Double-stranded siRNA analogue for inhibiting expression of HSD17B13 as well as preparation method and application of double-stranded siRNA analogue

Abstract

The invention relates to a double-stranded siRNA analogue for inhibiting expression of HSD17B13 and a preparation method and application thereof, the double-stranded siRNA analogue can inhibit expression of HSD17B13 and is used for treating diseases and symptoms related to HSD17B13, and the diseases and symptoms comprise NAFLD, NASH, hepatic fibrosis, or alcoholic liver diseases or non-alcoholic liver diseases such as liver cirrhosis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and more specifically, to a double-stranded siRNA analog for inhibiting HSD17B13 expression, and a preparation method and use thereof. Background Art

[0002] Hepatic lipid droplet protein 17β-hydroxysteroid dehydrogenase type 13 (commonly referred to as HSD17B13, 17β-HSD13, HSD17β13, 17β-HSD13, 17β-HSD type 13, or 17-HSD13) is a member of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The 17β-HSD family consists of 14 enzymes involved in the reduction or oxidation of sex hormones, fatty acids, and bile acids. Tissue distribution, subcellular localization, and catalytic preferences vary among various family members. The 17β-HSD family exhibits diverse substrate specificity, including steroids, lipids, and retinoids.

[0003] The 17β-HSD13 protein is distributed in a wide range of tissues in the body and is encoded by the HSD17B13 gene (alternatively referred to as the 17β-HSD13 gene). The highest expression level is found in hepatocytes of the liver, while it is also found in the ovaries, bone marrow, kidneys, brain, lungs, skeletal muscle, bladder, and testes. ▼ Lower levels can be detected in the liver. The function of 17β-HSD13 is not fully understood; however, several 17β-HSD family members, including 17β-HSD-4, -7, -10, and -12, have been shown to be involved in carbohydrate and fatty acid metabolism. This suggests that 17β-HSD13 may also play a role in lipid metabolism pathways. Hepatic upregulation of 17β-HSD13 has been reported in patients with fatty liver disease, supporting a role for this enzyme in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).

[0004] Wen Su et al. previously identified 17β-HSD13 as a lipid droplet (LD)-associated protein in NAFLD patients and reported that 17β-HSD13 is one of the most abundant LD proteins, specifically localized on the surface of LDs. (Wen Su et al., Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease, 111 PNAS 11437-11442 (2014)).

[0005] Studies have shown that HSD17B13 gene expression plays a key role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although international patent applications such as WO2019183329, WO2020061177, WO2020132564, and WO2021113820 disclose RNA compounds that can inhibit HSD17B13 gene expression, no drugs are currently approved for NASH or other diseases and conditions classified as NAFLD or ARLD. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a double-stranded siRNA analog for inhibiting the expression of HSD17B13, a preparation method thereof, and a use thereof. The double-stranded siRNA analog has a good inhibitory effect on the gene expression of HSD17B13.

[0007] In a first aspect, the present invention provides a double-stranded siRNA analog for inhibiting the expression of HSD17B13, wherein the double-stranded siRNA analog comprises a sense strand and an antisense strand, wherein the sense strand is at least partially complementary to the antisense strand, and the sense strand and the antisense strand are selected from the sequences shown in Table 1 and Table 2.

[0008] The sense strand and the antisense strand may be partially, substantially or completely complementary to each other, for example, the sense strand and the antisense strand may be 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary.

[0009] The lengths of the nucleotides of the sense strand and the antisense strand may be the same or different. For example, the sense strand includes 19 nucleotides and the antisense strand includes 21 nucleotides, or the sense strand includes 19 nucleotides and the antisense strand also includes 19 nucleotides, or the sense strand includes 21 nucleotides and the antisense strand includes 19 nucleotides, or the sense strand includes 21 nucleotides and the antisense strand includes 21 nucleotides.

[0010] As a preferred technical solution of the present invention, the sense strand and / or antisense strand contains at least one modified nucleotide.

[0011] Wherein, the modified nucleotide is selected from:

[0012] Alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acids (GNA); wherein the alkyl nucleotides are selected from methyl nucleotides and ethyl nucleotides; glycol nucleic acids include (S)-glycol nucleic acids ((S)-GNA) and (R)-glycol nucleic acids ((R)-GNA).

[0013] Wherein, the sense strand and antisense strand of the double-stranded siRNA analog each include at least one modified nucleotide. In a specific embodiment, the sense strand includes at least one modified nucleotide and the nucleotides of the antisense strand are unmodified, or the nucleotides of the sense strand are unmodified and the antisense strand includes at least one modified nucleotide. In a specific embodiment, every nucleotide of the sense strand is modified and every nucleotide of the antisense strand is also modified.

[0014] As a preferred technical solution of the present invention, the sense strand or antisense strand comprises 2'-fluoro nucleotides, and the number of the 2'-fluoro nucleotides is no more than 8, preferably 3, 4 or 5. For example, the sense strand comprises 4 2'-fluoro nucleotides, and the antisense strand comprises 8 2'-fluoro nucleotides.

[0015] As a preferred technical solution of the present invention, the sense strand or antisense strand contains phosphorothioate nucleotides, and the number of the phosphorothioate nucleotides is no more than 4, preferably 3, and more preferably 2.

[0016] As a preferred technical solution of the present invention, the sense strand or the antisense strand includes at most one locked nucleic acid modification, ethylene glycol nucleic acid modification or vinyl phosphonate deoxyribonucleotide (VP) modification.

[0017] For example, one nucleotide of the sense chain is modified by a locked nucleic acid, while the nucleotide of the antisense chain is not modified by a locked nucleic acid; or, one nucleotide of the antisense chain is modified by a locked nucleic acid, while the nucleotide of the sense chain is not modified by a locked nucleic acid; or, neither the nucleotide of the sense chain nor the antisense chain is modified by a locked nucleic acid.

[0018] For another example, one nucleotide in the sense chain is modified with ethylene glycol nucleic acid, while the nucleotide in the antisense chain is not modified with ethylene glycol nucleic acid; or, one nucleotide in the antisense chain is modified with ethylene glycol nucleic acid, while the nucleotide in the sense chain is not modified with ethylene glycol nucleic acid; or, neither the nucleotide in the sense chain nor the antisense chain is modified with ethylene glycol nucleic acid.

[0019] For another example, one nucleotide in the sense strand is modified with VP, while the nucleotides in the antisense strand are not modified with VP; or one nucleotide in the antisense strand is modified with VP, while the nucleotides in the sense strand are not modified with VP; or neither the nucleotides in the sense strand nor the antisense strand are modified with VP.

[0020] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand includes at most one deoxynucleotide. The 2'-deoxynucleotide is selected from 2'-deoxyadenosine-3'-phosphate, 2'-deoxycytidine-3'-phosphate, 2'-deoxyguanosine-3'-phosphate, and 2'-deoxythymidine-3'-phosphate. For example, one nucleotide in the sense strand is modified with a 2'-deoxynucleotide, and the nucleotides in the antisense strand are not modified with a 2'-deoxynucleotide; or, one nucleotide in the antisense strand is modified with a 2'-deoxynucleotide, and the nucleotides in the sense strand are not modified with a 2'-deoxynucleotide; or, one nucleotide in the sense strand is modified with a 2'-deoxynucleotide, and the nucleotides in the antisense strand are modified with a 2'-deoxynucleotide; or, neither the nucleotides in the sense strand nor the antisense strand are modified with a 2'-deoxynucleotide.

[0021] As a preferred technical solution of the present invention, the modified nucleotides are selected from the sequences in Tables 4-16.

[0022] ◆As a preferred technical solution of the present invention, the double-stranded siRNA analog is connected to a targeting ligand.

[0023] As a preferred technical solution of the present invention, the targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

[0024] As a preferred technical solution of the present invention, the targeting ligand is selected from:

[0025]

[0026] As a preferred technical solution of the present invention, the targeting ligand is connected to the 3' or 5' end of the sense strand.

[0027] As a preferred technical solution of the present invention, the targeting ligand is connected to the 3' or 5' end of the antisense strand.

[0028] In some embodiments, the targeting ligand is connected to the 5' end of the sense strand. In some embodiments, the targeting ligand is connected to the 3' end of the sense strand. In some embodiments, the targeting ligand can also be internally connected to nucleotides on the sense strand and / or antisense strand of the double-stranded siRNA analog. In some embodiments, the targeting ligand can also be connected to the double-stranded siRNA analog via a linker, for example, the targeting ligand can also be connected to the 3' or 5' end of the sense strand via a linker, or the targeting ligand can also be connected to the 3' or 5' end of the antisense strand via a linker, or the targeting ligand can also be internally connected to nucleotides on the sense strand and / or antisense strand of the double-stranded siRNA analog via a linker.

[0029] As a preferred technical solution of the present invention, the siRNA analog is selected from any one of the siRNA compounds shown in Table 19.

[0030] In a second aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of the HSD17B13 gene, wherein the pharmaceutical composition comprises the double-stranded siRNA analog as described above.

[0031] As a preferred technical solution of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0032] In a third aspect, the present invention further provides a use of the double-stranded siRNA analog as described above in the preparation of a method for treating a disease, disorder or symptom mediated at least in part by the expression of the HSD17B13 gene.

[0033] As a preferred technical solution of the present invention, the disease is selected from NAFLD, NASH, liver fibrosis, or alcoholic liver disease or non-alcoholic liver disease such as cirrhosis.

[0034] The double-stranded siRNA analogue for inhibiting HSD17B13 gene expression provided by the present invention has good inhibitory activity on HSD17B13 and can be used to prevent and / or treat related diseases mediated by HSD17B13 gene expression. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0036] As used herein, the term "comprising" is used to mean, and is used interchangeably with, the phrase "including, but not limited to," unless the context clearly dictates otherwise.

[0037] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0038] As used herein, the terms "sequence" and "nucleotide sequence" mean the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0039] In the present invention, the term "siRNA analog" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid chains, which have "sense" and "antisense" orientations relative to the target RNA.

[0040] As used herein, "complementary" has the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of that strand can be inferred from the sequence of its complementary strand.

[0041] The term "antisense strand" generally refers to a strand of an RNAi agent that includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" generally refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not fully complementary to the target sequence, mispairing can occur in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end.

[0042] The term "sense strand" generally refers to a strand of an RNAi agent that includes a region that is substantially complementary to the region of the antisense strand as defined herein. The "sense" strand is sometimes referred to as a "sense" strand, a "passenger" strand, or an "anti-guide" strand. By virtue of their sequence, the antisense strand targets the desired mRNA, while the sense strand targets different targets. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. The incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications or using 5' end caps on the sense strand.

[0043] In the present invention, modified nucleotides include but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), inverted abasic deoxyribose residues (invAb), and glycol nucleic acids (GNA).

[0044] Among them, alkyl-modified nucleotides, such as 2'-methyl nucleotides, 2'-ethyl nucleotides,

[0045] 2'-methoxy modified nucleotides, e.g. 2'-methoxyethyl nucleotides, e.g. 2'-fluoronucleotides, e.g. 5'-C-methylphosphononucleotides, e.g. Vinylphosphonate deoxyribonucleotides (VP), e.g. Phosphorothioate nucleotide(s), structure: Phosphate nucleotides (p), the structure is: 2'-deoxyribonucleotides, such as: Inverted abasic deoxyribose residues (invAb), for example: Glycol nucleic acids (GNAs), including (S)-glycol nucleic acids ((S)-GNAs), for example: and (R)-glycol nucleic acids ((R)-GNA), such as:

[0046] Among them, Base represents a base, R represents an alkyl group, Me represents a methyl group, and Et represents an ethyl group.

[0047] The term "locked nucleic acid" is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acid to siRNA has been shown to increase siRNA stability in serum. ▲and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0048] Representative U.S. patents for preparing locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125 and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0049] The locked nucleic acid structure is as follows:

[0050]

[0051] In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0052] The term "morpholino" means a sugar surrogate having the formula:

[0053]

[0054] In certain embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0055] In the present invention, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides. Lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorine-modified nucleotide; LNA indicates that the nucleotide adjacent to the right is a locked nucleic acid (LNA) modified; GNA indicates that the nucleotide adjacent to the left is a GNA modified; lowercase letter s indicates that the two nucleotides to the left and right of the letter are connected by a thiophosphate group; VP indicates that the nucleotide to the right of the letter VP is a vinyl phosphate-modified nucleotide. invAb represents an inverted abasic deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents a deoxyadenine nucleotide; dT represents a deoxythymine nucleotide; dU represents a deoxyuridine nucleotide; dC represents a deoxycytosine nucleotide; and dG represents a deoxyguanine nucleotide.

[0056] It should be emphasized that the "modification" of nucleotides in the present disclosure includes but is not limited to the above examples, and nucleotides can also be replaced with other nucleotides, such as (S)-glycerol nucleic acid.

[0057] The term "targeting ligand" can include naturally occurring substances, such as proteins (e.g., human serum albumin (HAS), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene acid-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0058] Targeting ligands can also be cell or tissue targeting agents that bind to a specific cell type, such as renal cells, such as lectins, glycoproteins, lipids or proteins, such as antibodies. The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0059] Targeting ligands can also be proteins, for example, glycoproteins, or peptides, for example, molecules with specific affinity for co-ligands, or antibodies, for example, antibodies that bind to a given cell type, for example, hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, for example, lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-KKB.

[0060] The targeting ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, e.g., by disrupting the cytoskeleton of the cell (e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments). The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, erythrosine A, phalloidin, swinholide A, indanocine, or myoservin.

[0061] Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to prepare a single dose form is generally the amount of the compound that produces the therapeutic effect. Generally speaking, in percent units, this amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0062] The term "prevention and / or treatment" includes not only preventing and / or curing a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, and / or treating a disease. ▼The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant. The invention relates to a method for treating a disease or a condition that is present in an animal, animal or plant.

[0063] The terms "disease" or "disorder" are used interchangeably and generally refer to any deviation of a subject from its normal state, such as any change in the state of the body or certain organs that prevents or disrupts the performance of a function and / or causes symptoms such as discomfort, dysfunction, suffering, or even death in those who are afflicted or exposed thereto. A disease or condition may also be referred to as a distemper, an ailment, an ailment, a malady, a disorder, a sickness, an illness, a complaint, an inderdisposion, or an affectation.

[0064] The term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms and includes any level of inhibition.

[0065] The phrase "inhibiting the expression of HSD17B13" in the present invention includes inhibiting the expression of any HSD17B13 gene (e.g., mouse HSD17B13 gene, rat HSD17B13 gene, monkey HSD17B13 gene, or human HSD17B13 gene) as well as variants or mutants of the HSD17B13 gene encoding the HSD17B13 protein.

[0066] "Inhibiting the expression of HSD17B13" includes any level of inhibition of the HSD17B13 gene, such as at least partially suppressing the expression of the HSD17B13 gene, such as inhibiting at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0067] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared by reacting a compound having a specific substituent discovered in the present invention with a pharmaceutically acceptable acid or base.

[0068] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical medicine. For additional information about carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0069] The term "pharmaceutically acceptable excipient" is a substance intentionally included in a drug delivery system other than the active pharmaceutical ingredient (API, therapeutic product, such as HSD17B13). Excipients do not exert or are not intended to exert a therapeutic effect at the intended dose. Excipients may serve the following functions: a) aid in the handling of the drug delivery system during preparation; b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) enhance any other properties of the API in terms of overall safety, efficacy, or delivery during storage or use.

[0070] Among them, excipients include (but are not limited to): absorption enhancers, anti-adherents, defoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavorings, glidants, wetting agents, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavoring agents and fragrances.

[0071] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0072] Example 1 Synthesis of Targeting Ligand

[0073] Step A: Hydroxyproline amine (3.00 g, 7.15 mmol) and monomethyl dodecanedioate (1.748 g, 7.15 mmol) were placed in N,N-dimethylformamide (DMF) (50 mL). The peptide coupling reagent (HBTU) (3.25 g, 8.56 mmol) and N,N-diisopropylethylamine (DIEA) (3.7 mL, 21.24 mmol) were added and the reaction was stirred overnight.

[0074] The reaction mixture was poured into an ice-water mixture and extracted with dichloromethane (DCM). The mixture was washed with bicarbonate solution, water, brine, and dried over sodium sulfate. The solvent was removed and the residue was purified by chromatography (eluting with 50% ethyl acetate / hexane, ethyl acetate, and then 5% methanol / dichloromethane) to afford the desired compound 115 as a white solid (4.30 g, 93%). MS: C 39 H 51 NO7, calculated as 645.37, found as 646.35 (M+H).

[0075]

[0076] Step B: Compound 101 (4.25 g, 6.58 mmol) was dissolved in a mixture of tetrahydrofuran / methanol / water (50 mL, 2:1:1). Lithium hydroxide (LiOH) (1.90 g, 45.2 mmol) was added and the mixture was stirred overnight.

[0077] After checking with thin layer chromatography silica gel plate (TLC), acetic acid was added to neutralize the reaction mixture. The solvent was removed and the residue was extracted with dichloromethane (DCM). Triethanolamine (TEA, excess) was added to the dichloromethane (DCM) solution and the solution was filtered through a small silica gel pad to obtain the desired product 102.▲ Its triethanolamine (TEA) salt (4.15 g, 86%). MS: C 38 H 49 NO7, calculated value is 631.35; measured value is 630.34 (MH).

[0078]

[0079] Step C: Compound 102 (1.30 g, 2.06 mmol) and a peptide coupling reagent (HBTU) (0.821 g, 1.05 eq.) were added to N,N-dimethylformamide (DMF) (30 mL). To this N,N-diisopropylethylamine (DIEA) (1.07 ml, 3 eq.) was added and the reaction mixture was stirred for 3-4 minutes. A solution of the amine (3.00 g, 1.58 mmol) was added, followed by 1 eq. of DIEA. The reaction mixture was stirred at room temperature overnight.

[0080] The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (DCM) and washed with bicarbonate and water. The dichloromethane (DCM) was dried over sodium sulfate and the solvent was removed. The residue was purified by chromatography (eluting first with ethyl acetate and then with 5-20% methanol / dichloromethane) to give the product 103 as a white solid (3.35 g, 88%). MS: for C 117 H 175 N 11 O 42 , the calculated value is 2406.19; the measured value is 2429.10 (M+Na).

[0081]

[0082] Among them, ligand 104 (L96) can be linked to siRNA via a phosphate group, a phosphorothioate group, or another linking group.

[0083] For the specific synthesis route, please refer to the document with International Patent Publication No. WO2009073809.

[0084] Example 2 Synthesis of siRNA analogs

[0085] siRNA was prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark). The specific synthesis route can be found in patent document CN201980061674.3, and the siRNA analog sequences are shown in Tables 1 and 2.

[0086] Table 1 is a list of siRNA analog sequences

[0087]

[0088]

[0089] Table 2

[0090]

[0091]

[0092]

[0093]

[0094] Example 3 In vitro testing of HSD17B13 siRNA analogs in HuH7 cells

[0095] HSD17B13 cDNA (GenBank accession number NM_178135.5) was cloned into the reporter-based screening plasmid psiCHECK2 (Promega-C8021) to produce Renilla luciferase / HSD17B13 fusion mRNA. HuH7 cells were cultured in DMEM (Gibco-10313021) supplemented with 10% fetal bovine serum (ExCell Bio-FSP500), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), and 1% penicillin-streptomycin (HyClone-SV30010). HSD17B13-psiCHECK2 plasmid, siRNA analogs and Lipo2000 (Invitrogen-11668019) transfection reagent diluted in Opti-MEM (Gibico-11058021) were added to the HuH7 cell suspension and the cells were transfected at a rate of 1×10 5 Cells were plated at a density of 100 μg / ml in 96-well plates to give a final concentration of 1 nM or 0.02 nM of siRNA analogs. After 24 hours of culture, relative levels of Renilla luciferase were measured using a dual-luciferase reporter assay (Promega-E2920) normalized to the level of constitutively expressed firefly luciferase also present on the psiCHECK2 plasmid.

[0096] The inhibition rate of HSD17B13 gene mediated by siRNA was calculated according to the following formula.

[0097] HSD17B13 gene inhibition rate (%) = (1-relative level of Renilla luciferase in the sample / relative level of Renilla luciferase in the control group) × 100.

[0098] The calculation results are shown in Table 3.

[0099] Table 3 shows the inhibition efficiency of HSD17B13

[0100]

[0101] From the experimental results in Table 3, it can be seen that the siRNA sequence of the present invention has a good inhibitory activity on the HSD17B13 gene.

[0102] Example 4 Synthesis of modified sequences of siRNA analogs

[0103] The modified siRNA analogs were prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark). The specific synthesis route can be found in patent document CN201980061674.3. The sequences of the modified siRNA analogs of sequences S1, S2, S3, S4, S5, S21, S22, S24, S25, S26, and S27 are shown in Tables 4-15, respectively. The modified sequences of the sequences in Table 2 are shown in Table 16.

[0104] Table 4 is a modified sequence list of sequence S1

[0105]

[0106]

[0107] Table 5 is a modified sequence list of sequence S2

[0108]

[0109]

[0110] Table 6 is a modified sequence list of sequence S3

[0111]

[0112] Table 7 is a modified sequence list of sequence S4

[0113]

[0114]

[0115] Table 8 is a modified sequence list of sequence S5

[0116]

[0117] Table 9 is a modified sequence list of sequence S21

[0118]

[0119]

[0120] Table 10 is a modified sequence list of sequence S22

[0121]

[0122] Table 11 is a modified sequence list of sequence S24

[0123]

[0124]

[0125] Table 12 is a modified sequence list of sequence S25

[0126]

[0127] Table 13 is a modified sequence list of sequence S26

[0128]

[0129]

[0130] Table 14 is a modified sequence list of sequence S27

[0131]

[0132] Table 15 is a modified sequence list of S63 and S64

[0133]

[0134]

[0135] Table 16 is a modified sequence list of the sequence in Table 2

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] Wherein, A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; T = thymidine-3'-phosphate; Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Gs = guanosine- 3'-phosphorothioate; Ams = 2'-O-methyladenosine-3'-phosphorothioate; Ums = 2'-O-methyluridine-3'-phosphorothioate; Cms = 2'-O-methylcytidine-3'-phosphorothioate; Gms = 2'-O-methylguanosine-3'-phosphorothioate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate Ester; Gf = 2'-fluoroguanosine-3'-phosphate; Afs = 2'-fluoroadenosine-3'-phosphorothioate; Ufs = 2'-fluorouridine-3'-phosphorothioate; Cfs = 2'-fluorocytidine-3'-phosphorothioate; Gfs = 2'-fluoroguanosine-3'-phosphorothioate; dA = 2'-deoxyadenosine-3'-phosphate; dC = 2'-deoxycytidine-3'-phosphate; dG = 2'-deoxyguanosine-3'-phosphorothioate ester; dT = 2'-deoxythymidine-3'-phosphate; m = 2'-O methyl; f = 2'-fluoro; s = phosphorothioate linkage; (LNA) = locked nucleic acid ribonucleotide; GNA = glycol nucleic acid ribonucleotide; VP = vinylphosphonate deoxyribonucleotide; Im = hypoxanthine 2'-OMe ribonucleoside; If = hypoxanthine 2'-F ribonucleoside; Ims = 2'-OMe hypoxanthine-3'-phosphorothioate.

[0145] Example 5 In vitro testing of HSD17B13-modified siRNA in Huh7 cells

[0146] HSD17B13 cDNA (GenBank accession number NM_178135.5) was cloned into the reporter-based screening plasmid psiCHECK2 (Promega C802★1) to produce Renilla luciferase / HSD17B13 fusion mRNA. HuH7 cells were cultured in DMEM (Gibco 10313021) supplemented with 10% fetal bovine serum (ExCell Bio FSP500), 1% glutamine (Gibco 35050061), 1% non-essential amino acids (Gibco 11140050), and 1% penicillin-streptomycin (HyClone SV30010). HSD17B13 psiCHECK2 plasmid, siRNA, and Lipo2000 (Invitrogen 11668019) transfection reagent diluted in OptiMEM (Gibco 11058021) were added to the HuH7 cell suspension and transfected at a rate of 1×10 5 Cells were plated at a density of 100 μg / ml in 96-well plates to give a final siRNA concentration of 1 nM, 0.1 nM, or 0.02 nM. After 24 hours of culture, relative levels of Renilla luciferase normalized to the level of constitutively expressed firefly luciferase also present on the psiCHECK2 plasmid were measured using a dual-luciferase reporter assay (Promega E2920).

[0147] The inhibition rate of HSD17B13 gene mediated by siRNA was calculated according to the following formula.

[0148] HSD17B13 gene inhibition rate (%) = (1-relative level of Renilla luciferase in the sample / relative level of Renilla luciferase in the control group) × 100.

[0149] The test results are shown in Tables 17 and 18.

[0150] Table 17 shows the test results of the modified siRNA

[0151]

[0152] Table 18

[0153]

[0154] As shown in Tables 17 and 18, the modified siRNA sequences of the present invention have good inhibitory activity against HSD17B13 mRNA.

[0155] Example 6 Synthesis of siRNA Compounds

[0156] Modified siRNAs were prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark). The targeting ligand L96 from Example 1 was then attached to the 3' end of the sense strand of the modified siRNAs listed in Tables 4-16. The specific synthesis routes can be found in patent document CN201980061674.3, and the specific structures of the siRNA compounds are shown in Table 19.

[0157] Table 19 is siRNA analogs

[0158]

[0159]

[0160]

[0161] Example 7 Evaluation of the in vivo activity of siRNA analogs in mice using the HSD17B13-SEAP system

[0162] To evaluate the in vivo activity of siRNA compounds, 6-8 week old Balb / C mice were injected with the HSD17B13-SEAP system plasmid by hydrodynamic tail vein injection (HDI) at least 7 days in advance, and transient transfection was achieved in mice using this plasmid. The plasmid contains the SEAP (secreted human placental alkaline phosphatase) reporter gene, and the HSD17B13 cDNA sequence (GenBank NM_178135.5) is inserted into the 3'UTR of the SEAP gene. 20 ug of the plasmid in saline at a total volume of 10% of the mouse body weight was injected into the mouse via the tail vein within 3-5 seconds to achieve the construction of HSD17B13-SEAP model mice. Subsequent treatment of mice with siRNA compounds will inhibit the expression of HSD17B13 and SEAP expression. Before the administration of siRNA (day -1), the mouse was treated with Phospha-Light TM The SEAP reporter gene assay system (Invitrogen) was used to measure baseline SEAP expression in mouse serum. Mice were grouped according to baseline SEAP levels. Serum was collected from mice on days 4, 5, 7, 8, 14, 15, 21, 22, 28, 29, 35, and 36 after administration, and SEAP expression was measured at each time point.

[0163] Normalization is achieved by dividing the SEAP expression level of a specific mouse at a certain time point by the mouse's baseline SEAP expression level, which is called the "SEAP expression normalization ratio" for that mouse. The inhibition rate of HSD17B13-SEAP at each time point was calculated according to the following formula: HSD17B13-SEAP inhibition rate (%) = (1 - SEAP expression normalization ratio of the specific mouse / average SEAP expression normalization ratio of the control group of mice) * 100.

[0164] Using the method described above, HSD17B13-SEAP mice were constructed by HDI. The siRNA compounds shown in Table 19 were then administered by subcutaneous injection at doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg. Serum SEAP levels were continuously monitored at different time points, and the average inhibition rate of each group of mice at the corresponding time point was calculated. The calculation results are shown in Tables 20 and 21, respectively.

[0165] Table 20 shows the in vivo activity of siRNA compounds administered at 3 mg / kg

[0166]

[0167]

[0168] ▼ Table 21 shows the in vivo activity of siRNA compounds administered at 10 mg / kg

[0169]

[0170] As shown in Tables 20 and 21, the siRNA compounds of the present invention have good inhibitory activity against HSD17B13 mRNA.

[0171] Example 8 Evaluation of the in vivo activity of siRNA compounds in mice using an adeno-associated virus expression system

[0172] To evaluate the in vivo activity of HSD17B13 siRNA compounds, C57BL / 6J mice infected with adeno-associated virus (AAV) were used (4-9 mice per group). Each mouse was injected with 2×10 ^11 or 5x10 ^11Viral particle counts of AAV expressing human HSD17B13. 14 days after viral infection, mice were administered 1 mg / kg, 3 mg / kg, or 10 mg / kg of siRNA compounds or saline (vehicle group). 15 days after administration, human HSD17B13 expression in mouse livers was detected by RT-PCR. Expression levels were assessed by normalizing the relative expression levels of human HSD17B13 mRNA to the corresponding mouse Gapdh mRNA, and the human HSD17B13 inhibition rate was calculated using the following formula:

[0173] Human HSD17B13 inhibition rate (%) = (1-relative expression level of human HSD17B13 mRNA in the sample / average relative expression level of human HSD17B13 mRNA in the vehicle group) × 100.

[0174] The inhibitory activity of a compound is the average inhibition rate of HSD17B13 in mice in the corresponding group.

[0175] Table 22 shows the inhibitory activity of siRNA compounds at a dose of 3 mg / kg against HSD17B13

[0176] Patent Number Inhibition rate (%) Patent Number Inhibition rate (%) Patent Number Inhibition rate (%) S1-21 58.58 S64-11 61.32 S64-4 58.26 S1-22 62.43 S64-12 61.31 S64-8 59.93 S1-24 57.62 S64-13 63.50 S64-9 55.07 S63-4 53.57 S64-15 61.70 S64-10 52.80 S64-1 62.23

[0177] As shown in Table 22, the siRNA compound of the present invention has good inhibitory activity against HSD17B13.

[0178] Example 9 In vivo activity evaluation in cynomolgus monkeys

[0179] To evaluate the in vivo efficacy of the siRNA compounds of the present invention, male cynomolgus monkeys were used to test the in vivo activity of the siRNA compounds listed in Table X. The specific procedures were as follows: 21 days prior to administration of the siRNA compounds (day -21), a small amount of liver tissue from the left and right lobes of the cynomolgus monkeys was obtained by liver puncture. Endogenous HSD17B13 expression in the monkey liver tissue was detected by RT-PCR, and the ACTB mRNA level in the corresponding tissue was normalized to determine the baseline relative expression of HSD17B13. On day 0, each group of cynomolgus macaques (n=4) received a subcutaneous injection of a 3 mg / kg dose of an siRNA compound. Liver tissue was then collected by liver puncture on days 7, 28, and 51, and the relative expression of endogenous HSD17B13 in the animals was measured. The change in HSD17B13 expression relative to baseline was calculated using the following formula: Inhibition rate relative to baseline (%) = (1 - HSD17B13 expression level of a specific cynomolgus macaque at a specific time point / HSD17B13 expression level of the same cynomolgus macaque on day -21) * 100%. The in vivo efficacy of the siRNA compound was assessed by calculating the average relative baseline inhibition rate of HSD17B13 for the animals in the corresponding groups at different time points. The relative baseline inhibition rate of HSD17B13 by the siRNA compound of the present invention was greater than 70% on days 28 and 51. The results demonstrated that the siRNA compound of the present invention exhibited significant in vivo inhibitory activity against HSD17B13.

[0180] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A double-stranded siRNA analog for inhibiting HSD17B13 expression, characterized in that: The double-stranded siRNA analog includes a sense strand and an antisense strand, wherein the sense strand is at least partially complementary to the antisense strand, and the sense strand and the antisense strand are selected from the sequences shown in Table 1 and Table 2.

2. The double-stranded siRNA analog according to claim 1, characterized in that The sense strand and / or antisense strand comprises at least one modified nucleotide selected from: Alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphonate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acids (GNA).

3. The double-stranded siRNA analog according to claim 1 or 2, characterized in that The modified nucleotides are selected from the sequences in Tables 4-16.

4. The double-stranded siRNA analog according to claim 1, characterized in that The double-stranded siRNA analog is linked to a targeting ligand.

5. The double-stranded siRNA analog according to claim 4, characterized in that The targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

6. The double-stranded siRNA analog according to claim 4, characterized in that The targeting ligand is selected from:

7. The double-stranded siRNA analog according to claim 4, characterized in that The targeting ligand is linked to the 3' or 5' end of the sense strand or the antisense strand.

8. The double-stranded siRNA analog according to claim 4, characterized in that The siRNA analog is selected from any one of the siRNA compounds shown in Table 19.

9. A pharmaceutical composition for inhibiting HSD17B13 gene expression, characterized in that: The pharmaceutical composition comprises the double-stranded siRNA analog according to any one of claims 1 to 8.

10. Use of the double-stranded siRNA analog according to any one of claims 1 to 8 and the pharmaceutical composition according to claim 9 for the preparation of a pharmaceutical composition for treating a disease, disorder or symptom mediated at least in part by HSD17B13 gene expression, wherein the disease is selected from NAFLD, NASH, liver fibrosis, or alcoholic liver disease or non-alcoholic liver disease such as cirrhosis.

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