[0316] Group 2: Conjugate control group, on day 1 and day 15, mice were subcutaneously injected with 1.8 mg / mL solution of Conjugate 1 at a dose of 5 mL / kg mouse body weight, respectively. The dose was a single dose of 9 mpk (mg / kg) per mouse.

[0317] Group 3: CpG 7909 control group, 50 μg of CpG 7909 sodium salt solution was injected into the peritoneal cavity of mice at a dose of 200 μL per mouse on days 29, 43, and 57, respectively.

[0318] Group 4: AH preparation control group, on days 29, 43, and 57, 50 μg of AH preparation solution was injected into the peritoneal cavity of mice at a dose of 200 μL per mouse.

[0319] Group 5: Pharmaceutical composition group 2, on day 1 and day 15, the mice were subcutaneously injected with a 0.6 mg / mL concentration of component III solution at a dose of 5 mL / kg mouse body weight, with a single dose of 3 mpk per mouse; and on days 29, 43, and 57, the mice were intraperitoneally injected with 50 μg of component II solution at a dose of 200 μL per mouse.

[0320] Group 6: Group 3 of the pharmaceutical composition, on day 1 and day 15, mice were subcutaneously injected with a 1.8 mg / mL concentration of component I solution at a dose of 5 mL / kg mouse body weight, with a single dose of 9 mpk per mouse; and on days 29, 43, and 57, mice were intraperitoneally injected with 50 μg of component II solution and 50 μg of AH preparation solution at a dose of 200 μL per mouse, respectively.

[0321] [2] Blood sample collection and quantitative detection of HBV virus-related indicators during the medication period:

[0322] For each mouse in Group 1, Group 2, Group 3 and Group 5, whole blood samples were collected from each mouse 1 day and 3 days before administration, and on days 8, 15, 22, 29, 36, 43, 57, 64, 71, 78 and 85 to prepare serum for HBV DNA, HBsAg, HBeAg and HBsAb detection; for each mouse in Group 4 and Group 6, whole blood samples were collected from each mouse 1 day and 3 days before administration, and on days 8, 15, 22, 29, 36, 43 and 57 to prepare serum for HBV DNA, HBsAg, HBeAg and HBsAb detection. The experimental results are shown in Figures 4 to 6.

[0323] Figure 4 shows a line graph of HBV DNA levels in the serum of mice from Groups 1 to 6 during the dosing period. The blank control group (Group 1) and the AH formulation control group (Group 4) showed almost no decrease in HBV DNA levels. The CpG 7909 control group (Group 3) showed only a slight decrease in HBV DNA levels, with the maximum decrease being 1.53 log. 10 (IU / mL). On the other hand, the conjugate control group (Group 2) achieved a maximum decrease of 2 log on day 22 after administration. 10 IU / mL, that is, the maximum HBV DNA inhibition rate reached 99%.

[0324] Compared with the control group, the disclosed pharmaceutical composition group 2 (Group 5) showed an exceptionally high HBV DNA inhibition effect. Component III was administered to mice at a 3 mpk dose on day 1 and day 15, respectively. On day 29, after the first administration of component II, HBV DNA levels rapidly decreased and remained at low levels for up to 85 days after the first dose. The maximum reduction in HBV DNA in the pharmaceutical composition group 2 (Group 5) was achieved on day 64, with a 4.48 log reduction in HBV DNA. 10 IU / mL, that is, the maximum HBV DNA inhibition rate reached 99.9967%. Moreover, compared with the conjugate control group with a single RNAi agent dose of 9 mpk, the pharmaceutical composition 2 of the present disclosure further significantly reduced the HBV DNA level at a single RNAi agent dose of 3 mpk, with a maximum reduction of 2.76 log 10 IU / mL, that is, compared with the use of RNAi agents alone, on the basis of the significant reduction in HBV DNA levels in the conjugate group, compared with the conjugate control group (Group 2), the HBV DNA level was further reduced by 99.83%. The reduction in HBV DNA levels far exceeded the sum of the inhibitory effects of using siRNA conjugates or immune response regulators alone.

[0325] For Group 3 (Group 6), 9 mpk of Component I was administered to mice on day 1 and day 15, respectively. On day 29, after the first administration of AH formulation and Component II, HBV DNA levels decreased rapidly and reached the maximum reduction on day 36, with a 4.36 log reduction in HBV DNA. 10 IU / mL, that is, the maximum HBV DNA inhibition rate reached 99.9956%. Compared with the conjugate control group, the pharmaceutical composition 3 of the present disclosure further significantly reduced the HBV DNA level, with a maximum reduction of 2.61log 10 IU / mL, that is, compared with the use of RNAi agent alone, the HBV DNA level in the conjugate group was further reduced by 99.75% based on the significant reduction in HBV DNA level.

[0326] Figure 5 is a line graph showing the changes in HBsAg levels in the serum of mice in groups 1 to 6 over time during the administration period, wherein the blank control group (group 1) and the AH formulation control group (group 4) showed almost no decrease in HBsAg levels. The CpG 7909 control group (group 3) showed only a slight decrease in HBV DNA levels, with the maximum decrease not exceeding 0.3 log 10 On the other hand, the conjugate control group (Group 2) reached the maximum reduction on the 29th day after administration, with a maximum reduction of 2.24 log (IU / mL) compared with the blank control group. 10 IU / mL, that is, the maximum HBsAg inhibition rate reached 99.4246%.

[0327] Compared with the above-mentioned control group, similar to the results of HBV DNA, the pharmaceutical composition group 2 (group 5) of the present disclosure also unexpectedly showed an abnormally high HBsAg inhibitory effect. On the 1st and 15th days, the mice were given a 3 mpk dose of component III. On the 29th day, the HBsAg level decreased rapidly after the first administration of component II and remained stable at a low level during the 85-day experimental period. The pharmaceutical composition group 2 (group 5) reached the maximum reduction on the 64th day, with HBsAg reduced by 3.32 log 10 IU / mL, that is, the maximum HBsAg inhibition rate reached 99.9521%. Furthermore, compared with the conjugate control group 2, the pharmaceutical composition 2 of the present disclosure was unexpectedly able to further significantly reduce the HBsAg level, with a maximum reduction of 1.31log 10IU / mL, that is, compared with the use of RNAi agents alone, the conjugate group further reduced the HbsAg level by 95.1022% based on the already significant reduction in HbsAg levels, and the dose of the siRNA conjugate used was significantly reduced, thereby further reducing the cost of medication and any safety risks associated with drug dosage.

[0328] For Group 3 (Group 6), 9 mpk of Component I was administered to mice on day 1 and day 15, respectively. On day 29, after the first administration of AH formulation and Component II, HBV DNA levels decreased rapidly and reached the maximum reduction on day 36, with a 2.89 log reduction in HBV DNA. 10 IU / mL, that is, the maximum HBV DNA inhibition rate reached 99.87%. Compared with the conjugate control group, the pharmaceutical composition 3 of the present disclosure further significantly reduced the HBV DNA level, with a maximum reduction of 0.91log 10 IU / mL, that is, compared with the use of RNAi agent alone, the HBV DNA level in the conjugate group was further reduced by 87.69% based on the significant reduction in HBV DNA level.

[0329] Figure 6 is a line graph showing the changes in HBeAg levels in the serum of mice in groups 1 to 6 over time during the administration period. The blank control group (group 1), the CpG 7909 control group (group 3), and the AH formulation control group (group 4) showed almost no decrease in HBeAg levels. The conjugate control group (group 2) achieved the maximum decrease on day 64 after administration, with a maximum decrease of approximately 1.04 log (P < 0.05). 10 IU / mL, that is, the maximum HbeAg inhibition rate reached 90.88%.

[0330] Compared with the above control group, the pharmaceutical composition group 2 (Group 5) of the present disclosure showed an HBeAg inhibition effect similar to that of the conjugate control group (Group 2), and the pharmaceutical composition group 3 (Group 6) showed a better HBeAg inhibition effect than the conjugate control group, with the HBeAg level being reduced by about 0.325 log compared with the conjugate control group. 10 IU / mL further decreased.

[0331] In summary, the pharmaceutical composition provided by the present disclosure can induce HBsAb production in mice, indicating that while effectively inhibiting HBV antigens and DNA, it can also stimulate the immune response of mice, showing excellent prospects for achieving functional cure of hepatitis B.

[0332] Some embodiments of the present disclosure are described in detail above. However, the present disclosure is not limited to the specific details of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.

[0333] It should also be noted that the various specific technical features described in some of the above embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0334] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A pharmaceutical composition comprising a pharmaceutically active ingredient, wherein: The active components of the drug are composed of an RNAi agent and an immune response regulator, and the RNAi agent and the immune response regulator exist independently; the RNAi agent refers to one or more of an siRNA composition, an siRNA conjugate and a pharmaceutically acceptable salt thereof; the siRNA composition contains siRNA and a pharmaceutically acceptable carrier; the siRNA conjugate contains an siRNA group and a conjugated group conjugated to the siRNA group; the siRNA group refers to a group formed after removing one or more atoms or groups from siRNA, and the siRNA is an siRNA that can inhibit HBV mRNA; based on siRNA, the weight ratio of the RNAi agent to the immune response regulator is (0.5-5000):

1.

2. The pharmaceutical composition according to claim 1, wherein The weight ratio of the RNAi agent to the immune response regulator is (0.5-2000):1; (1-400):1; (2-150):1; or (2.4-50):

1.

3. The pharmaceutical composition according to claim 1, wherein In terms of siRNA, the dosage ratio of the RNAi agent to the immune response regulator is (0.02-540) mg / kg subject body weight: 1 mg; (0.03-400) mg / kg subject body weight: 1 mg; (0.06-380) mg / kg subject body weight: 1 mg; (0.06–180) mg / kg subject body weight: 1 mg; (0.1-80) mg / kg subject body weight: 1 mg; or (0.2-30) mg / kg subject body weight: 1 mg.

4. The pharmaceutical composition according to claim 1, wherein The RNAi agent refers to a siRNA conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA group, the linker and the targeting group are sequentially connected.

5. The pharmaceutical composition according to claim 4, wherein The RNAi agent refers to a siRNA conjugate having a structure as shown in formula (308) or a pharmaceutically acceptable salt thereof: in, n1 is an integer selected from 1-3, n3 is an integer selected from 0-4; Each m1, m2 or m3 is independently an integer selected from 2-10; R 10 , R 11 , R 12 , R 13 , R 14 or R 15 Each is independently H, or is selected from the group consisting of: C1-C 10 Alkyl, C1-C 10 Haloalkyl and C1-C 10 Alkoxy; R3 has the structure shown in formula (A59): Wherein, E1 is OH, SH or BH2, and Nu represents the siRNA group; R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C5-C 10 Cycloalkylene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein R2 may optionally have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl); Each L1 is independently a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein L1 may optionally have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl); represents the site of covalent attachment of a group; Each M1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

6. The pharmaceutical composition according to claim 5, wherein The RNAi agent refers to a conjugate having a structure as shown in formula (403) or a pharmaceutically acceptable salt thereof: Wherein, Nu represents the siRNA group.

7. The pharmaceutical composition according to claim 4, wherein The RNAi agent refers to a conjugate having a structure as shown in formula (305) or a pharmaceutically acceptable salt thereof: Wherein, Nu represents the siRNA group.

8. The pharmaceutical composition according to claim 1, wherein The siRNA composition comprises siRNA, a key lipid, an auxiliary lipid and a pegylated lipid, wherein the key lipid has a structure as shown in formula (214) or formula (215): The auxiliary lipid is cholesterol, a cholesterol analog and / or a cholesterol derivative; The PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000; The molar ratio of the organic amine, the auxiliary lipid and the PEGylated lipid is (19.7-80): (19.7-80): (0.3-50); or, the molar ratio of the organic amine, the auxiliary lipid and the PEGylated lipid is (50-70): (20-40): (3-20).

9. The pharmaceutical composition according to claim 1, wherein The siRNA comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are both composed of 19 nucleotides, each nucleotide in the nucleotide sequence I and the nucleotide sequence II is a modified or unmodified nucleotide, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, the nucleotide sequence II is at least partially reverse complementary to the first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence with a length of 19 nucleotides in HBV mRNA.

10. The pharmaceutical composition according to claim 9, wherein The sense strand and antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 20-26 nucleotides.

11. The pharmaceutical composition according to claim 9, wherein The nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1, and has no more than 3 nucleotide differences, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:2, and has no more than 3 nucleotide differences: 5'-CCUUGAGGCAUACUUCAAZ1-3' (SEQ ID NO:1); 5'-Z2UUGAAGUAUGCCUCAAGG-3' (SEQ ID NO:2); Wherein, Z1 is A, Z2 is U, the nucleotide sequence I contains a nucleotide Z3 corresponding to Z1, the nucleotide sequence II contains a nucleotide Z4 corresponding to Z2, and Z4 is the first nucleotide at the 5' end of the antisense strand; Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:3, and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:4, and differs by no more than 3 nucleotides: 5'-GUGUGCACUUCGCUUCACZ5-3' (SEQ ID NO: 3); 5'-Z6GUGAAGCGAAGUGCACAC-3' (SEQ ID NO: 4); Wherein, Z5 is A, Z6 is U, the nucleotide sequence I contains a nucleotide Z7 corresponding to Z5, the nucleotide sequence II contains a nucleotide Z8 corresponding to Z6, and Z8 is the first nucleotide at the 5' end of the antisense strand; Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:5, and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:6, and differs by no more than 3 nucleotides: 5'-GGACUUCUCUCAAUUUUCZ9-3' (SEQ ID NO: 5); 5′-Z 10 GAAAAUGAGAGAGUCC-3'(SEQ ID NO:6)? Among them, Z9 is U, Z 10 A, the nucleotide sequence I contains a nucleotide Z corresponding to position Z9 11 The nucleotide sequence II contains a position corresponding to Z 10 The nucleotide Z 12 , the Z 12 is the first nucleotide at the 5' end of the antisense strand; Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:7, and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:8, and differs by no more than 3 nucleotides: 5'-CUGUAGGCAUAAAUUGGUZ 13 -3'(SEQ ID NO:7); 5'-Z 14 ACCAAUUUAUGCCUACAG-3'(SEQ ID NO:8); Among them, Z 13 A, Z 14 is U, the nucleotide sequence I contains a position corresponding to Z 13 The nucleotide Z 15 The nucleotide sequence II contains a position corresponding to Z 14 The nucleotide Z 16 , the Z 16 is the first nucleotide at the 5' end of the antisense strand; Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:9, and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:10, and differs by no more than 3 nucleotides: 5'-GUGCACUUCGCUUCACZ 17 -3'(SEQ ID NO:9); 5'-Z 18 ACCAAUUUAUGCCUACAG-3'(SEQ ID NO:10); Among them, Z 17 A, Z 18 is U, the nucleotide sequence I contains a position corresponding to Z 17 The nucleotide Z 19 The nucleotide sequence II contains a position corresponding to Z 18 The nucleotide Z 20 , the Z 20 It is the first nucleotide at the 5' end of the antisense strand.

12. The pharmaceutical composition according to claim 1, wherein The immune response regulator is selected from one or more of an adjuvant or an immunostimulant, the adjuvant is selected from one or more of agents capable of promoting an immune response, and the immunostimulant is selected from one or more of independently administrable agents capable of stimulating an immune response; Alternatively, the adjuvant is selected from the group consisting of pathogen components, particulate adjuvants, and combination adjuvants; Alternatively, the pathogen component is selected from monophosphatidyl lipid A (MPL), poly (I:C), polyICLC adjuvant, CpG DNA, c-di-AMP, c-di-GMP, c-di-CMP; short, blunt-ended 5′-triphosphate dsRNA (3pRNA) RIG-1 ligand and emulsion; Alternatively, the particulate adjuvant is selected from alum, virosomes, cytokines.

13. The pharmaceutical composition according to claim 12, wherein The immune response regulator is selected from one or more TLR agonists; or, the immune response regulator is selected from one or more TLR9 agonists.

14. The pharmaceutical composition according to claim 13, wherein The immune response regulator is selected from one or more of alum adjuvant, CpG DNA or pharmaceutically acceptable salts thereof; or, the CpG DNA contains a nucleotide sequence as shown in SEQ ID NO 25 or SEQ ID NO 26: 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO 25) 5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO 26); Wherein, each nucleotide in the CpG DNA is a deoxynucleotide, and each nucleotide in the CpG DNA is connected by a phosphorothioate bond.

15. The pharmaceutical composition according to claim 1, wherein The RNAi agent is a conjugate of the structure shown in formula (403), its sodium salt or a partial sodium salt thereof, wherein Nu represents a siRNA group, the siRNA group has a sense strand as shown in SEQ ID NO: 11 and an antisense strand as shown in SEQ ID NO: 12, and the siRNA group is formed by removing a hydrogen atom from the 3' hydroxyl group of the 3' terminal nucleotide of the sense strand; 5'-CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 11); 5'-VP- UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm-3' (SEQ ID NO: 12); The immune response regulator is the CpG DNA shown in SEQ ID NO: 25, its sodium salt or its partial sodium salt and / or alum adjuvant.

16. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition further contains an auxiliary agent, which is combined with the RNAi agent and / or the immune response modifier to form a pharmaceutical preparation, and the auxiliary agent is selected from one or more of a solvent, a pharmaceutically acceptable carrier, and a pharmaceutically acceptable excipient; Alternatively, based on the siRNA in the RNAi agent, the weight ratio of the RNAi agent to the auxiliary agent is 1:(1-600), or 1:(1-50); the weight ratio of the immune response regulator to the auxiliary agent is 1:(1-5000), or 1:(1-500).

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein The pharmaceutically acceptable salt of one or more of the siRNA, the siRNA conjugate and the CpG DNA is a water-soluble salt or a partial salt; or, the pharmaceutically acceptable salt is an alkali metal salt or a partial alkali metal salt.

18. The use according to claim 1, wherein the RNAi agent is in the form of a preparation for subcutaneous injection, and the immune response regulator is in the form of a preparation for intraperitoneal injection or subcutaneous injection.

19. Use of the pharmaceutical composition according to any one of claims 1 to 18 in the preparation of a medicament for treating diseases associated with hepatitis B virus infection.

20. The use according to claim 19, wherein The disease related to hepatitis B virus infection is inflammation caused by hepatitis B virus infection, one or more of liver fibrosis, liver proliferative disease, liver failure and hepatocellular carcinoma; or inflammation caused by hepatitis B virus infection refers to hepatitis B and / or hepatitis D.

21. A method for treating a disease associated with hepatitis B virus infection, the method comprising administering to a subject an effective amount of the pharmaceutical composition according to any one of claims 1 to 17.

22. The method of claim 21, wherein: The method comprises one or more treatment courses, wherein the RNAi agent and the immune response modifier are each independently administered one or more times in a treatment course; Alternatively, in one course of treatment, after administering an effective amount of the RNAi agent to the subject, the immune response regulator is administered for the first time; or, in one course of treatment, after administering 0.01-27 mg / kg of the RNAi agent to the subject, 0.03-3 mg of the immune response regulator is administered for the first time; Alternatively, in one course of treatment, the subject is administered 0.1-9 mg / kg of the subject's body weight of the RNAi agent each time in 1-5 administrations, and then the immune response modifier is administered 1-5 times, each time at 0.05-1 mg.

23. The method of claim 22, wherein: In terms of siRNA, the single-administration dosage of the RNAi agent is 0.05 mg-200 mg, and the single-administration dosage of the immune response regulator is 0.05-2 mg; Alternatively, calculated on the basis of siRNA, the single-administration dosage of the RNAi agent is 4mg-200mg, and the single-administration dosage of the immune response regulator is 0.1mg-2mg; alternatively, calculated on the basis of siRNA, the single-administration dosage of the RNAi agent is 6mg-180mg, and the single-administration dosage of the immune response regulator is 0.12mg-1mg.

24. The method of claim 22, wherein: The single dose of the RNAi agent is 0.05-3.5 mg / kg of the subject's body weight, calculated as siRNA, and the single dose of the immune response regulator is 0.1-2 mg; or, the single dose of the RNAi agent is 0.1-3 mg / kg of the subject's body weight, calculated as siRNA, and the single dose of the immune response regulator is 0.12-1 mg; Alternatively, calculated on the basis of siRNA, the single-administration dose of the RNAi agent is 2.5-12 mg / kg of the subject's body weight, and the single-administration dose of the immune response regulator is 20-200 μg; alternatively, calculated on the basis of siRNA, the single-administration dose of the RNAi agent is 3-9 mg / kg of the subject's body weight, and the single-administration dose of the immune response regulator is 30-50 μg.

25. The method according to any one of claims 22 to 24, wherein the administration is multiple times, the interval between each administration of the RNAi agent is 5 days to 60 weeks, and the interval between each administration of the immune response regulator is 1 day to 60 days; or The interval between each administration of the RNAi agent is 10 days to 40 weeks, and the interval between each administration of the immune response regulator is 5 days to 45 days; or The immune response regulator is administered after administering an effective amount of the RNAi agent, and the administration interval between the RNAi agent and the immune response regulator is 5 days to 2 months, or the administration interval between the RNAi agent and the immune response regulator is 7 days to 45 days.

26. The method of claim 23, wherein: The first single dose of the immune response regulator is administered after administration of 12 mg-200 mg of the RNAi agent; or, the first single dose of the immune response regulator is administered after administration of 0.3 mg-0.5 mg of the RNAi agent.

27. The method according to any one of claims 21 to 26, wherein: The disease related to hepatitis B virus infection is inflammation caused by hepatitis B virus infection, one or more of liver fibrosis, liver proliferative disease, liver failure and hepatocellular carcinoma; or inflammation caused by hepatitis B virus infection refers to hepatitis B and / or hepatitis D.

28. A kit comprising the pharmaceutical composition according to any one of claims 1 to 17.

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