Novel double-stranded siRNA (small interfering ribonucleic acid) as well as conjugate and application thereof

Abstract

The invention provides a novel double-stranded siRNA (small interfering Ribonucleic Acid) as well as a conjugate and application thereof, and the double-stranded siRNA and the conjugate thereof can inhibit the expression of an APOC3 gene and can be used for preparing a medicine for treating and / or preventing APOC3 related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of small nucleic acid drugs, and the object of the present invention is to provide a novel double-stranded siRNA, its conjugate and its use. The double-stranded siRNA and its conjugate of the present invention can be prepared into a drug for treating and / or preventing APOC3-related diseases. Background Art

[0002] Apolipoprotein C-III (also known as APOC3, apoC-III, APOC-III and APOC-III) encoded by the human apolipoprotein C-III gene has recently become a promising target for treating diseases related to hypertriglyceridemia. Elevated serum triglyceride (TG) levels have been identified as an independent risk factor for cardiovascular diseases and as a contributing factor in the development of atherosclerosis. Individuals with severe hypertriglyceridemia (usually >1000 mg / dL) are also at risk of recurrent pancreatitis. Triglycerides are transported in the blood as the main component of very low density lipoprotein cholesterol (VLDL-C) and chylomicron particles (known as TG-rich lipoproteins). Lipoproteins consist of a hydrophobic core of triglycerides and cholesterol esters and a hydrophilic outer layer of phospholipids, cholesterol and apolipoproteins. APOC3 is one of these apolipoproteins.

[0003] APOC3 is mainly synthesized in the liver and plays an important role in the production, metabolism and clearance of TG-rich lipoproteins in plasma. Currently, several gain-of-function polymorphisms have been identified in the promoter region of the APOC3 gene, which are presumed to be contributing factors in the development of hypertriglyceridemia. Increased APOC3 synthesis in the liver promotes the secretion of TG-rich VLDL-C. In addition, excessive APOC3 inhibits the activities of lipoprotein lipase and hepatic lipase, further increasing serum TG levels by delaying the catabolism of TG-rich lipoproteins. In addition, elevated APOC3 also delays the hepatic clearance of TG-rich lipoproteins and their remnant particles by interfering with the binding of TG-rich lipoproteins to hepatic receptors.

[0004] An increase in APOC3 levels causes the development of hypertriglyceridemia or high (hyper) blood concentrations (emia) of triglycerides. Elevated triglyceride levels are associated with a variety of diseases, including cardiovascular disease, atherosclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes (insulin resistance), hypertension, and skin lesions (xanthomas). Very high triglyceride levels also increase the risk of acute pancreatitis. Therefore, modulating APOC3 metabolism may be a novel and important therapeutic approach for managing hypertriglyceridemia and related diseases. RNA interference (RNAi) refers to the phenomenon of highly conserved homologous mRNA degradation induced by small interfering ribonucleic acid (siRNA) in the process of evolution. It is of great significance to develop siRNAs targeting APOC3 gene expression. Summary of the Invention

[0005] The present invention provides a novel double-stranded siRNA and its conjugate, which can achieve cleavage of the RNA transcript of the APOC3 gene mediated by the RNA-induced silencing complex (RISC). The present invention also provides the use of the double-stranded siRNA and its conjugate in the preparation of a drug for treating and / or preventing APOC3-related diseases (such as dyslipidemia). The double-stranded siRNA and its conjugate can inhibit or reduce APOC3 gene expression, and are used to inhibit APOC3 gene expression by cleavage of the RNA transcript of the APOC3 gene mediated by the RNA-induced silencing complex (RISC). The siRNA and its conjugate of the present invention have good APOC3 inhibitory activity both in vitro and in vivo, have high in vivo delivery efficiency and good stability, and have high gene expression inhibitory activity and / or low toxicity to APOC3.

[0006] On the one hand, the present invention provides a double-stranded siRNA, its conjugate or salt, which comprises a sense strand and an antisense strand forming a double-stranded region. The sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 2, or a nucleotide sequence having 0, 1, 2, 3, 4 or 5 nucleotide differences therefrom; wherein, the detailed information of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 2 can be found in Table 1 of the specification of the present invention.

[0007] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 9 to SEQ ID NO: 10, or a nucleotide sequence having 0, 1, 2, 3, 4 or 5 nucleotide differences therefrom;

[0008] Among them, the detailed information of the nucleotide sequences shown in SEQ ID NO:9 to SEQ ID NO:10 can be found in Table 1 of the specification of the present invention.

[0009] In some embodiments of the present invention, the double-stranded siRNA, its conjugate or salt is used for APOC3 gene expression.

[0010] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the double-stranded siRNA is a modified double-stranded siRNA.

[0011] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA with a conjugating group.

[0012] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

[0013] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the length of the sense strand is 19, 20, 21, 22 or 23 nucleotides long, and the length of the sense strand does not exceed 23 nucleotides.

[0014] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the length of the antisense strand is 21, 22 or 23 nucleotides long.

[0015] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the length of the sense strand does not exceed 23 nucleotides, and the length of the sense strand does not exceed 23 nucleotides.

[0016] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the sense strand comprises a 3'-overhang and / or a 5'-overhang, and each of the 3'-overhang and the 5'-overhang independently comprises 1, 2 or 3 nucleotides.

[0017] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the antisense strand comprises a 3'-overhang and / or a 5'-overhang, and each of the 3'-overhang and the 5'-overhang independently comprises 1, 2 or 3 nucleotides.

[0018] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA, its conjugate or salt comprises one of the double-stranded siRNAs shown in siRNA ID NO:1 to siRNA ID NO:2. Among them, the nucleotide sequence information of the double-stranded siRNAs shown in siRNA ID NO:1 to siRNA ID NO:2 can be found in Table 1 of the specification of the present invention.

[0019] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand and the antisense strand each independently comprise at least one modified nucleotide, and the modified nucleotides each independently are selected from at least one of the following:

[0020] Nucleotides with unnatural bases, deoxynucleotides, 2'-fluoro-modified nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-O-alkyl-modified nucleotides (such as 2'-methoxy-modified nucleotides), 2'-O-alkoxyalkyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, locked nucleic acids (LNA), unlocked nucleic acid-modified nucleotides (UNA), 2'-allyl-modified nucleotides, abasic nucleotides, invAb (i.e., invB)-modified nucleotides, morpholino-modified nucleotides, tetrahydropyran-modified nucleotides, cyclohexenyl-modified nucleotides, PEG-modified nucleotides, 5'-aminophosphate-modified nucleotides, phosphorothioate-linkage-modified nucleotides (such as: 5'-thiophosphate-linkage-modified nucleotides and / or the 2'-thiophosphate-linkage-modified nucleotides), 5'-methylphosphonate-modified nucleotides (such as 5'-(E)-VP-modified nucleic acids), 5'-vinylphosphate-modified nucleic acids, 5'-phosphate mimetic-modified nucleotides, TNA-modified nucleotides, PNA-modified nucleotides, D-FNA-modified nucleotides, HNA-modified nucleotides, FNA-modified nucleotides, bcDNA-modified nucleotides, tcDNA-modified nucleotides, S-MC-modified nucleotides, N-MC-modified nucleotides, 2'-F-NMC-modified nucleotides, 5-methylcytosine-modified nucleotides, 5-methyluracil-modified nucleotides, 2,6-diamino-modified adenine-modified nucleotides, and glycol nucleic acids (GNA). Among them, the 2'- refers to the 2-position of ribose. For example, 2'-fluoro-modified nucleotides refer to nucleotides in which the 2-position of ribose is substituted by fluorine. Again, for example, 2'-methoxy-modified nucleotides refer to nucleotides in which the 2-position of ribose is substituted by methoxy.

[0021] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, each of the sense strand and the antisense strand independently comprises at least one modified nucleotide, and each of the modified nucleotides is independently selected from at least one of the following:

[0022] 2'-methoxy-modified nucleotide, 2'-fluoro-modified nucleotide, 2'-O-methoxyethyl-modified nucleotide, 2'-methoxyethyl-modified nucleotide, 5'-thiophosphate-linked modified nucleotide, 2'-thiophosphate-linked modified nucleotide, 2'-deoxy-modified nucleotide, 2'-amino-modified nucleotide, 2'-hydroxy-modified nucleotide, locked nucleic acid-modified nucleotide, unlocked nucleic acid (UNA), glycol nucleic acid (GNA), 5'-vinyl phosphate-modified nucleotide, 5'-(E)-VP-modified nucleotide, 5'-thiophosphate-linked modified nucleotide or 2'-thiophosphate-linked modified nucleotide, invAb-modified nucleotide (equivalent to InvB in the sequence of the present invention), i-substituted nucleotide and Y-substituted nucleotide, wherein, the Y is the i is wherein, the 2'- refers to the 2-position of ribose, for example, 2'-fluoro-modified nucleotide refers to a nucleotide in which the 2-position of ribose is substituted by fluorine, and further, 2'-methoxy-modified nucleotide refers to a nucleotide in which the 2-position of ribose is substituted by methoxy.

[0023] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, each of the modified nucleotides independently exists at one or more positions selected from the following:

[0024] the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st positions starting from the nucleotide at the 5'-end of the sense strand.

[0025] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, each of the modified nucleotides independently exists at one or more positions selected from the following:

[0026] the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd and 23rd positions starting from the nucleotide at the 5'-end of the antisense strand.

[0027] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 5'-thiophosphate linkage modification and the 2'-thiophosphate linkage modification independently exist at one or more positions selected from the following:

[0028] Between the 1st-2nd, 2nd-3rd, 3rd-4th, 4th-5th, 5th-6th, 6th-7th, 7th-8th, 8th-9th, 9th-10th, 10th-11th, 11th-12th, 12th-13th, 13th-14th, 14th-15th, 15th-16th, 16th-17th, 17th-18th, 18th-19th, 19th-20th, and 20th-21st positions counted from the nucleotide at the 5'-end of the sense strand as the starting point.

[0029] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 5'-thiophosphate linkage modification and / or the 2'-thiophosphate linkage modification independently exist at one or more positions selected from the following:

[0030] Between the 1st-2nd, 2nd-3rd, 3rd-4th, 4th-5th, 5th-6th, 6th-7th, 7th-8th, 8th-9th, 9th-10th, 10th-11th, 11th-12th, 12th-13th, 13th-14th, 14th-15th, 15th-16th, 16th-17th, 17th-18th, 18th-19th, 19th-20th, 20th-21st, 21st-22nd, and 22nd-23rd positions counted from the nucleotide at the 5'-end of the antisense strand as the starting point.

[0031] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 2'-fluoro-modified nucleotide, the 2'-methoxy-modified nucleotide, and Y independently exist at one or more positions selected from the following:

[0032] The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st positions counted from the nucleotide at the 5'-end of the sense strand as the starting point.

[0033] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 2'-fluoro-modified nucleotide, the 2'-methoxy-modified nucleotide, and Y independently exist at one or more positions selected from the following:

[0034] The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, and 23rd positions starting from the nucleotide at the 5'-end of the antisense strand.

[0035] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the 2'-fluoro-modified nucleotides are each independently present at one or more positions selected from the following:

[0036] The 5th, 7th, 8th, 9th, 10th, and 11th positions starting from the nucleotide at the 5'-end of the sense strand.

[0037] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the 2'-fluoro-modified nucleotides are each independently present at one or more positions selected from the following:

[0038] The 2nd, 6th, 8th, 9th, 12th, 14th, and 16th positions starting from the nucleotide at the 5'-end of the antisense strand.

[0039] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the 5'-thiophosphate linkage modification and the 2'-thiophosphate linkage modification are each independently present at one or more positions selected from the following:

[0040] Between the 1st - 2nd, 2nd - 3rd, and 3rd - 4th positions starting from the nucleotide at the 5'-end of the sense strand.

[0041] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the 5'-thiophosphate linkage modification and the 2'-thiophosphate linkage modification are each independently present at one or more positions selected from the following:

[0042] Between the 1st - 2nd, 2nd - 3rd, and 3rd - 4th positions starting from the nucleotide at the 3'-end of the sense strand.

[0043] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the 5'-thiophosphate linkage modification and the 2'-thiophosphate linkage modification are each independently present at one or more positions selected from the following:

[0044] Between the 1st - 2nd, 2nd - 3rd, and 3rd - 4th positions starting from the nucleotide at the 5'-end of the antisense strand.

[0045] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 5'-thiophosphate group linkage modification and the 2'-thiophosphate group linkage modification independently exist at one or more positions selected from the following:

[0046] Between the 1st-2nd, 2nd-3rd, and 3rd-4th positions starting from the nucleotide at the 3'-end of the antisense strand.

[0047] The "at least one modified nucleotide" according to the present invention refers to that in the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modified nucleotides, and / or the antisense strand includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modified nucleotides.

[0048] The "one or more positions" in the "each independently optionally exists at one or more positions selected from the following" or "each independently exists at one or more positions selected from the following" according to the present invention refers to the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 positions of modification, where optionally means that it may be modified or may not be modified, that is, 0 modifications.

[0049] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded region is 17-23 nucleotide pairs long.

[0050] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand contains one of the nucleotide sequences shown in SEQ ID NO:3 to SEQ ID NO:5, the length of the sense strand does not exceed 21 nucleotides, and the detailed information of SEQ ID NO:3 to SEQ ID NO:5 is shown in Table 1 of the specification of the present invention.

[0051] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand contains one of the following sequences:

[0052] csusuaaaAfgGfGfAfcaguauusa

[0053] InvBsasgggacAfgUfAfUfucucagusa and

[0054] InvBscsuuaaaAfgGfGfAfcaguauusa, and the length of the sense strand does not exceed 21 nucleotides.

[0055] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NOs: 11 to 12, the length of the antisense strand does not exceed 23 nucleotides, and the detailed information of the nucleotide sequences of SEQ ID NOs: 11 to 12 can be found in Table 1 of the specification of the present invention.

[0056] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA, its conjugate or salt comprises one of the double-stranded siRNAs shown in siRNA ID NOs: 3 to 5, and the detailed information of the nucleotide sequences of siRNA ID NOs: 3 to 5 can be found in Table 1 of the specification of the present invention.

[0057] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA with a conjugating group.

[0058] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded region is 17, 18, 19, 20, 21, 22 or 23 nucleotide pairs long.

[0059] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, in the double-stranded siRNA conjugate, the 3'-end or 5'-end of the sense strand and antisense strand of the double-stranded siRNA are each independently conjugated with a conjugating group.

[0060] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, in the double-stranded siRNA conjugate, the 3'-end of the sense strand of the double-stranded siRNA is conjugated with the conjugate.

[0061] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, in the double-stranded siRNA conjugate, the 3'-end and 5'-end of the sense strand of the double-stranded siRNA are each independently conjugated with the conjugating group through a phosphodiester bond or a phosphorothioate bond.

[0062] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugating group includes GalNAc or its derivative.

[0063] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugating group is GalNAc or its derivative connected by a divalent, trivalent or tetravalent branched linker.

[0064] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention,

[0065] the conjugating group is DAW40007-4, L-96, NAG37 or its stereoisomer, wherein the structures of the conjugating groups DAW40007-4, L-96 and NAG37 are respectively:

[0066]

[0067]

[0068] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the antisense strand contains two phosphorothioate backbone modifications between the three terminal nucleotides at the 3'-end and 5'-end respectively.

[0069] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand contains two phosphorothioate backbone modifications between the three terminal nucleotides at the 3'-end and 5'-end.

[0070] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand contains one of the nucleotide sequences shown in SEQ ID NO: 6 to SEQ ID NO: 8, the length of the sense strand does not exceed 23 nucleotides, and the detailed nucleotide information of SEQ ID NO: 6 to SEQ ID NO: 8 can be found in Table 1 of the specification of the present invention.

[0071] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the antisense strand contains one of the nucleotide sequences shown in SEQ ID NO: 11 to SEQ ID NO: 12, the length of the antisense strand does not exceed 25 nucleotides, and the detailed nucleotide information of SEQ ID NO: 11 to SEQ ID NO: 12 can be found in Table 1 of the specification of the present invention.

[0072] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA, its conjugate or salt comprises one of the nucleotide sequences shown in siRNA ID NO:6 to siRNA ID NO:8; wherein, the length of the sense strand does not exceed 23 nucleotides, the length of the antisense strand does not exceed 25 nucleotides, and the detailed nucleotide information of siRNA ID NO:6 to siRNA ID NO:8 can be found in Table 1 of the specification of the present invention.

[0073] In some embodiments of the double-stranded siRNA, its conjugate or salt, double-stranded siRNA conjugate or its salt according to the present invention, the double-stranded siRNA or its conjugate or salt further comprises (R)- and (S)-enantiomers, diastereoisomers, and / or their racemic mixtures.

[0074] In some embodiments of the double-stranded siRNA, its conjugate or salt, double-stranded siRNA conjugate or its salt according to the present invention, the phosphorothioate moiety of the double-stranded siRNA or its conjugate comprises (R)- and (S)-enantiomers, diastereoisomers, and / or their racemic mixtures.

[0075] On the other hand, the present invention provides a pharmaceutical composition comprising the double-stranded siRNA, its conjugate or its salt, double-stranded siRNA conjugate or its salt according to the present invention and a pharmaceutically acceptable carrier.

[0076] In some embodiments, the pharmaceutical composition according to the present invention may be an injection solution.

[0077] In some embodiments, the injection solution according to the present invention can be used for subcutaneous, intramuscular or intravenous injection.

[0078] On the other hand, the present invention also provides a method for inhibiting the expression of APOC3 gene in a patient, which comprises administering to the patient the double-stranded siRNA and double-stranded siRNA conjugate or its composition (i.e., double-stranded RNAi agent) according to the present invention, and the nucleic acid ligand conjugate or its composition can be a therapeutically effective amount.

[0079] In some embodiments, the double-stranded siRNA, double-stranded siRNA conjugate or its composition is administered at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg, or at a dose of 10 mg / kg to 30 mg / kg, or at a dose of 3 mg / kg, or at a dose of 10 mg / kg.

[0080] In some embodiments, the double-stranded siRNA, double-stranded siRNA conjugate or composition thereof is administered twice a week at a dose of 0.5 mg / kg, or once every other week at a dose of 10 mg / kg, or once a week at a dose of 0.5 - 1 mg / kg.

[0081] In some embodiments, the double-stranded siRNA, double-stranded siRNA conjugate or composition thereof is administered subcutaneously or intravenously.

[0082] In some embodiments, the double-stranded siRNA, double-stranded siRNA conjugate or composition thereof is administered in two or more doses.

[0083] In another aspect, the present invention provides the use of the double-stranded siRNA, its conjugate and its salt, the double-stranded siRNA conjugate or its salt, and the pharmaceutical composition described in the present invention in the preparation of a drug for treating and / or preventing APOC3-related diseases.

[0084] In some embodiments of the use described in the present invention, the APOC3-related diseases are hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome or familial partial lipodystrophy

[0085] Definitions and General Terms of the Present Invention

[0086] In the present invention, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0087] Unless otherwise specified, in the present invention, the term "Small interfering RNA (siRNA)" refers to a double-stranded RNA comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently about 17 to 30 nucleotides in length. siRNA mediates the targeted cleavage of RNA transcripts of the RISC pathway by forming a RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and the conversion into proteins.

[0088] The double-stranded region of the double-stranded siRNA of the present invention can be 12-30 nucleotide pairs in length. For example, the duplex region can be 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length or 21-23 nucleotide pairs in length. In another embodiment, the duplex region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotide pairs in length.

[0089] Unless otherwise stated, in the present invention, the term "antisense strand (or guide strand)" refers to a sequence 17-30 nucleotides in length that includes a region that is substantially complementary to the target sequence. The "sense strand (or lagging strand)" refers to a sequence 17-30 nucleotides in length that is substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the internal or terminal regions of the molecule, where the most tolerated mismatches exist within the terminal regions, for example, within 5, 4, 3 or 2 nucleotides at the 5'- and / or 3'-ends of the siRNA.

[0090] It should be noted that the "substantially complementary" or "at least partially complementary" of the antisense strand to the mRNA means that the antisense strand contains a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest.

[0091] The term "nucleotide overhang" or "protruding end" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of the siRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of the siRNA duplex extends beyond the 5'-end of the other strand, and vice versa. The siRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 or more nucleotides. The nucleotide overhang can include or be composed of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs can be on the sense strand, antisense strand or any combination thereof. Additionally, one or more nucleotides of the overhang can be present at the 5'-end, 3'-end or both ends of the antisense or sense strand of the siRNA.

[0092] The conjugating groups described in the present invention include pharmaceutically acceptable conjugating groups. Generally speaking, pharmaceutically acceptable conjugating groups include pharmaceutically acceptable targeting molecules and optionally a linker. For the types of exemplary conjugating groups, linkers, and targeting molecules, reference can be made to the disclosures of WO2015006740A2 and CN114555188A. Exemplary conjugating groups include but are not limited to L96, NAG37, or DAW40007-4 described in the present invention.

[0093] Unless otherwise specified, "conjugation" means that two or more chemical moieties each having a specific function are connected to each other in a covalent linkage manner; correspondingly, "conjugate" means a compound formed by covalent connection between individual chemical moieties.

[0094] The double-stranded siRNA conjugate described in the present invention is a compound formed by connecting double-stranded siRNA and a pharmaceutically acceptable conjugating group, and the double-stranded siRNA and the pharmaceutically acceptable conjugating group are covalently linked.

[0095] When used for the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well-known in the pharmaceutical field. The said methods include the step of combining the active ingredient with excipients constituting one or more accessory components. Generally, the composition is prepared by uniformly and sufficiently combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0096] In the present invention, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" described in the present invention refers to those listed in the generally recognized pharmacopoeia for use in animals, especially in humans.

[0097] In the present invention, the term "pharmaceutically acceptable carrier" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form.

[0098] In addition to any conventional carrier, the scope that is incompatible with the RNAi (such as siRNA) of the present invention, for example, producing any adverse biological effects or interacting with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope contemplated by the present invention.

[0099] In some embodiments, for the pharmaceutical composition according to the present invention, the pharmaceutically acceptable carrier may be various carriers conventionally used in the art. For example, it may include at least one of a pH buffer, a protecting agent, and an osmotic pressure regulator. The pH buffer may be acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5 - 8.5 and / or a phosphate buffer with a pH of 5.5 - 8.5, preferably a phosphate buffer with a pH of 5.5 - 8.5. The protecting agent may be at least one of inositol, sorbitol, and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protecting agent may be 0.01 - 30% by weight (such as 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, or any value between any two of the above values). The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200 - 700 milliosmoles per kilogram. Those skilled in the art can determine the content of the osmotic pressure regulator according to the required osmotic pressure.

[0100] In the present invention, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect, which may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. The "treatment" used in the present invention covers diseases of mammals, especially humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is prone to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, such as blocking the development of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease. The "treatment" used in the present invention covers any administration of a drug, an RNAi reagent, or an siRNA to an individual to treat, cure, alleviate, improve, reduce, or inhibit the disease of the individual, including but not limited to administering a drug containing the RNAi reagent, siRNA, or siRNA conjugate of the present invention to an individual in need.

[0101] The "RNAi reagent" described in the present invention refers to a reagent containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can degrade or inhibit the transcription and translation of a target messenger RNA (mRNA) in a sequence-specific manner. In the present invention, the RNAi reagent may act through the RNA interference mechanism (i.e., by interacting with the RNA interference pathway constitutive mechanism (RNA-induced silencing complex or RISC) of mammalian cells to induce RNA interference), or act through any other mechanism or pathway.

[0102] Unless otherwise specified, in the context of the present invention, the capital letters C, G, U, and A represent the bases of natural nucleotides; lowercase letters represent bases with a 2'-O-methyl modification on the ribose of the nucleotide, such as c, g, u, and a represent 2'-OMe (2'-O-methyl)C, 2'-OMe G, 2'-OMe U, and 2'-OMe A, respectively; the letter f to the right of a capital letter represents a base with a 2'-fluorine modification on the ribose of the nucleotide, such as Cf, Gf, Uf, and Af represent 2'-F (2'-fluorine)C, 2'-F G, 2'-F U, and 2'-F A, respectively; "s" represents that the two nucleotide residues adjacent to "s" on the left and right are linked by a phosphorothioate group. For example, "gsu" represents that the g and u residues are linked by a phosphorothioate group; Tgn represents a thymine-diol nucleotide residue, and its structure is Y in the double-stranded siRNA or its conjugate of the present invention represents i represents

[0103]

[0104] TNA, PNA, D-FNA, ANA-5, HNA-5, FANA-5, ANA-6, HNA-6, FANA-6, bcDNA, tcDNA, S-MC, N-MC, and 2'-F-NMC of the present invention have the following structures respectively:

[0105]

[0106] Among them, B is a base (including natural bases (A, U, G, C, or T) or modified bases), and each of TNA, PNA, D-FNA, ANA-5, HNA-5, FANA-5, ANA-6, HNA-6, FANA-6, bcDNA, tcDNA, S-MC, N-MC, and 2'-F-NMC is independently linked to the rest or the conjugate group through a phosphodiester bond or a phosphorothioate bond.

[0107] The invAb described in the present invention represents a reverse abasic deoxynucleoside residue, and it has the following structure: and

[0108] The structure of the unlocked nucleic acid-modified nucleoside (UNA) of the present invention is The structure of the glycol nucleic acid-modified nucleoside (GNA) described is Among them, B is a base (including natural bases (A, U, G, C, or T) or modified bases).

[0109] In the present invention, A, U, G, C or T is a base or a nucleoside containing a base. Whether it is specifically a base or a nucleoside containing a base should be understood according to the common knowledge in the art. For example, "in the present invention" where B is a base (A, U, G, C or T) or a modified base", A, U, G, C or T here should be understood as bases A, U, G, C or T without sugar; for another example, A, U, G, C or T in Table 1 of the present invention should be understood as nucleosides containing bases A, U, G, C or T.

[0110] In the present invention, "phosphate group", "phosphate ester group", "phosphate ester bond" can be used interchangeably, including mono-phosphate, di-phosphate or tri-phosphate. The "phosphate ester group" in "phosphorothioate group" also has the same meaning. Unless otherwise specified, the phosphate ester group between natural nucleotides is a di-phosphate group.

[0111] In the invention, "deoxynucleotide" refers to a nucleotide after the hydroxyl group in the pentose of the nucleotide is deoxygenated, and the position of deoxygenation can be 2'-OH or 3'-OH.

[0112] In some optional embodiments of the present invention, the deoxynucleotide includes 3'-deoxy-modified nucleotides and 2'-deoxy-modified nucleotides.

[0113] In the present invention, "2'-deoxy modification" means that the hydroxyl group (2'-OH) in the pentose of the nucleotide is deoxygenated to hydrogen (2'-H), and "3'-deoxy modification" means that the hydroxyl group (3'-OH) in the pentose of the nucleotide is deoxygenated to hydrogen (3'-H).

[0114] In the present invention, "2'-X modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by X (2'-X). For example, "2'-fluoro modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by fluorine (2'-F); "2'-amino modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by an amino group (2'-NH2); "2'-O-allyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by an allyloxy group (2'-OCH2CH=CH2); "2'-alkyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by an alkyl group (2'-alkyl), wherein the alkyl group may be substituted by a suitable substituent, such as being substituted by an alkoxy group, and the 2'-substituted alkyl-modified nucleotide may be a 2'-alkoxyalkyl-modified nucleotide (such as a 2'-methoxyethyl-modified nucleotide), wherein the hydroxyl group (2'-OH) in the pentose of the nucleotide is replaced by an alkoxyalkyl group (2'-alkoxyalkyl); "2'-O-alkyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by an alkoxy group (2'-alkoxy), wherein the alkoxy group may be substituted by a suitable substituent, such as being substituted by an alkoxy group, and the 2'-substituted alkoxy-modified nucleotide may be a 2'-alkoxyalkoxy-modified nucleotide (such as a 2'-methoxyethoxy-modified nucleotide), "2'-methoxy modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by a methoxy group (2'-OCH3), "2'-O-methoxyethyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide by a methoxyethoxy group (2'-MOE, 2'-O-CH2CH2OCH3).

[0115] In the present invention, "locked nucleic acid" refers to a nucleotide obtained after modification by linking the carbon atoms at the 2' and 4' positions on the pentose of the nucleotide together.

[0116] In the present invention, "5'-X modification" refers to the replacement of the phosphate ester (5'-PO(OH)2) in the pentose of a nucleotide by X (5'-X). For example, "5'-aminophosphate modification" refers to the replacement of the phosphate ester group (5'-PO(OH)2) in the pentose of a nucleotide by an aminophosphate group, "5'-thiophosphate modification" refers to the replacement of the phosphate ester group (5'-PO(OH)2) in the pentose of a nucleotide by a thiophosphate group, "5'-methylphosphonate modification" refers to the replacement of the phosphate ester group (5'-PO(OH)2) in the pentose of a nucleotide by a methylphosphonate group, "5'-phosphate mimetic modification" refers to the replacement of the phosphate ester group (5'-PO(OH)2) in the pentose of a nucleotide by a phosphate mimetic.

[0117] In the present invention, "5'-methylcytosine modification" or "5-methylcytosine modification" refers to the methylation of the carbon atom at the 5th position of cytosine; "5'-methyluracil modification" or "5-methyluracil modification" refers to the methylation of the carbon atom at the 5th position of uracil.

[0118] In the present invention, in 2'-fluorinated modified nucleotides, 2'-aminated modified nucleotides, 2'-O-allyl modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-alkoxyalkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-allyl modified nucleotides, the "2'" refers to the group at the 2nd position of ribose being modified (replaced) by the corresponding group, where "2'" and "2’" can be interchanged. Similarly, according to the understanding of the knowledge in the art, in the present invention, "5'" and "5’" can be interchanged, and "3'" and "3’" can be interchanged.

[0119] As used in the present invention, "chemical modification" or "modification" means a structure that has a chemical difference compared to the naturally occurring counterpart, including all changes by chemical means, such as the addition or removal of a chemical moiety, or the replacement of one chemical moiety with another.

[0120] The compounds described in the present invention can be asymmetric. For example, they can have one or more stereoisomers. Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are included in the present invention. The compounds containing asymmetric carbon atoms described in the present invention can be isolated in optically pure form or in racemic form. The optically pure form can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.

[0121] In addition, it should be noted that unless otherwise explicitly indicated, in the present invention, the description methods "each... independently", "... independently of each other", and "... independently" can be interchanged and should be understood in a broad sense. It can either mean that among different modification options, the specific options expressed between the same symbols do not affect each other, or it can also mean that among the same modification options, the specific options expressed between the same symbols do not affect each other.

[0122] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, in which the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereomers are resolved by conventional methods known in the art, and the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (such as formation of carbamates from amines).

[0123] The present invention also includes isotopically labeled compounds having the same structure as those compounds described in the present invention, but in which one or more atoms are replaced by atoms having an atomic weight or mass number different from the atomic weight or mass number commonly found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0124] Unless otherwise specified, when a position is specifically designated as deuterium (D), that position is understood to have a deuterium abundance greater than the natural abundance of deuterium (which is 0.015%) by at least 1000-fold (i.e., at least 10% deuterium incorporation). The deuterium abundance of the compounds in the examples can be at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold or higher than the natural abundance of deuterium. The present invention also includes various deuterated forms of the compound of formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of the compounds of the present invention by referring to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of the compounds of the present invention, or they can be synthesized using conventional techniques with deuterated reagents, including but not limited to deuterated borane, tetrahydrofuran solution of trideuterated borane, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, etc.

[0125] The conjugating group described in the present invention can enhance the delivery of a therapeutic agent to a specific target location (e.g., a specific organ or tissue) within an object such as a human or an animal. In some embodiments of the present invention, the conjugating group can enhance the targeted delivery of an inhibitory double-stranded siRNA. In some embodiments of the present invention, the conjugating group can enhance the delivery of an inhibitory double-stranded siRNA to the liver.

[0126] The conjugating group described in the present invention can be directly or indirectly linked to a compound, such as a therapeutic agent, e.g., an inhibitory double-stranded siRNA, e.g., the 3' or 5' end of an inhibitory double-stranded siRNA. In some embodiments of the present invention, the conjugating group is linked to the 3' end of the sense strand of the double-stranded siRNA. In some embodiments of the present invention, the conjugating group is linked to the 5' end of the sense strand of the double-stranded siRNA. In some embodiments, the conjugating group disclosed in the present invention is linked to an inhibitory double-stranded siRNA at the 3' end of the sense strand of the double-stranded siRNA via a phosphate, phosphorothioate or phosphonate group.

[0127] The term "composition" refers to a mixture of a drug containing one or more compounds described in the present invention (such as the double-stranded siRNA or its conjugate) or its physiologically pharmaceutically acceptable salt or precursor, and other components, including but not limited to pharmaceutically acceptable carriers and / or excipients. The purpose of the composition is to facilitate the administration to an organism, facilitate the absorption of the active ingredient and then exert its biological activity.

[0128] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable vehicle" includes, but is not limited to, any acceptable adjuvant, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent or emulsifying agent that has been approved for use in humans or domestic animals.

[0129] Unless otherwise specified, the "compounds", "nucleic acid conjugates", "double-stranded siRNA conjugates", "double-stranded siRNAs", and "nucleic acids" of the present invention can each independently exist in the form of salts, mixed salts, or non-salts (e.g., free acids or free bases). When in the form of salts or mixed salts, they can be pharmaceutically acceptable salts.

[0130] The term "hydroxy protecting group" refers to a labile chemical moiety that protects a hydroxy group from unwanted reactions during one or more synthetic procedures. After the one or more synthetic procedures, the hydroxy protecting group can be selectively removed. Hydroxy protecting groups known in the art are generally described in T.H. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York (1999). Examples of hydroxy protecting groups of the present invention include, but are not limited to, C 1-10 alkyl methyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-furyloxycarbonyl, allyloxycarbonyl, acetyl (Ac or -C(O)CH3), formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl (Bz or -C(O)C6H5), C 1-10 alkyl (methyl, tert-butyl, etc.), 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, C 6-10 aryl C 1-4 alkyl (such as benzyl, phenethyl, etc.), p-methoxybenzyl diphenylmethyl, triphenylmethyl (triphenylmethyl or trityl), tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, mesyl, tosyl, C 1-10Alkylsilyls (such as trimethylsilyl (TMS or -Si(CH3)3), triethylsilyl, triisopropylsilyl, MMTr, DMTr, or 4',4',4'-trimethoxytrityl, etc.).

[0131] The term "amino protecting group" refers to an unstable chemical moiety that protects an amino group from unwanted reactions during a synthetic procedure. After one or more such synthetic procedures, the amino protecting group, as described herein, can be selectively removed. Amino protecting groups known in the art are generally described in T.H. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York (1999). Examples of amino protecting groups include, but are not limited to, acetyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, and benzyloxycarbonyl, etc.

[0132] The term "solid support" specifically refers to any particle, bead, or surface on which oligonucleotide synthesis can occur. For example, both inorganic solid supports and organic solid supports can be optionally used in the embodiments of the present invention. The inorganic solid support is preferably selected from silica gel and controlled-pore glass beads (Controlled-pore glass, abbreviated as CPG). The organic solid support is a resin, preferably a macroporous resin, more preferably highly cross-linked polystyrene, Tentagel (a graft copolymer composed of a low cross-linked polystyrene matrix with polyethylene glycol (PEG or POE) grafted thereon), polyvinyl acetate (PVA), Poros - a copolymer of polystyrene / divinylbenzene, amino polyethylene glycol, and cellulose, etc. Preferred embodiments of the present invention utilize a CPG-based solid support. Many other commercially available solid supports fall within the scope of the present invention.

[0133] Unless otherwise specified, in the case of conflict or inconsistency between the sequence information in the specification of the present invention and the sequence information in the Sequence Listing (ST26 Sequence Listing), the sequence information recorded in the specification shall prevail.

[0134] Drug compositions, formulations, administrations, and methods of treating diseases of the nucleic acid conjugates of the present invention

[0135] The effective amount of the nucleic acid conjugate (such as the siRNA conjugate or its pharmaceutical composition as described in the present invention) can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art based on various factors (such as through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc.

[0136] The pharmaceutical composition or drug as described in the present invention comprises a pharmacologically effective amount of at least one APOC3 double-stranded siRNA agent and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance intentionally included in a drug delivery system in addition to the active pharmaceutical ingredient (API, therapeutic product, such as the APOC3 double-stranded siRNA agent of the present invention). An excipient does not play or is not intended to play a therapeutic role at the expected dosage. Excipients can function to a) assist in the processing of the drug delivery system during manufacture, b) protect, support or enhance the stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other properties of the overall safety and effectiveness of API delivery during storage or use. A pharmaceutically acceptable excipient can be or not be an inert substance.

[0137] Excipients can include, but are not limited to: absorption enhancers, anti-adhesives, anti-foaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents and wetting agents.

[0138] A pharmaceutical composition suitable for injection comprises a sterile aqueous solution (water-soluble). For subcutaneous or intravenous administration, suitable carriers can include physiological saline, bacteriostatic water, ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the production and storage conditions and should prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol) and their suitable mixtures.

[0139] A sterile injectable solution can be prepared by incorporating the required amount of the active compound, with or without one or a combination of the ingredients listed above, into a suitable solvent and then filtering the solution under sterile conditions. Generally, a dispersion is prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above.

[0140] APOC3 RNAi agents can be formulated into compositions in unit dosage form for ease of administration and uniformity of dosage. A unit dosage form refers to a physically discrete unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. In some embodiments, the unit dosage is from about 1 mg to about 100 mg of the APOC3 nucleic acid conjugate drug substance. In some embodiments, the unit dosage is from about 10 mg to about 100 mg of the APOC3 nucleic acid conjugate drug substance. In some embodiments, the unit dosage is from about 10 mg to about 50 mg of the APOC3 RNAi drug substance. In some embodiments, the unit dosage is about 10 mg of the APOC3 nucleic acid conjugate drug substance. In some embodiments, the unit dosage is about 25 mg of the APOC3 RNAi drug substance. In some embodiments, the unit dosage is about 50 mg of the APOC3 nucleic acid conjugate drug substance. In some embodiments, the unit dosage is about 100 mg of the APOC3 RNAi drug substance. In some embodiments, the unit dosage is from about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 or 98 to about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100 mg of the APOC3 nucleic acid conjugate drug substance.

[0141] It should be understood that in some cases, the initial dose administered can be increased beyond the upper limit levels described above to rapidly achieve the desired blood level or tissue level, or in some cases, the initial dose can be less than the optimal value. For example, in some embodiments, an initial dose or a first dose of about 1 mg to about 100 mg of an APOC3 nucleic acid conjugate drug substance is administered, followed by a second dose of about 1 to 100 mg of an APOC3 nucleic acid conjugate drug substance administered about 1 month later, and thereafter an additional dose is administered once every three months (e.g., once per calendar quarter or once every 12 weeks (q12w)).

[0142] The drug can be administered to a subject by any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal and sublingual administration), preferably intravenous administration.

[0143] In some embodiments, the pharmaceutical composition can be administered by intravenous infusion over a period of time, such as within a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about 25 minute time period. For example, the administration can be repeated regularly, such as weekly, biweekly (i.e., every two weeks), for one month, two months, three months, four months, or longer. After an initial treatment regimen, the treatment can be given at a lower frequency. For example, after administering weekly or biweekly for three months, the administration can be repeated monthly for six months, one year, or longer.

[0144] In some embodiments, the pharmaceutical composition can be administered by subcutaneous administration. The pharmaceutical composition can be administered once daily, or the nucleic acid conjugate can be administered as two, three, or more sub-doses at appropriate intervals during the day, or even delivered using continuous infusion or via a controlled release formulation. In such cases, the amount of nucleic acid conjugate contained in each sub-dose must be correspondingly less to obtain the total daily dose. Composite dosage units can also be used to deliver over several days, for example, using conventional sustained release formulations that provide a continuous release of the nucleic acid conjugate over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents to specific sites, such as can be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. A higher dose (i.e., a loading dose) can be administered initially, followed by a lower dose over a continuous period.

[0145] In some embodiments, a single dose of the pharmaceutical composition can be long-acting such that subsequent doses are administered at intervals of no more than 3, 4 or 5 days, or at intervals of no more than 1, 2, 3 or 4 weeks. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In other embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once every two months. In certain embodiments, the nucleic acid conjugate is administered from about once a month to about once every three months (i.e., about once every quarter).

[0146] The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and lipid-containing formulations. These compositions can be produced from a variety of components, including but not limited to preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Particularly preferred are formulations that target the liver when treating liver disorders (such as liver cancer).

[0147] The pharmaceutical preparations of the present invention (which can conveniently be in unit dosage form) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include steps such as combining these active ingredients with the pharmaceutical carrier or excipient(s). Generally, these preparations are prepared by uniformly and finely combining these active ingredients with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0148] The compositions of the present invention can be formulated into any of many possible dosage forms, such as but not limited to tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous media or mixed media. The aqueous suspension can further contain substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension can also contain stabilizers.

[0149] The pharmaceutical compositions disclosed in the present invention include preparations suitable for parenteral administration. The preparations can conveniently be in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of the active ingredient combined with the excipient substances to prepare a single dose form is generally the amount of siRNA that produces a therapeutic effect. Generally, on a percentage basis, this amount is about 1% to about 99% active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0150] The siRNA conjugate or its pharmaceutical composition of the present invention provides a significant TG-lowering effect and is used for treating APOC3-related diseases and disorders, such as hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome (FCS), chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndromes including familial partial lipodystrophy, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, hypertriglyceridemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, and other dyslipidemia and metabolism-related disorders and diseases. In some embodiments, the methods disclosed in the invention can treat APOC3-related diseases or disorders by significantly reducing TG levels and thus reducing the risk of developing hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome (FCS), chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndromes including familial partial lipodystrophy, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, hypertriglyceridemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, or other dyslipidemia or metabolism-related disorders and diseases. In the present invention, the "pharmaceutical composition" can refer to being used for the treatment of diseases and can also be used for in vitro cell culture experiments.

[0151] General synthetic methods of the compounds, double-stranded siRNAs, and double-stranded siRNA conjugates of the present invention

[0152] Generally, the compounds and nucleic acid conjugates of the present invention can be prepared by the methods described in the present invention. The following reaction schemes and examples are used to further illustrate the content of the present invention.

[0153] For the embodiments described below, unless otherwise indicated, all temperatures are in degrees Celsius (°C). The chromatographic column used is a silica gel column. The silica gel (200 - 300 mesh) was purchased from Qingdao Marine Chemical Factory, and all NH2 CPGs were purchased from Hebei Dina Xingke. Nuclear magnetic resonance spectra were recorded in CDC13, DMSO-d6, CD3OD, or acetone-d6 as solvents (in ppm), using TMS (0 ppm) or chloroform (7.25 ppm) as reference standards. When multiple peaks appear, the following abbreviations will be used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets), br.s (broadened singlet), q (quartet). The coupling constant J is expressed in Hertz (Hz).

[0154] Low-resolution mass spectrometry (MS) data was determined using a spectrometer of Agilent 6320 series LC-MS equipped with a G1312A binary pump and a G1316A TCC (column temperature maintained at 30 °C), with a G1329A autosampler and a G1315B DAD detector applied for analysis, and an ESI source applied for the LC-MS spectrometer.

[0155] High-resolution mass spectrometry (MS) data was determined using a spectrometer of Agilent 6130 series LC-MS equipped with a G1311A quaternary pump and a G1316A TCC (column temperature maintained at 30 °C), with a G1329A autosampler and a G1315D DAD detector applied for analysis, and an ESI source applied for the HR-MS spectrometer.

[0156] The following abbreviations are used throughout this invention:

[0157] DCM Dichloromethane DMTrCl 4,4'-Dimethoxytrityl chloride TFA Trifluoroacetic acid TFA Trifluoroacetic acid

[0158] Py Pyridine i-Pr Isopropyl

[0159] PE Petroleum ether EA Ethyl acetate

[0160] TBAF Tetrahydrofuran solution of tetrabutylammonium fluoride HOBT 1-Hydroxybenzotriazole

[0161] ACN Acetonitrile DMAP 4-Dimethylaminopyridine

[0162] Ac2O Acetic anhydride, MsCl Methanesulfonyl chloride

[0163] Boc tert-Butyloxycarbonyl, TMSOTf Trimethylsilyl trifluoromethanesulfonate MeOH Methanol, THF Tetrahydrofuran

[0164] DMSO Dimethyl sulfoxide, mL Milliliter

[0165] DMF N,N-Dimethylformamide, min Minute

[0166] DCM Dichloromethane, M, N, mol / L Mole per liter DIPEA N,N-Diisopropylethylamine, h Hour

[0167] TiPDSCl2 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane, RT, rt Room temperature

[0168] HBTU Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Detailed implementation manners

[0169] The solutions of the present invention will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only for explaining the present invention and should not be regarded as limiting the scope of the present invention. In particular, the synthesis of small nucleic acids and nucleic acid conjugates can be obtained by adjusting the synthesis according to the examples of the present invention or the routine in the art. For those not specifically noted in the examples about the technical or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchases.

[0170] Preparation examples

[0171] In the following preparation examples, the inventors took some compounds of the present invention as examples and described in detail the preparation process of the compounds of the present invention, where is CPG.

[0172] Example 1: Synthesis of conjugate (Compound DAW40007-3)

[0173]

[0174]

[0175] Step 1: Synthesis of Compound 2-2

[0176] Compound 2-1 (2.50 g, 28.05 mmol) and triethylamine (7.8 mL, 56.1 mmol) were dissolved in DCM (120 mL), and benzyl chloroformate (9.57 g, 56.1 mmol) was added dropwise at 0 °C. After the addition, the reaction mixture was warmed to room temperature and stirred for 20 h. Saturated ammonium chloride solution (50 mL) was added to dilute, and the layers were separated. The aqueous phase was discarded, and the organic phase was concentrated. The residue obtained was purified by silica gel column chromatography (MeOH / DCM (V / V) = 1 / 30) to give white solid compound 35 (2.96 g, 47.2%).

[0177] MS (ESI, pos. ion) m / z: 224.2 [M+H] + ;

[0178] 1 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 4.2 Hz, 5H), 5.11 (s, 2H), 5.00 (s, 1H), 4.19–4.14 (m, 1H), 3.70–3.64 (m, 2H), 3.24 (t, J = 6.4 Hz, 2H), 3.15 (q, J = 4.6 Hz, 1H), 1.90 (dq, J = 14.2, 5.0, 4.3 Hz, 2H), 1.77–1.71 (m, 1H).

[0179] Step 2: Synthesis of Compound 2-4

[0180] Compound 2-3 (1.5 g, 4.56 mmol) and compound 2-2 (1.22 g, 5.47 mmol) were dissolved in 1,2-dichloroethane (30 mL), and then 3A molecular sieve (2.0 g) was added. The mixture was stirred at room temperature for 10 min. TMSOTf (0.51 g, 2.28 mmol) was added, and the reaction mixture was stirred at room temperature for 18 h. The reaction solution was poured into saturated sodium bicarbonate solution (100 mL), then extracted with DCM (100 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue obtained was purified by silica gel column chromatography (PE / EA (V / V) = 2 / 1 to EA) to give light brown oily compound 2-4 (1.8 g, 71.51%). MS (ESI, pos. ion) m / z: 553.3 [M+H] + .

[0181] Step 3: Synthesis of Compound 2-5

[0182] Compound 2-4 (0.57 g, 1.03 mmol) and palladium on carbon (0.11 g, 0.1 mmol, 10%) were added to THF (10 mL), followed by the addition of TFA (0.12 g, 1.03 mmol). Subsequently, the reaction system was purged with hydrogen three times, and then stirred at room temperature for 19 h under a hydrogen atmosphere. After completion of the reaction, the mixture was filtered through diatomaceous earth, and the solvent was evaporated under reduced pressure to obtain a light brown oily compound 2-5 (0.55 g, 100.32%). MS (ESI, pos. ion) m / z: 419.3 [M-TFA+H] + .

[0183] Step 4: Synthesis of Compound 2-7

[0184] Compound 2-6 (0.46 g, 2.29 mmol, purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.) and compound 2-5 (1.16 g, 2.18 mmol) were dissolved in DCM (30 mL). HOBT (0.46 g, 3.44 mmol), HBTU (1.30 g, 3.44 mmol) and DIPEA (2.66 mL, 16.03 mmol) were added successively, and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction, water (20 mL) and DCM (50 mL×2) were added successively. The organic phase was washed with saturated sodium bicarbonate solution (30 mL) and saturated brine (20 mL) successively, and the solvent was evaporated under reduced pressure. The resulting residue was separated and purified by silica gel column chromatography (MeOH / EA (v / v) = 1 / 20) to obtain a white solid 2-7 (1.0 g, yield 72.7%).

[0185] MS (ESI, pos. ion) m / z: 602.3 [M+H] + ;

[0186] 1 H NMR (400 MHz, CD3OD) δ 5.39–5.33 (m, 1H), 5.07 (dd, J = 11.2, 3.4 Hz, 1H), 4.58 (d, J = 8.4 Hz, 1H), 4.21–4.09 (m, 3H), 4.04 (t, J = 6.7 Hz, 1H), 3.89 (dt, J = 10.4, 5.1 Hz, 1H), 3.60–3.50 (m, 1H), 3.30–3.13 (m, 2H), 2.16 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H), 1.94 (s, 3H), 1.67–1.56 (m, 4H), 1.48 (s, 9H), 1.41–1.37 (m, 2H), 1.02–0.98 (m, 2H).

[0187] Step 5: Synthesis of Compound 2-8

[0188] Compound 2-7 (0.72 g, 1.17 mmol) was dissolved in DCM (8 mL), and then TFA (0.87 mL, 11.7 mmol) was added. The reaction mixture was stirred at 25 °C for 16 h, and the solvent was concentrated to obtain compound 2-8 as a brown oil (0.74 g, 103.1%).

[0189] MS (ESI, pos. ion) m / z: 502.2 [M-TFA+H] + ;

[0190] 1 H NMR (400 MHz, CD3OD) δ 5.36 (d, J = 3.3 Hz, 1H), 5.08 (dd, J = 11.3, 3.3 Hz, 1H), 4.58 (d, J = 8.4 Hz, 1H), 4.16–4.10 (m, 3H), 4.08–4.02 (m, 1H), 3.92–3.85 (m, 1H), 3.58–3.50 (m, 1H), 3.26–3.20 (m, 2H), 2.16 (s, 3H), 2.04 (d, J = 5.2 Hz, 6H), 1.98 (s, 3H), 1.97 (s, 3H), 1.61–1.56 (m, 4H), 1.54–1.52 (m, 2H), 1.42–1.38 (m, 2H).

[0191] Step 6: Synthesis of Compound 2-10

[0192] Compound 2-9 (0.19 g, 0.30 mmol) and compound 2-8 (0.50 g, 0.99 mmol) were dissolved in DCM (30 mL), and then HOBT (0.17 g, 1.26 mmol), HBTU (0.48 g, 1.26 mmol) and DIPEA (0.5 mL, 3.0 mmol) were added in sequence. The reaction mixture was stirred at 30 °C for 3 h. After the reaction was completed, water (20 mL) was added to quench the reaction, and then it was extracted with DCM (100 mL×2). The organic phases were combined, washed successively with saturated sodium bicarbonate solution (40 mL) and saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was separated and purified by silica gel column chromatography (DCM / MeOH (v / v) = 10 / 1) to obtain compound 2-10 as a white solid (0.23 g, yield 37%). MS (ESI, pos. ion) m / z: 1046.3 [M / 2+H] + ;

[0193] 11H NMR (400 MHz, CD3OD) δ 7.78 (t, J = 5.8 Hz, 2H), 7.38–7.36 (m, 3H), 5.36 (d, J = 3.4 Hz, 3H), 5.13 (s, 2H), 5.08 (dd, J = 11.2, 3.4 Hz, 3H), 4.58 (d, J = 8.4 Hz, 3H), 4.21–4.08 (m, 9H), 4.04 (t, J = 6.7 Hz, 3H), 3.93–3.82 (m, 3H), 3.73–3.66 (m, 12H), 3.60–3.52 (m, 3H), 3.29–3.18 (m, 6H), 2.52 (t, J = 6.0 Hz, 6H), 2.38 (t, J = 7.4 Hz, 2H), 2.20 (t, J = 7.7 Hz, 2H), 2.16 (s, 9H), 2.04 (s, 9H), 1.97 (s, 9H), 1.95 (s, 9H), 1.62–1.56 (m, 12H), 1.50–1.42 (m, 6H), 1.35–1.27 (m, 16H), 1.04–0.97 (m, 6H).

[0194] Step 7: Synthesis of Compound 2-11

[0195] Compound 2-10 (0.20 g, 0.094 mmol) was dissolved in methanol (10 mL), then Pd / C (10 mg, 10%) was added. The mixture was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 11 h under a hydrogen atmosphere. After the reaction was completed, the reaction mixture was filtered through diatomaceous earth, and the filtrate was evaporated to dryness under reduced pressure to obtain the white solid compound 2-11 (0.19 g, 100%).

[0196] MS (ESI, pos. ion) m / z: 1001.1, [M / 2 + H] + 。

[0197] Step 8: Synthesis of Compound DAW40007-1

[0198] Compound 2-11 (0.28 g, 0.14 mmol) was dissolved in DCM (20 mL). HOBT (0.038 g, 0.28 mmol), HBTU (0.080 g, 0.21 mmol), DIPEA (0.054 g, 0.42 mmol) and compound 13 (0.068 g, 0.16 mmol) were added successively. The reaction mixture was stirred at room temperature for 13 h. After the reaction was completed, the reaction was quenched by adding water (10 mL), and the mixture was extracted with DCM (20 mL). The organic phase was washed with saturated sodium bicarbonate solution (10 mL), and the solvent was evaporated under reduced pressure. The obtained residue was dissolved in acetonitrile (10 mL) and separated by a reverse-phase preparative column (acetonitrile / water solution (v / v) = 43% to 60%, 50 min). Table salt was added to the solution containing the product after preparative separation to saturate the solution, and the organic phase was separated. The aqueous phase was extracted with acetonitrile (100 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was added to acetonitrile (30 mL), dried over sodium sulfate again, filtered, and the filtrate was concentrated under reduced pressure to obtain a light yellow solid compound DAW40007-1 (0.080 g, yield 24%).

[0199] MS(ESI,neg.ion)m / z:2400.18[M-H] - ;

[0200] 11H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 3H), 7.81 (d, J = 9.2 Hz, 3H), 7.57 (t, J = 6.0 Hz, 3H), 7.35–7.26 (m, 4H), 7.20 (td, J = 8.9, 3.0 Hz, 5H), 6.95 (s, 1H), 6.88 (ddd, J = 8.8, 5.8, 2.2 Hz, 4H), 5.22 (d, J = 3.4 Hz, 3H), 4.97 (dd, J = 11.2, 3.5 Hz, 4H), 4.49 (d, J = 8.5 Hz, 3H), 4.40 (d, J = 4.8 Hz, 1H), 4.15 (s, 1H), 4.07–4.00 (m, 9H), 3.88 (dt, J = 11.2, 8.8 Hz, 3H), 3.74 (s, 9H), 3.59–3.48 (m, 12H), 3.17 (dd, J = 8.8, 5.0 Hz, 1H), 3.10–2.95 (m, 8H), 2.35 (t, J = 6.3 Hz, 6H), 2.10 (s, 9H), 2.08 (s, 3H), 2.04 (d, J = 4.7 Hz, 2H), 2.00 (s, 9H), 1.89 (s, 9H), 1.78 (s, 9H), 1.40 (d, J = 12.2 Hz, 17H), 1.31–1.16 (m, 18H), 0.79 (q, J = 3.2 Hz, 6H).

[0201] Step 9: Synthesis of Compound DAW40007-2

[0202] Compound DAW40007-1 (0.080 g, 0.033 mmol) was dissolved in DCM (10 mL). DIPEA (0.029 mL, 0.17 mmol), succinic anhydride (0.008 g, 0.083 mmol) and DMAP (0.014 g, 0.12 mmol) were added. The mixture was stirred at 40 °C for 5 h. Succinic anhydride (10 mg) was added. After continuing the reaction for 16 h, DCM (10 mL), succinic anhydride (10 mg) and DIPEA (0.05 mL) were added. Stirring was continued for 9 h. Then succinic anhydride (10 mg) was added. After reacting for another 10 h, it was diluted with DCM (20 mL), washed with saturated sodium bicarbonate solution (10 mL), the aqueous phase was discarded, the organic phase was dried with anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure to obtain white solid compound DAW40007-2 (0.08 g, 96.07%). MS (ESI, neg. ion) m / z: 2500.12 [M-H] - .

[0203] Step 10: Synthesis of Compound DAW40007-3

[0204] Dissolve compound DAW40007-2 (0.08 g, 0.032 mmol), HBTU (0.015 g, 0.04 mmol) and DIPEA (0.011 mL, 0.064 mmol) in ACN (5 mL), and stir at room temperature for 5 min. Subsequently, transfer it to a solid-phase synthesizer containing 0.35 g of H2N-CPG (purchased from Hebei Dina Xingke), and shake for 22.5 h. Filter by suction, wash the filter cake with DCM / MeOH (V / V = 9 / 1, 10 mL) and DCM (10 mL), and drain. Dilute the obtained filter cake in 25% Ac2O / Py solution (5 mL) and stir for reaction for 3 h. Filter, wash the filter cake successively with DCM / MeOH (V / V = 9 / 1, 10 mL) and DCM (10 mL), and dry under reduced pressure to obtain white solid DAW40007-3 (0.357 g). The measured loading amount is 14.95 μmol / g.

[0205] Synthesis of compound L96-DMTr-CPG:

[0206] Compound L96-DMTr-CPG is prepared according to the method described in patent application WO2014025805A1.

[0207]

[0208] Synthesis of compound 38:

[0209] Compound 38 is prepared according to the method described in patent application WO2018044350A1.

[0210]

[0211] The conjugating group of the present invention can be linked (conjugated) to the siRNA molecule according to the methods well-known in the art. For example, after compound DAW40007-3, L96-DMTr-CPG and compound 38 are respectively linked to siRNA and the protecting groups are removed, conjugating groups with DAW40007-4, L96 and NAG37 structures are respectively formed.

[0212] Example 3: Synthesis of double-stranded siRNA and double-stranded siRNA conjugate

[0213] 1. Synthesis of double-stranded siRNA without conjugated group

[0214] The synthesis steps of the sense strand and the antisense strand of the siRNA according to the present invention are as follows:

[0215] The synthesis was completed according to the theoretical yield of 1umol. Weigh 1umol of solid support CPG (purchased from Hebei Dina Xingke). All RNA phosphoramidite monomers and auxiliary reagents were commercially obtained, and all phosphoramidite monomers were provided in 0.1M anhydrous acetonitrile solution. For oligonucleotides with phosphate backbone thiolation modification, 0.1M DDTT solution was used as the thiolation reagent. 5-Ethylthio-1H-tetrazole acetonitrile solution (0.25M) was used as an activator (purchased from Suzhou Kelema), 0.02M iodine pyridine / water solution was used as an oxidant, and 3% trichloroacetic acid in dichloromethane was used as a deprotection reagent. They were placed in the designated reagent position corresponding to the KA-H8 model DNA / RNA automatic synthesizer. Set the synthesis program and enter the specified oligonucleotide base sequence. After checking that everything is correct, start the cyclic oligonucleotide synthesis. The coupling time for each step is 6 minutes, and the thiolation time is 6 minutes. After automatic circulation, an oligonucleotide containing solid support CPG is obtained.

[0216] The CPG-containing nucleotides obtained above were blown dry with dry argon, then transferred to a 2 mL EP tube and 28% aqueous ammonia solution (1.8 mL) was added and heated at 55°C for 5 to 18 hours. Filter, wash the filter cake with water (0.5 mL), combine the filtrates, and concentrate under reduced pressure to obtain a white or yellow colloidal solid. After reverse phase preparative purification, the prepared solution was concentrated and passed through a gel column to remove excess salt to obtain the oligonucleotides. The concentration of the obtained oligonucleotides was determined by a micro-UV spectrophotometer (SPECTRO statNano). Mass spectrometry detection and analysis were performed on an Agilent 6530 LC-MS Q-Tof system. After primary scanning, the nucleic acid molecular weight was calculated after deconvolution.

[0217] Annealing step:

[0218] The double-stranded siRNA sense strand synthesized above was mixed with the antisense strand synthesized above in equimolar amounts, heated to 95°C, maintained at this temperature for 10 minutes, and then slowly cooled to room temperature. The target double-stranded siRNA was then lyophilized.

[0219] 2. Synthesis of siRNA conjugate:

[0220] The synthesis of the antisense strand was obtained by referring to the above-mentioned synthesis method.

[0221] Synthesis of the positive chain: The general solid phase support CPG was replaced with the GalNAc solid phase support prepared by the present invention (such as Compound DAW40007-3), and the sense strand of the double-stranded siRNA connected to the conjugated group of the present invention was prepared with reference to the above-mentioned synthesis method.

[0222] Annealing step:

[0223] Mix the sense strand of the double-stranded siRNA obtained by the above synthesis with the antisense strand obtained by the above synthesis in equimolar amounts, heat to 95 °C, control the temperature for 10 min, and then slowly cool to room temperature. Subsequently, lyophilize to obtain the target siRNA conjugate.

[0224] The double-stranded siRNA or its conjugate obtained by the above synthesis is shown in Table 1.

[0225] Table 1: Double-stranded siRNA or its conjugate obtained by the synthesis of the present invention

[0226]

[0227] Unless otherwise specified, in the context of the present invention, the capital letters C, G, U, and A represent the bases of natural nucleotides; the lowercase letters represent the bases with the 2'-position of the nucleotide ribose modified by methoxy, such as c, g, u, and a represent 2'-OMe (2'-O-methyl)C, 2'-OMe G, 2'-OMe U, and 2'-OMe A, respectively; the letter f on the right of the capital letter represents the base with the 2'-position of the nucleotide ribose modified by fluorine, such as Cf, Gf, Uf, and Af represent 2'-F (2'-fluoro)C, 2'-F G, 2'-F U, and 2'-F A, respectively; "s" represents a phosphorothioate linkage between the two nucleotide residues adjacent to the left and right of "s". For example, "gsu" represents a phosphorothioate linkage between g and u residues; invB represents a reverse abasic deoxynucleoside residue, which has the following structure:

[0228] Example 4: Test of cell activity and cytotoxicity of the siRNA or its conjugate of the present invention (I) Pharmacodynamic effect on human hepatoma cell line

[0229] 1. Digest the HepG2 cells in good growth state, resuspend with medium, count, and then inoculate into a 96-well plate at a density of about 20,000 cells / well, and culture overnight at 37 °C and 5% CO2.

[0230] 2. Gradiently dilute the siRNA to be detected, and transfect each siRNA at two concentrations (10 nM, 0.4 nM) using Lipofectamine RNAiMAX (Thermo Fisher), and continue to culture for 24 hours.

[0231] 3. Extract the RNA of the sample using the CellAmp Direct Probe RT-qPCR Kit (Takara), perform reverse transcription on the extracted product to obtain cDNA. Using the cDNA obtained from the above reverse transcription as a template, detect the transcriptional level of the target gene using qPCR reaction. The forward primer for GAPDH used is 5’-AGCCTCAAGATCATCAGCAAT-3’, the reverse primer is: 5’-GTCATGAGTCCTTCCACGAT-3’, and the probe is 5’-FAM-CCACCAACTGCTTAGCACCCCTGGCC-TAMRA-3’; the forward primer for APOC3 used is 5’-GTTACATGAAGCACGCCAC-3’, the reverse primer is 5’-GTCTTTCAGGGAACTGAAGC-3’, and the probe is 5’-FAM-AGACCGCCAAGGATGCACTGAGCAGC-TAMRA-3’.

[0232] 4. By comparing with the control of the internal reference gene, compare the relative expression levels of Apoc3 mRNA in each group, and use the expression level of the untreated group for data normalization.

[0233] The experimental results show that the siRNA or its conjugate of the present invention has good inhibitory activity against APOC3. The experimental results of the inhibition of partially modified siRNA APOC3 are shown in Table A.

[0234] Table A: Experimental results of the inhibition of partially modified siRNA APOC3 of the present invention

[0235]

[0236] According to the technical knowledge in the art, the siRNA conjugate of the present invention is obtained by conjugating siRNA with a conjugation group of GalNAc or its derivative. The main function of the conjugation group is for delivery. When the siRNA has good activity, the corresponding siRNA conjugate can be expected to have similar activity. Those skilled in the art can select appropriate conjugation groups according to the technical knowledge in the art, such as GalNAc or its derivatives like L96 and DAW40007-4.

[0237] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A double-stranded siRNA, a conjugate or a salt thereof, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 2, or a nucleotide sequence having no more than 5 nucleotide differences therefrom, Optionally, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 9 to SEQ ID NO: 10, or a nucleotide sequence having no more than 5 nucleotide differences therefrom; Optionally, the double-stranded region is 17-23 nucleotide pairs in length.

2. The double-stranded siRNA, its conjugate or salt according to claim 1, characterized in that The length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides; Optionally, the sense strand and the antisense strand each independently comprise a 3' overhang and / or a 5' overhang, wherein the 3' overhang and the 5' overhang each independently comprise 1, 2 or 3 nucleotides.

3. The double-stranded siRNA, conjugate or salt thereof according to claim 1 or 2, characterized in that It contains one of the double-stranded siRNAs shown in siRNA ID NO: 1 to siRNA ID NO:

2.

4. The double-stranded siRNA, its conjugate or salt according to claim 1, characterized in that The sense strand and / or the antisense strand each independently comprises at least one modified nucleotide, and the modified nucleotides are each independently selected from at least one of the following: Non-natural base nucleotides, deoxynucleotides, 2'-fluoro modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-alkoxyalkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, locked nucleotides, unlocked nucleic acid modified nucleotides, 2'-allyl modified nucleotides, abasic nucleotides, invAb modified nucleotides, morpholino modified nucleotides, tetrahydropyran modified nucleotides, cyclohexenyl modified nucleotides, PEG modified nucleotides, 5'-phosphoramidate modified nucleotides, phosphorothioates ligated modified nucleotides, 5'-methylphosphonate modified nucleotides, 5'-vinyl phosphate modified nucleic acids, 5'-phosphate mimetic modified nucleotides, TNA modified nucleotides, PNA modified nucleotides, D-FNA modified nucleotides, HNA modified nucleotides, FNA modified nucleotides, bcDNA modified nucleotides, tcDNA modified nucleotides, S-MC modified nucleotides, N-MC modified nucleotides, 2'-F-NMC modified nucleotides, 5-methylcytosine modified nucleotides, 5-methyluracil modified nucleotides, 2,6-diamino modified adenine modified nucleotides and glycol nucleic acids, Preferably, the sense strand and the antisense strand each independently comprise at least one modified nucleotide, and the modified nucleotides are each independently selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 5'-phosphorothioate linked modified nucleotides, 2'-phosphorothioate linked modified nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-hydroxy modified nucleotides, locked nucleic acid modified nucleotides, unlocked nucleic acid modified nucleotides, ethylene glycol nucleic acids, 5'-vinyl phosphate modified nucleotides, 5'-(E)-VP modified nucleotides, 5'-phosphorothioate linked modified nucleotides, 2'-phosphorothioate linked modified nucleotides, invAb modified nucleotides, i substituted nucleotides and Y substituted nucleotides, wherein the Y is The i is 5. The double-stranded siRNA, its conjugate or salt according to claim 4, characterized in that The modified nucleotides are each independently present in one or more positions selected from the group consisting of: The nucleotides at the 5' end of the sense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 of the starting point; and / or The nucleotides at the 5' end of the antisense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the starting point; and / or The 5'-phosphorothioate linkage modification and the 2'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of: The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, and 20-21 of the starting point; and / or The nucleotides at the 5' end of the antisense strand are positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, between 18-19, 19-20, 20-21, 21-22, and between 22-23 of the starting point; Preferably, the 2'-fluoro modified nucleotides are each independently present at one or more positions selected from the following: The nucleotides at the 5' end of the sense strand are the 5th, 7th, 8th, 9th, 10th and 11th nucleotides of the starting point; and / or The nucleotides at the 5' end of the antisense strand are the 2nd, 6th, 8th, 9th, 12th, 14th and 16th positions of the starting point; Optionally, the 5'-phosphorothioate linkage modification or the 2'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of: The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3 and 3-4 of the starting point; and / or The nucleotides at the 5' end of the antisense strand are between positions 1-2, 2-3 and 3-4 of the starting point; and / or The nucleotides at the 3' end of the antisense strand are between positions 1-2, 2-3 and 3-4 of the starting point; Optionally, an invAb modified nucleotide is attached to the 5' end of the sense strand.

6. The double-stranded siRNA, conjugate or salt thereof according to claim 1, wherein the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 3 to SEQ ID NO: 5, and the length of the sense strand does not exceed 21 nucleotides; or The sense strand comprises one of the following sequences: csusuaaaAfgGfGfAfcaguauusa InvBsasgggacAfgUfAfUfucucagusa and InvBscsuuaaaAfgGfGfAfcaguauusa, the sense strand is no longer than 21 nucleotides; Optionally, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 11 to SEQ ID NO: 12, and the length of the antisense strand does not exceed 23 nucleotides; Preferably, the double-stranded siRNA, its conjugate or salt comprises one of the double-stranded siRNAs shown in siRNA ID NO: 3 to siRNA ID NO:

5.

7. The double-stranded siRNA, its conjugate or its salt according to claim 1 or 6, characterized in that The double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA to a conjugation group; optionally, in the double-stranded siRNA conjugate, the 3' end or 5' end of the sense strand or antisense strand of the double-stranded siRNA is conjugated to the conjugation group, preferably, in the double-stranded siRNA conjugate, the 3' end of the sense strand of the double-stranded siRNA is conjugated to the conjugation group; optionally, in the double-stranded siRNA conjugate, the 3' end and 5' end of the sense strand of the double-stranded siRNA are each independently conjugated to the conjugation group through a phosphate bond or a phosphorothioate bond.

8. The double-stranded siRNA, its conjugate or its salt according to claim 7, characterized in that The conjugated group includes GalNAc or a derivative thereof; preferably, the conjugated group is GalNAc or a derivative thereof connected by a divalent, trivalent or tetravalent branched linker; more preferably, the conjugated group is DAW40007-4, L-96, NAG37 or an isomer thereof, wherein the structures of the conjugated groups DAW40007-4, L-96 and NAG37 are respectively:

9. The double-stranded siRNA, its conjugate or salt according to claim 1, characterized in that The antisense strand comprises two thiolate backbone modifications between the three terminal nucleotides at each of the 3' and 5' ends; optionally, the sense strand comprises two thiolate backbone modifications between the three terminal nucleotides at the 3' and 5' ends.

10. The double-stranded siRNA, conjugate or salt thereof according to claim 1, wherein the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 6 to SEQ ID NO: 8, and the length of the sense strand does not exceed 23 nucleotides; Optionally, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 11 to SEQ ID NO: 12, and the length of the antisense strand does not exceed 25 nucleotides; Preferably, the double-stranded siRNA, its conjugate or salt comprises one of the nucleotide sequences shown in siRNA ID NO: 6 to siRNA ID NO: 8; wherein, The length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 25 nucleotides. 11 . A pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

12. Use of the double-stranded siRNA according to any one of claims 1 to 10, its conjugate or salt thereof, or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating and / or preventing an APOC3-related disease, preferably, the APOC3-related disease is hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome or familial partial lipodystrophy.

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