CN120608055A - Double-stranded siRNA analogs for inhibiting HSD17B13 expression and preparation methods and uses thereof - Google Patents

Abstract

The present invention relates to double stranded siRNA analogs for inhibiting expression of HSD17B13, which can inhibit expression of HSD17B13, and methods of making and using the same, for treating HSD17B 13-related diseases and conditions, including NAFLD, NASH, liver fibrosis, or alcoholic liver disease or non-alcoholic liver disease such as cirrhosis.

Description

Double-stranded siRNA analogue for inhibiting HSD17B13 expression and preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a double-chain siRNA analogue for inhibiting HSD17B13 expression, a preparation method and application thereof.

Background

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

The 17β -HSD13 protein is distributed in a wide range of tissues in vivo and is encoded by the HSD17B13 gene (alternatively referred to as the 17β -HSD13 gene). It is known that the highest expression level is found in hepatocytes of the liver, whereas lower levels can be detected in ovaries, bone marrow, kidneys, brain, lungs, skeletal muscles, bladder and testes _▼. The function of 17β -HSD13 is not fully understood, however, some 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. Liver upregulation of 17β -HSD13 has been reported in fatty liver patients, supporting the role of this enzyme in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).

Wen Su et al have previously identified 17β -HSD13 as a Lipid Droplet (LD) related protein in NAFLD patients, and reported 17β -HSD13 to be among one of the most abundantly expressed LD proteins that is specifically localized on the surface of LD. (Wen Su et al ,Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty live disease,111PNAS 11437-11442(2014)).

Research has shown that HSD17B13 gene expression plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Although international patent application publication nos. WO2019183329, WO2020061177, WO2020132564, WO2021113820, etc. disclose RNA compounds and are capable of inhibiting the expression of the HSD17B13 gene, no drug has been approved for NASH or other diseases and conditions listed as NAFLD or ARLD.

Disclosure of Invention

In view of the problems existing in the prior art, the invention provides a double-stranded siRNA analogue for inhibiting the expression of HSD17B13, a preparation method and application thereof, and the double-stranded siRNA analogue has a good inhibition effect on the gene expression of the HSD17B 13.

In a first aspect, the invention provides a double stranded siRNA analogue for inhibiting expression of HSD17B13, wherein the double stranded siRNA analogue comprises a sense strand and an antisense strand, wherein the sense strand is at least partially complementary to the antisense strand, the sense strand and the antisense strand being selected from the sequences shown in table 1 and table 2.

Wherein the sense strand and the antisense strand may be partially, substantially, or fully complementary to each other, e.g., the sense strand and the antisense strand may be 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary.

Wherein the length of the nucleotides of the sense strand and the antisense strand may be the same or different, for example, the sense strand comprises 19 nucleotides, the antisense strand comprises 21 nucleotides, or the sense strand comprises 19 nucleotides, the antisense strand also comprises 19 nucleotides, or the sense strand comprises 21 nucleotides, the antisense strand comprises 19 nucleotides, or the sense strand comprises 21 nucleotides, and the antisense strand comprises 21 nucleotides.

As a preferred embodiment of the invention, the sense strand and/or the antisense strand comprises at least one modified nucleotide.

Wherein the modified nucleotide is selected from the group consisting of:

Alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxy ethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxynucleotides, 5 '-methylphosphonate nucleotides, 5' -C-methylphosphonate nucleotides, 2 '-deoxy-2' -fluoronucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked Nucleic Acids (LNA), morpholino oligonucleotides (PMO), ethylene Glycol Nucleic Acids (GNA), wherein the alkyl nucleotides are selected from the group consisting of methyl nucleotides, ethyl nucleotides, ethylene glycol nucleic acids including (S) -ethylene glycol nucleic acids ((S) -GNA) and (R) -ethylene glycol nucleic acids ((R) -GNA).

Wherein both the sense strand and the antisense strand of the double stranded siRNA analog comprise at least one modified nucleotide, and in a specific embodiment, the sense strand comprises at least one modified nucleotide, the antisense strand has no modification of the nucleotides, or the sense strand has no modification of the nucleotides, and the antisense strand comprises at least one modified nucleotide. In a specific embodiment, each nucleotide of the sense strand is modified and each nucleotide of the antisense strand is also modified.

As a preferred embodiment of the present invention, the sense strand or antisense strand comprises 2 '-fluoro nucleotides, the number of said 2' -fluoro nucleotides being at most not more than 8, preferably 3, 4 or 5. For example, the sense strand comprises 42 '-fluoro nucleotides and the antisense strand comprises 8 2' -fluoro nucleotides.

As a preferred embodiment of the present invention, the sense strand or the antisense strand comprises phosphorothioate nucleotides, the number of phosphorothioate nucleotides being at most 4, preferably 3, more preferably 2.

As a preferred embodiment of the invention, the sense strand or the antisense strand comprises at most one locked nucleic acid modification, ethylene glycol nucleic acid modification or vinylphosphonate deoxyribonucleotide (VP) modification.

For example, one nucleotide of the sense strand is modified by a locked nucleic acid, one nucleotide of the antisense strand is not modified by a locked nucleic acid, one nucleotide of the antisense strand is modified by a locked nucleic acid, one nucleotide of the sense strand is not modified by a locked nucleic acid, or one nucleotide of both the sense strand and the antisense strand is not modified by a locked nucleic acid.

For another example, one nucleotide of the sense strand is modified with a glycol nucleic acid, one nucleotide of the antisense strand is not modified with a glycol nucleic acid, one nucleotide of the antisense strand is modified with a glycol nucleic acid, one nucleotide of the sense strand is not modified with a glycol nucleic acid, or one nucleotide of both the sense and antisense strands is not modified with a glycol nucleic acid.

For another example, one nucleotide of the sense strand is modified by VP, one nucleotide of the antisense strand is not modified by VP, one nucleotide of the antisense strand is modified by VP, one nucleotide of the sense strand is not modified by VP, or neither nucleotide of the sense strand nor nucleotide of the antisense strand is modified by VP.

As a preferred embodiment of the present invention, said sense strand and/or said antisense strand comprises at most one of said deoxynucleotides. Wherein the 2' -deoxynucleotide is selected from the group consisting of 2' -deoxyadenosine-3 ' -phosphate, 2' -deoxycytidine-3 ' -phosphate, 2' -deoxyguanosine-3 ' -phosphate, and 2' -deoxythymidine-3 ' -phosphate. For example, one nucleotide of the sense strand is modified with a 2' -deoxynucleotide, one nucleotide of the antisense strand is not modified with a 2' -deoxynucleotide, or one nucleotide of the sense strand is modified with a 2' -deoxynucleotide, one nucleotide of the antisense strand is modified with a 2' -deoxynucleotide, or both the sense strand and the antisense strand are not modified with a 2' -deoxynucleotide.

As a preferred embodiment of the present invention, the modified nucleotide is selected from the sequences in tables 4 to 16.

As a preferred embodiment of the present invention, the double-stranded siRNA analogue is linked to a targeting ligand.

As a preferred embodiment of the present invention, the targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

As a preferred embodiment of the present invention, the targeting ligand is selected from the group consisting of:

as a preferred embodiment of the invention, the targeting ligand is attached to the 3 'or 5' end of the sense strand.

As a preferred embodiment of the invention, the targeting ligand is attached to the 3 'or 5' end of the antisense strand.

In some embodiments, the targeting ligand is attached to the 5' end of the sense strand. In some embodiments, the targeting ligand is attached to the 3' end of the sense strand. In some embodiments, the targeting ligand can also be attached internally to nucleotides on the sense and/or antisense strand of the double stranded siRNA analog. In some embodiments, the targeting ligand can also be attached to the double stranded siRNA analogue via a linker, e.g., the targeting ligand can also be attached to the 3 'or 5' end of the sense strand via a linker, or the targeting ligand can also be attached to the 3 'or 5' end of the antisense strand via a linker, or the targeting ligand can also be attached internally to a nucleotide on the sense strand and/or the antisense strand of the double stranded siRNA analogue via a linker.

As a preferred embodiment of the present invention, the siRNA analogue is selected from the group consisting of siRNA compounds shown in any one of table 19.

In a second aspect, the invention provides a pharmaceutical composition for inhibiting HSD17B13 gene expression, the pharmaceutical composition comprising a double stranded siRNA analogue as described above.

As a preferred embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In a third aspect, the invention also provides the use of a double stranded siRNA analogue as described above in the preparation of a medicament for the treatment of a disease, disorder or condition mediated at least in part by HSD17B13 gene expression.

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

The double-chain siRNA analogue for inhibiting the expression of the HSD17B13 gene provided by the invention has better inhibition activity on the HSD17B13 and can be used for preventing and/or treating related diseases mediated by the HSD17B13 gene expression.

Detailed Description

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

The term "comprising" as used herein is intended to mean, and is used interchangeably with, the phrase "including (but not limited to)". Unless the context clearly indicates otherwise.

The term "or" is used herein to mean the term "and/or" and is used interchangeably with the term unless the context clearly indicates otherwise.

The terms "sequence" and "nucleotide sequence" as used herein mean the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

In the present invention, the term "siRNA analogue" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientations relative to a target RNA.

"Complementary" in the context of the present invention 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 the bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand is always paired with uracil on the other strand, and guanine is always paired with cytosine, the two strands are considered complementary to each other, and the sequence of the strand can be deduced from the sequence of its complementary strand.

The term "antisense strand" generally refers to the strand of an RNAi agent that includes a region substantially complementary to a target sequence. As used herein, the term "complementarity region" 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 perfectly complementary to the target sequence, the mismatch may be in the interior or terminal region of the molecule. Typically, the most tolerated mismatches are within the terminal region, e.g., 5, 4, 3 or 2 nucleotides at the 5 'end and/or 3' end.

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

In the present invention, modified nucleotides include, but are not limited to, alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxy ethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxynucleotides, 5 '-methylphosphonate nucleotides, 5' -C-methylphosphonate nucleotides, 2 '-deoxy-2' -fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked Nucleic Acids (LNA), morpholino oligonucleotides (PMO), inverted abasic deoxyriboresidues (invAb), ethylene Glycol Nucleic Acids (GNA).

Wherein alkyl modified nucleotides, such as 2' -methyl nucleotides,A 2' -ethyl nucleotide, which is a nucleotide,

2' -Methoxy modified nucleotide, for example,2' -Methoxyethyl nucleotide, for example,2' -Fluoronucleotides, for example,5' -C-methylphosphonate nucleotides, e.gVinyl phosphonate deoxyribonucleotides (VP), for example,Phosphorothioate nucleotide(s) of the structure: The phosphate nucleotide (p) has the structure: 2' -deoxyribonucleotides, for example: reverse abasic deoxyribose residues (invAb), for example: ethylene Glycol Nucleic Acid (GNA), including (S) -ethylene glycol nucleic acid ((S) -GNA), for example: and (R) -ethylene glycol nucleic acid ((R) -GNA), for example:

Wherein Base represents a Base, R represents an alkyl group, me represents a methyl group, and Et represents an ethyl group.

The term "locked nucleic acid" is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an additional bridge linking the 2 'carbon and the 4' carbon. This structure effectively "locks" the ribose in the 3' -endo structural conformation. Addition of locked Nucleic acids to siRNA has been shown to increase siRNA stability _▲ in serum and reduce off-target effects (Elmen (Elman), J.et al, (2005) NucleicAcids Research (Nucleic acids research) 33 (1): 439-447; mook (Mu Ke), OR. et al, (2007) MolCancTher (molecular cancer therapeutics) 6 (3): 833-843; grunwiller (Grinival), A.et al, (2003) Nucleic ACIDS RESEARCH (Nucleic acids research) 31 (12): 3185-3193).

Representative U.S. patents for preparing locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. 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.

The locked nucleic acid structure is as follows:

In certain embodiments, the sugar substitute 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. Pat. Nos. 5,698,685;5,166,315;5,185,444; and 5,034,506).

The term "morpholino" means a sugar substitute having the formula:

In certain embodiments, morpholino groups may be modified, for example, by adding or altering various substituents according to the morpholino structures above. Such sugar substitutes are referred to herein as "modified morpholino groups".

In the present invention, capital letters C, G, U, A denote the base composition of nucleotides unless otherwise specified. The lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy modified nucleotide, the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro modified nucleotide, the LNA indicates that one nucleotide adjacent to the right side of the letter f is a Locked Nucleic Acid (LNA) modified nucleotide, the GNA indicates that one nucleotide adjacent to the left side of the letter m is a GNA modified nucleotide, the lower case letter s indicates that phosphorothioate group connection is formed between the left nucleotide and the right nucleotide of the letter, and the VP indicates that one nucleotide on the right side of the letter VP is a vinyl phosphate modified nucleotide. invAb denotes an inverted abasic deoxyribonucleotide, dN denotes any deoxyribonucleotide, dA denotes deoxyadenine nucleotide, dT denotes deoxythymine nucleotide, dU denotes deoxyuracil nucleotide, dC denotes deoxycytosine nucleotide, and dG denotes deoxyguanine nucleotide.

It is emphasized that "modifications" of the nucleotides described in the present disclosure include, but are not limited to, the examples described above, but that the nucleotides may also be replaced with other nucleotides, e.g., (S) -glycerol nucleic acids, etc.

The term "targeting ligand" may 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 may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of the polyamino acid include Polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, a styrene acid-maleic anhydride copolymer, a poly (L-lactide-co-glycolide) copolymer, a divinyl ether-maleic anhydride copolymer, an N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), an N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

The targeting ligand may also bind to a cell or tissue targeting agent, e.g., lectin, glycoprotein, lipid or protein, e.g., antibody, of a designated cell type, e.g., kidney cells. The targeting group may be thyroid stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein a, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, cholic acid, folic acid, vitamin B12, vitamin a, biotin, or RGD peptide mimetic.

The targeting ligand may also be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule having a specific affinity for a secondary ligand, or an antibody, e.g., an antibody that binds to a designated cell type, e.g., a hepatocyte. Ligands may also include hormones and hormone receptors. They may also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. The ligand may be, for example, lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-KKB.

The targeting ligand may be a substance, e.g., a drug, that may increase uptake of the iRNA agent into the cell, e.g., by disrupting the cytoskeleton of the cell (e.g., by disrupting cell microtubules, microfilaments, and/or intermediate filaments). The drug may be, for example, taxol (taxon), vincristine, vinblastine, cytochalasin, nocodazole, microfilament-promoting polymerizer (iaplakinolide), halichondrin a, phalloidin, marine moss (swinholide) a, indanrox (indanocine) or drug (myoservin).

Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient which can be combined with the carrier material to prepare a single dosage form is generally that amount of the compound which produces the therapeutic effect. Generally, the amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%, in one percent.

The term "preventing and/or treating" includes not only preventing and/or treating a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression _▼ of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and/or alleviating one or more symptoms associated with a disease, reducing the severity and/or duration of a disease and/or any symptoms associated therewith and/or preventing further increases in the severity of a disease and/or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by a disease, and any pharmacological effects that are generally beneficial to the patient being treated. The nucleic acid or pharmaceutical composition of the application forms a viable therapeutic agent without the need to achieve complete cure or eradication of any symptoms or manifestations of the disease. As recognized in the relevant art, drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered useful therapeutic agents. Similarly, a prophylactically administered treatment constitutes a viable prophylactic agent and need not be completely effective in preventing the onset of the condition. It may be sufficient to simply reduce the impact of the disease in the subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of disease occurrence or exacerbation.

The terms "disease" or "disorder" are used interchangeably and generally refer to any deviation of a subject from a normal state, such as any change in the state of the body or certain organs, which impedes or disrupts performance of the function, and/or causes symptoms such as discomfort, dysfunction, pain, or even death in a person suffering from or in contact with the disease. The disease or disorder may also be referred to as a disorder (distemp), malaise (ailing), ailment (ailment), disease (malady), disorder (disorder), disease (sickness), illness (illness), physical malaise (complaint), inderdisposion, or affectation.

The term "inhibit" may be used interchangeably with "reduce," "silence," "down-regulate," "refrain" and other similar terms and includes any level of inhibition.

The phrase "inhibiting expression of HSD17B 13" in the present invention includes inhibiting expression of any HSD17B13 gene (e.g., a mouse HSD17B13 gene, a rat HSD17B13 gene, a monkey HSD17B13 gene, or a human HSD17B13 gene) along with a variant or mutant of the HSD17B13 gene encoding the HSD17B13 protein.

"Inhibiting expression of HSD17B 13" includes inhibiting any level of HSD17B13 gene, e.g., at least partially inhibiting expression of HSD17B13 gene, e.g., 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%.

The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, prepared from a compound of the present invention having a particular substituent found therein and a pharmaceutically acceptable acid or base.

The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium capable of delivering an effective amount of the active agents of the present invention, which does not interfere with the biological activity of the active agents and which does not have toxic or side effects to the host or patient, representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. Such matrices include suspending agents, viscosity enhancers, transdermal enhancers, and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts. For additional information on the vector, reference may 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.

The term "pharmaceutically acceptable excipient" is a substance that is intentionally included in a drug delivery system in addition to an active pharmaceutical ingredient (API, therapeutic product, e.g., HSD17B 13). Excipients do not play a therapeutic role at the intended dose or not. Excipients may function to a) aid in handling of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) aid in product identification, and/or d) enhance any other attribute of the API in terms of overall safety, effectiveness or delivery during storage or use.

Among the excipients include, but are not limited to, absorption enhancers, anti-tackifiers, defoamers, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, glidants, humectants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, wetting agents, lubricants such as sodium and magnesium lauryl sulfate, flavoring agents, and flavoring agents.

The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a disorder, disease or condition of interest.

Example 1 Synthesis of targeting ligands

Step A hydroxyproline amine (3.00 g,7.15 mmol) and dodecanedioic acid monomethyl ester (1.748 g,7.15 mmol) were placed together in N, N-Dimethylformamide (DMF) (50 mL). To 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 stirred overnight.

The reaction mixture was poured into an ice-water mixture and extracted with Dichloromethane (DCM). Washed with bicarbonate solution, water, brine and dried over sodium sulfate. The solvent was removed and the residue purified by chromatography (50% ethyl acetate/hexane, ethyl acetate, then 5% methanol/dichloromethane) to give the desired compound 115 as a white solid (4.30 g, 93%). MS: C ₃₉H₅₁NO₇, calculated as 645.37, found 646.35 (M+H).

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.

Checked by 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 a Dichloromethane (DCM) solution and the solution was filtered through a small pad of silica gel to give the desired product 102 as _▲ its Triethanolamine (TEA) salt (4.15 g, 86%). MS: C ₃₈H₄₉NO₇, calculated 631.35, found 630.34 (M-H).

Step C Compound 102 (1.30 g,2.06 mmol) was added to N, N-Dimethylformamide (DMF) (30 mL) along with peptide coupling reagent (HBTU) (0.823 g,1.05 eq.). To the N, N-Diisopropylethylamine (DIEA) (1.07 ml,3 eq) was added and the reaction mixture was stirred for 3-4 minutes. A solution of amine (3.00 g,1.58 mmol) was added followed by 1eq DIA. The reaction mixture was stirred at room temperature overnight.

The solvent was removed under reduced pressure and the residue was dissolved in Dichloromethane (DCM), washed with bicarbonate and water. 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 5-20% methanol in dichloromethane) to give product 103 as a white solid (3.35 g, 88%). MS calculated for C ₁₁₇H₁₇₅N₁₁O₄₂ is 2406.19 and found 2429.10 (M+Na).

Wherein ligand 104 (L96) can be linked to the siRNA through a phosphate group, a phosphorothioate group, or another linking group.

For specific synthetic routes, reference is made to the document WO 2009073809.

EXAMPLE 2 Synthesis of siRNA analogues

SiRNA was prepared using OligoMakerApS RNA synthesizer (denmark) for specific synthetic routes, see patent document CN201980061674.3, and siRNA analogue sequences are shown in tables 1 and 2.

Table 1 shows the sequence listing of siRNA analogs

TABLE 2

Example 3 in vitro testing of HSD17B13siRNA analogs in HuH7 cells

The HSD17B13 cDNA (GenBank accession No. NM-178135.5) was cloned into reporter-based screening plasmid psiCHECK2 (Promega-C8021) to produce Renilla luciferase/HSD 17B13 fusion mRNA. HuH7 cells were cultured in DMEM (Gibco-10313021) medium containing 10% fetal bovine serum (Excell Bio-FSP 500), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), 1% penicillin-streptomycin (HyClone-SV 30010). HSD17B13-psiCHECK2 plasmid, siRNA analogs, and Lipo2000 (Invitrogen-11668019) transfection reagent diluted with Opti-MEM (Gibico-11058021) were added to the HuH7 cell suspension and plated in 96-well plates at a cell density of 1X 10 ⁵/ml to give a final concentration of siRNA analogs of 1nM or 0.02nM. After 24 hours of incubation, the relative levels of renilla luciferase normalized to the levels of constitutively expressed firefly luciferase also present on psiCHECK2 plasmid were measured using a dual luciferase reporter assay (Promega-E2920).

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

HSD17B13 gene inhibition (%) = (1-sample renilla luciferase relative level/control renilla luciferase relative level) ×100.

The calculation results are shown in Table 3.

Table 3 shows the inhibition efficiency of HSD17B13

As shown in the experimental results of Table 3, the siRNA sequence of the present invention has excellent inhibitory activity on HSD17B13 gene.

EXAMPLE 4 Synthesis of modified sequences of siRNA analogues

The siRNA analogues containing modifications were prepared using OligoMakerApS RNA synthesizer (Denmark) and specific synthetic routes are described in patent document CN201980061674.3, and the modified siRNA analogues with sequences S1, S2, S3, S4, S5, S21, S22, S24, S25, S26 and S27 are shown in tables 4-15, respectively, and the modified sequences of the sequences shown in Table 2 are shown in Table 16.

Table 4 is a modified sequence table of the sequence S1

Table 5 is a modified sequence table of the sequence S2

Table 6 is a modified sequence table of sequence S3

Table 7 is a modified sequence table of the sequence S4

Table 8 is a modified sequence table of sequence S5

Table 9 is a modified sequence table of the sequence S21

Table 10 is a modified sequence table of sequence S22

Table 11 is a modified sequence table of the sequence S24

Table 12 is a modified sequence table of sequence S25

Table 13 is a modified sequence table of sequence S26

Table 14 is a modified sequence table of sequence S27

Table 15 is a modified sequence listing of S63 and S64

Table 16 is a modified sequence listing of the sequences of Table 2

Wherein a=adenosine-3 '-phosphate, u=uridine-3' -phosphate, c=cytidine-3 '-phosphate, g=guanosine-3' -phosphate, t=thymine-3 '-phosphate, am=2' -O-methylcytidine-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-methylguanosine-3' -phosphorothioate, ums =2 '-O-methyluridine-3' -phosphorothioate, s=2 '-O-methylcytidine-3' -phosphorothioate, gms =2 '-O-methylguanosine-3' -phosphorothioate, af=2 '-fluoroadenosine-3' -phosphate, uf=2 '-fluorouridine-3' -phosphate, cf=2 '-O-methylguanosine-3' -phosphorothioate, gs=2 '-methylguanosine-3' -phosphorothioate, fs=2 '-fluoroguanosine-3' -phosphorothioate, cf=2 '-fluoroguanosine-3' -phosphate =2 '-fluoroguanosine-3' -phosphorothioate, da=2 '-deoxyadenosine-3' -phosphate, dc=2 '-deoxycytidine-3' -phosphate, dg=2 '-deoxyguanosine-3' -phosphate, dt=2 '-deoxythymidine-3' -phosphate, m=2 '-O-methyl, f=2' -fluoro, s=phosphorothioate linkage, (LNA) =locked nucleic acid ribonucleotide, gna=ethylene glycol nucleic acid nucleotide, vp=vinylphosphonate deoxyribonucleotide, im=hypoxanthine 2'-OMe ribonucleoside, if=hypoxanthine 2' -F ribonucleoside, ims=2 '-OMe hypoxanthine-3' -phosphorothioate.

Example 5 in vitro testing of HSD17B13 modified siRNA in Huh7 cells

The HSD17B13 cDNA (GenBank accession No. NM-178135.5) was cloned into reporter-based screening plasmid psiCHECK2 (PromegaC. Quadrature.1) to produce a renilla luciferase/HSD 17B13 fusion mRNA. HuH7 cells were cultured in DMEM (Gibco 10313021) medium containing 10% fetal bovine serum (ExCell BioFSP500,500), 1% glutamine (Gibco 35050061), 1% non-essential amino acids (Gibco 11140050), 1% penicillin streptomycin (HyCloneSV 30010). HSD17B13psiCHECK2 plasmid, siRNA, and Lipo2000 (Invitrogen 11668019) transfection reagent diluted with OptiMEM (Gibico 11058021) were added to the HuH7 cell suspension and plated in 96-well plates at a cell density of 1X 10 ⁵/ml to give final siRNA concentrations of 1nM, 0.1nM, or 0.02nM. After 24 hours of incubation, the relative levels of renilla luciferase normalized to the levels of constitutively expressed firefly luciferase also present on psiCHECK2 plasmid were measured using a dual luciferase reporter assay (PromegaE 2920).

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

HSD17B13 gene inhibition (%) = (1-sample renilla luciferase relative level/control renilla luciferase relative level) ×100.

The test results are shown in tables 17 and 18.

Table 17 shows the test results of modified siRNA

TABLE 18

As shown in tables 17 and 18, the modified siRNA sequences of the present invention have good inhibitory activity on HSD17B13 mRNA.

EXAMPLE 6 Synthesis of siRNA Compounds

Modified siRNA was prepared using OligoMakerApS RNA synthesizer (denmark) and then targeting ligand L96 of example 1 was ligated to the 3' end of the sense strand containing modified siRNA in tables 4-16. Among them, specific synthetic routes can be referred to patent document CN201980061674.3, and specific structures of siRNA compounds are shown in suggestion table 19.

Table 19 shows siRNA analogues

Example 7 evaluation of in vivo Activity of siRNA analogues in mice Using HSD17B13-SEAP System

To assess the in vivo activity of siRNA compounds, balb/C mice aged 6-8 weeks at least 7 days in advance were subjected to transient transfection in vivo by hydrodynamic tail vein injection (HDI) of HSD17B13-SEAP system plasmid. The plasmid contains the SEAP (secreted human placental alkaline phosphatase) reporter gene, with the HSD17B13 cDNA sequence (GenBankNM _ 178135.5) inserted within the 3' UTR of the SEAP gene. The construction of HSD17B13-SEAP model mice was accomplished by injecting into the mice a total volume of 10% of the mice weight in physiological saline containing 20ug of the plasmid via the tail vein for 3-5 seconds. siRNA compounds from post-treatment mice inhibit HSD17B13 expression with concomitant inhibition of SEAP expression. Prior to siRNA administration (day-1), baseline expression levels of SEAP in mouse serum were measured using the Phospha-Light ^TM SEAP reporter gene detection system (Invitrogen) and grouped according to baseline SEAP average levels. Mouse serum was collected on days 4,5, 7, 8, 14, 15, 21, 22, 28, 29, 35, and 36 after dosing, respectively, and SEAP expression levels at each time point were measured.

Normalization is achieved by dividing the SEAP expression level at a time point for a particular mouse by the baseline SEAP expression level for that mouse, referred to as the "SEAP expression normalization rate" for that mouse. The inhibition rate of HSD17B13-SEAP at each time point was calculated as follows, inhibition rate (%) = (1-average of normalized rate of SEAP expression in specific mice/normalized rate of SEAP expression in control mice) ×100 for HSD17B 13-SEAP.

HSD17B13-SEAP mice were constructed by HDI using the method described above, siRNA compounds shown in Table 19 were administered subcutaneously at 1mg/kg, 3mg/kg or 10mg/kg doses, serum SEAP levels at different time points were continuously measured, and the average inhibition rates at the time points corresponding to each group of mice were calculated, and the calculation results are shown in tables 20 and 21, respectively.

Table 20 shows the in vivo Activity of siRNA Compounds administered at 3mg/kg

_▼ Table 21 shows the in vivo Activity of siRNA Compounds administered at 10mg/kg

As can be seen from tables 20 and 21, the siRNA compounds of the present invention have a good inhibitory activity against HSD17B13 mRNA.

Example 8 evaluation of in vivo Activity of siRNA Compounds in mice Using adeno-associated Virus expression System

To assess the in vivo activity of HSD17B13 siRNA compounds, C57BL/6J mice (4-9 per group) infected with adeno-associated virus (AAV) were used. Each mouse was injected with 2X10 ^{^11} or 5X10 ^{^11} viral particle numbers of AAV expressing human HSD17B13 via the tail vein prior to dosing. 14 days after infection with the virus, mice were administered with siRNA compounds or physiological saline (vehicle group) at a dose of 1mg/kg, 3mg/kg or 10 mg/kg. 15 days after administration, human HSD17B13 expression in mouse liver was detected by RT-PCR. Expression levels were assessed by normalizing the relative expression levels of human HSD17B13 mRNA corresponding to mouse GAPDH MRNA and the human HSD17B13 inhibition rate calculation was performed by the following formula:

human HSD17B13 inhibition (%) = (1-average of relative expression level of sample human HSD17B13 mRNA/relative level of vehicle group human HSD17B13 mRNA) ×100.

The inhibitory activity of a compound is the average of the inhibition rates of the compound for the mice HSD17B13 grouped accordingly.

Table 22 shows the inhibitory activity of siRNA compounds at a dose of 3mg/kg on HSD17B13

Patent number	Inhibition ratio (%)	Patent number	Inhibition ratio (%)	Patent number	Inhibition ratio (%)
						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

As can be seen from Table 22, the siRNA compounds of the present invention had excellent inhibitory activity against HSD17B 13.

Example 9 evaluation of in vivo Activity in cynomolgus monkey

To evaluate the in vivo efficacy of the siRNA compounds of the invention, the in vivo activity study of the siRNA compounds in table X was tested using male cynomolgus monkeys by obtaining small amounts of tissues of left and right liver by liver puncture 21 days (day-21) prior to administration of the siRNA compounds, detecting endogenous HSD17B13 expression in monkey liver tissues by RT-PCR, and normalizing ACTB mRNA levels in the corresponding tissues to determine the baseline relative expression amount of HSD17B 13. On day 0, each group of cynomolgus monkeys (n=4) was injected with 3mg/kg of siRNA compound, and liver tissue was collected by liver puncture on days 7, 28, and 51, and the relative expression amount of endogenous HSD17B13 was examined, and the change in the expression level of HSD17B13 was calculated from the baseline by the following formula, i.e., inhibition (%) = (1-HSD 17B13 expression level of a specific cynomolgus monkey at a specific time point/HSD 17B13 expression level of the cynomolgus monkey on day-21) ×100%. In vivo efficacy of siRNA compounds was assessed by calculating the mean value of HSD17B13 relative to baseline inhibition of animals correspondingly grouped at different time points, with the siRNA compounds of the invention having greater than 70% HSD17B13 relative to baseline inhibition on both day 28 and day 51. The results show that the siRNA compounds of the invention have good in vivo inhibition activity on HSD17B 13.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A double-stranded siRNA analogue for inhibiting HSD17B13 expression, wherein said double-stranded siRNA analogue comprises a sense strand and an antisense strand, wherein said sense strand is at least partially complementary to said antisense strand, said sense strand and said antisense strand being selected from the sequences shown in tables 1 and 2.

2. The double stranded siRNA analogue of claim 1, wherein said sense strand and/or antisense strand comprises at least one modified nucleotide selected from the group consisting of:

Alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxy ethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxynucleotides, 5 '-methylphosphonate nucleotides, 5' -C-methylphosphonate nucleotides, 2 '-deoxy-2' -fluoronucleotides, vinyl phosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked Nucleic Acids (LNA), morpholino oligonucleotides (PMO), ethylene Glycol Nucleic Acids (GNA).

3. The double stranded siRNA analogue according to claim 1 or 2, wherein said modified nucleotide is selected from the sequences in tables 4-16.

4. The double stranded siRNA analogue according to claim 1, wherein said double stranded siRNA analogue has a targeting ligand attached.

5. The double stranded siRNA analogue according to claim 4, wherein said targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

6. The double stranded siRNA analogue according to claim 4, wherein said targeting ligand is selected from the group consisting of:

7. The double stranded siRNA analogue according to claim 4, wherein said targeting ligand is attached to the 3 'or 5' end of said sense strand or said antisense strand.

8. The double stranded siRNA analogue according to claim 4, wherein said siRNA analogue is selected from the group consisting of the siRNA compounds shown in any one of table 19.

9. A pharmaceutical composition for inhibiting HSD17B13 gene expression, comprising the double stranded siRNA analogue of any one of claims 1-8.

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