Treatment of Nonalcoholic Fatty Liver Disease

Abstract

Methods for treating non-alcoholic fatty liver disease by reducing HSD17B13 expression in a human subject in need of treatment are described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 329,081, filed April 8, 2022, and U.S. Provisional Application No. 63 / 401,858, filed August 29, 2022, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy is named 70162WO01_SL.xml, is approximately 115,715 bytes in size, and was created on March 7, 2023.

[0003] FIELD OF THEINVENTION Provided herein is a method for treating nonalcoholic fatty liver disease (NAFLD) that can reduce (e.g., inhibit or silence) the gene expression and protein expression of the hepatic lipid droplet protein 17β-hydroxysteroid dehydrogenase type 13 (also known as HSD17B13, 17β-HSD13, HSD17β13, 17β-HSD13, 17β-HSD13, or 17Β-HSD13). Summary of the Invention

[0004] Quick Overview In some aspects, the present disclosure provides a method for treating non-alcoholic fatty liver disease in a human subject, comprising administering to the human subject a compound or a pharma- ceutically acceptable salt thereof at a dose of about 25 to about 200 mg, calculated on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide bound to a ligand, the double-stranded oligonucleotide reduces expression of HSD17β13 in the human subject, and the ligand is [ka] or a pharma- ceutically acceptable salt thereof. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 shows the effect of test compounds on SD17β13 mRNA expression at day 71 compared to baseline in patients with NASH (nonalcoholic steatohepatitis) or NAFLD. Boxes indicate Q1, median, and Q3. Crosses indicate mean values, whiskers extend to minimum and maximum values. [Figure 2A] 2A and 2B show the effect of test compounds on liver enzymes, ALT (alanine aminotransferase) and AST (aspartate aminotransferase), respectively, in patients with NASH or NAFLD. [Figure 2B] 2A and 2B show the effect of test compounds on liver enzymes, ALT (alanine aminotransferase) and AST (aspartate aminotransferase), respectively, in patients with NASH or NAFLD. [Figure 3A] Figures 3A and 3B show the effect of test compounds on messenger RNA knockdown at day 17 compared to baseline by genotype in NASH or NAFLD patients with HSD17β13 rs72613567 (Figure 3A, boxes represent Q1, median, and Q3, crosses represent mean values, whiskers extend to minimum and maximum values) and PNPLA3 rs738409 (Figure 3B, boxes represent Q1, median, and Q3, diamonds represent mean values, whiskers extend to minimum and maximum values). [Figure 3B]Figures 3A and 3B show the effect of test compounds on messenger RNA knockdown at day 17 compared to baseline by genotype in NASH or NAFLD patients with HSD17β13 rs72613567 (Figure 3A, boxes represent Q1, median, and Q3, crosses represent mean values, whiskers extend to minimum and maximum values) and PNPLA3 rs738409 (Figure 3B, boxes represent Q1, median, and Q3, diamonds represent mean values, whiskers extend to minimum and maximum values). [Figure 4] FIG. 4 shows the clinical trial design and dosing schedule of the test compounds. [Figure 5A] 5A-5D show the chemical structures of the test compounds described in Example 2 in free acid form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 5B] 5A-5D show the chemical structures of the test compounds described in Example 2 in free acid form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 5C] 5A-5D show the chemical structures of the test compounds described in Example 2 in free acid form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 5D] 5A-5D show the chemical structures of the test compounds described in Example 2 in free acid form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 6A] 6A-6D show the chemical structures of the test compounds described in Example 2 in sodium salt form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 6B] 6A-6D show the chemical structures of the test compounds described in Example 2 in sodium salt form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 6C] 6A-6D show the chemical structures of the test compounds described in Example 2 in sodium salt form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 6D]6A-6D show the chemical structures of the test compounds described in Example 2 in sodium salt form. Circled numbers indicate the connection points of structural portions that have been split across four pages due to their length. [Figure 7] FIG. 7 shows a simulation of target engagement based on the clinical results of Example 2 to further support a dose of 200 mg every 12 weeks or 3 months. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description In some examples, the treatments disclosed herein can reduce hepatocyte production of HSD17β13 and provide therapeutic and / or protective effects in human subjects with or suspected of having NAFLD and potentially other chronic liver diseases. In some examples of human patients with or suspected of having NASH (e.g., NAFLD with high ALT), the treatments can result in a reduction, e.g., a near-maximal reduction (e.g., at least about 90% reduction), of HSD17β13 mRNA expression at dose levels of about 25 to about 200 mg based on a biopsy 71 days after administration. For doses disclosed herein, e.g., 100 mg and 200 mg, the treatments inhibit HSD17β13 in subjects with PNPLA3 rs738409 CG or GG genotypes associated with increased risk of NASH cirrhosis, and such inhibitory effects can be substantially the same as in subjects lacking the PNPLA3 mutation. In some instances, reduced HSD17β13 expression may lead to or be associated with reduced ALT and AST, surrogate markers of hepatic inflammation in NASH.

[0007] Unless otherwise specified, the term "about" is used herein to mean plus or minus 5% of the referenced numerical value.

[0008] Unless otherwise specified, the terms "a," "an," and "the" include singular and plural referents.

[0009] Compounds for Use The compounds used to treat human subjects disclosed herein may comprise, for example, RNA interference (RNAi) drugs "HSD17B13 RNAi drugs" that reduce the expression of HSD17B13. In some cases, they comprise HSD17β13-targeting double-stranded oligonucleotides linked to galactose-containing ligands that can be preferentially taken up by hepatocytes to promote hepatic specificity, and then the compounds engage the cellular RNAi machinery to target and degrade HSD17β13 mRNA, thereby reducing the amount of free HSD17β13 mRNA for translation. These factors may minimize the off-target effects or extrahepatic inhibition of the compounds, contributing to the acceptable safety profile of the compounds. In some cases, the compounds herein mimic LOF (loss-of-function) mutations in NASH to target engagement with knockdown of HSD17β13.

[0010] As used herein, the terms "reducing", "silencing", "inhibiting", "downregulating" or "knockdown" with respect to the expression of HSD17B13 refer to the expression of a gene, as measured by the level of mRNA (messenger RNA) transcribed from the gene or the level of protein (or protein subunit) translated from the mRNA, in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, being reduced when the cell, group of cells, tissue, organ, or subject is treated with a drug, compared to a second cell, group of cells, tissue, organ, or subject that has not been so treated. Reduction of HSD17B13 may be measured by any suitable assay or method known in the art. Non-limiting examples of assays include microarrays, Affymetrix gene chips, and qRT-PCR (quantitative reverse transcriptase polymerase chain reaction) for the quantification of HSD17B13 gene, as well as gel electrophoresis and mass spectrometry such as Western blot for the measurement of HSD17B13 protein, and the assays shown in International Publication No. 2020 / 061177 (Patent Application No. PCT / US2019 / 051707), the entirety of which is incorporated herein by reference. The normal human reference HSD17B13 mRNA gene transcript can be transcript variant A, GenBank NM_178135.4. In some cases, the reduction in gene expression is measured by comparing the baseline level of HSD17B13 mRNA or HSD17B13 protein in a human subject before the treatment disclosed herein with the HSD17B13 mRNA or HSD17B13 protein level after administration of the compound.

[0011] The compounds disclosed herein may contain one or more asymmetric centers (also called chiral centers) and therefore may exist as individual enantiomers, diastereoisomers, or other forms of stereoisomers, or mixtures thereof. Chiral centers, such as chiral carbon atoms, may also be present in substituents, such as alkyl groups. If the stereochemistry of a chiral center is not shown in a chemical structure, the structure is intended to encompass stereoisomers and all mixtures thereof. In some cases, the compounds disclosed herein that contain one or more chiral centers can be used as racemic mixtures and racemates, enantiomerically enriched mixtures, or racemic modifications, including enantiomerically pure individual stereoisomers. In some cases, the compounds disclosed herein are provided as salts, salt mixtures, or free acids.

[0012] Individual stereoisomers containing one or more asymmetric centers can be resolved by methods known to those skilled in the art. For example, such resolution can be carried out (1) by the formation of diastereoisomeric salts, complexes or other derivatives, (2) by selective reaction with stereoisomer-specific reagents, for example by enzymatic oxidation or reduction, or (3) by gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support such as silica with chiral ligands or in the presence of a chiral solvent. It will be understood that if the desired stereoisomer is converted to another chemical entity by one of the above-mentioned resolution procedures, additional steps will be required to liberate the desired form. Alternatively, a particular stereoisomer can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation. In some examples, the compounds for use herein comprise a sense strand (also called passenger strand) and an antisense strand (also called guide strand) that are annealed into a duplex.

[0013] In some cases, the compound comprises a double-stranded oligonucleotide, for example, comprises an RNA, a chemically modified RNA, a DNA, or a chemically modified DNA oligonucleotide molecule, each of which can have two complementary sequence strands.In some cases, the double-stranded oligonucleotide can degrade or inhibit the translation of messenger RNA (mRNA) transcript of HSD17B13 mRNA in a sequence-specific manner. In some cases, the compounds disclosed herein may function through the RNA interference machinery (i.e., RNA interference via interaction with the RNA interference pathway apparatus (RNA-induced silencing complex or RISC) of mammalian cells, or any other mechanism or pathway. Although HSD17B13 RNAi agents, as that term is used herein, are believed to function primarily through the RNA interference mechanism, the disclosed RNAi agents are not constrained or limited to a particular pathway or mechanism of action. In some cases, the compounds comprise short or small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), or Dicer substrates. In some cases, the compounds for use herein include an antisense strand having a region complementary to at least a portion of HSD17B13 mRNA.

[0014] In some cases, the compounds for use herein have a sense strand that is 15-49 nucleotides long and an antisense strand that is 18-49 nucleotides long. In some cases, the sense strand and the antisense strand are independently 18-26 nucleotides long. In some cases, the sense strand and the antisense strand are independently 21-26 nucleotides long. In some cases, the sense strand and the antisense strand are independently 21-24 nucleotides long. In some cases, the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some cases, the antisense strand is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some cases, the sense strand and the antisense strand are both 21 nucleotides long. The sense and antisense strands may be the same or different lengths. The sense and antisense strands may also form overhanging nucleotides at one or both ends of the nucleotide strand.

[0015] Compounds suitable for use herein can be covalently linked to a ligand that includes one or more N-acetyl-galactosamine moieties. The N-acetyl-galactosamine moieties can facilitate targeting of the HSD17B13 RNAi agent to the asialoglycoprotein receptor (ASGPR), which is readily present on the surface of liver cells, resulting in internalization of the HSD17B13 RNAi agent by endocytosis or other means.

[0016] Modified nucleotides and modified internucleoside linkages The compounds disclosed herein, for example, HSD17B13 RNAi agents, can comprise modified nucleotides, which can increase serum stability while retaining the activity of the RNAi agent and minimize the possibility of activating interferon activity in humans.As used herein, "modified nucleotide" refers to a nucleotide other than ribonucleotides (2'-hydroxyl nucleotides).In some cases, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimetics, 2'-modified nucleotides, inverted nucleotides, nucleotides comprising modified nucleobases, bridged nucleotides, peptide nucleic acids (PNAs), 2',3' cellonucleotide mimetics (unlocked nucleobase analogues), locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me,2'-fluoro nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides. In some cases, a modified nucleotide of an HSD17B13 RNAi agent is a 2'-modified nucleotide (i.e., a nucleotide having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring. 2'-modified nucleotides include, but are not limited to, 2'O methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides (commonly referred to as simply 2'-fluoro nucleotides), 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides. All nucleotides of a given RNAi agent need not be uniformly modified. In some cases, one or more modifications can be incorporated in a single HSD17B13 RNAi agent, or even in that single nucleotide.The sense and antisense strands of HSD17B13 RNAi agents can be synthesized and / or modified by methods known in the art. Modifications at one nucleotide can be independent of modifications at another nucleotide.

[0017] In some cases, the nucleobases disclosed herein (often simply referred to as "bases") can be modified. Natural nucleobases can include the basic purine bases adenine and guanine and the basic pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. The synthesis of such modified nucleobases (including phosphoramidite compounds that include modified nucleobases) is known in the art.

[0018] Modified nucleobases include, for example, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2 aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5 hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2 -thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5 propynyluracil, 5 propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4 thiouracil, 8-halo, 8 amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7 deazaguanine, 7 deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0019] In some cases, all or substantially all nucleotides of the compounds disclosed herein, such as HSD17B13 RNAi agents, are modified nucleotides.As used herein, substantially all nucleotides of the RNAi agent are modified nucleotides, and both sense strand and antisense strand have no more than 1 (i.e., 0, 1, 2, 3, or 4) nucleotides of ribonucleotides (i.e., unmodified).As used herein, substantially all nucleotides of the sense strand are modified nucleotides, and two (i.e., 0, 1, or 2) nucleotides of the sense strand are ribonucleotides.As used herein, substantially all nucleotides of the antisense strand are modified nucleotides, and two (i.e., 0, 1, or 2) nucleotides of the sense strand are ribonucleotides.As used herein, substantially all nucleotides of the antisense strand are modified nucleotides, and two (i.e., 0, 1, or 2) nucleotides of the sense strand are ribonucleotides.

[0020] In some cases, one or more nucleotides of a compound disclosed herein, e.g., an HSD17B13 RNAi agent, are linked by a non-standard bond or backbone (i.e., a modified internucleoside bond or a modified backbone). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkyl phosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkyl phosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates with inverted polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some cases, the modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. In some cases, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate backbones and sulfonamide backbones, amide backbones, and mixed N, O, S, and CH backbones. 2 Other frameworks having components are included.

[0021] In some cases, the sense strand of a compound disclosed herein, e.g., an HSD17B13 RNAi agent, can include 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of an HSD17B13 RNAi agent can include 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand can independently include 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some cases, the sense strand of an HSD17B13 RNAi agent can include 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of an HSD17B13 RNAi agent can include 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand can independently include 1, 2, 3, or 4 phosphorothioate linkages.

[0022] In some cases, the sense strand of a compound disclosed herein, e.g., an HSD17B13 RNAi agent, comprises at least two phosphorothioate internucleoside linkages. In some cases, the at least two phosphorothioate internucleoside linkages are between nucleotides 1-3 from the 3' end of the sense strand. In some cases, the at least two phosphorothioate internucleoside linkages are between nucleotides 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5' end of the sense strand. In some cases, phosphorothioate internucleoside linkages are used to link the terminal nucleotides of the sense strand to cap residues at the 5' end, 3' end, or both the 5' and 3' ends of the nucleotide sequence. In some cases, phosphorothioate internucleoside linkages are used to link the ligand to the sense strand.

[0023] In some cases, the antisense strand of a compound disclosed herein, e.g., an HSD17B13 RNAi agent, comprises three or four phosphorothioate internucleoside linkages. In some cases, the antisense strand comprises three phosphorothioate internucleoside linkages. In some cases, these three phosphorothioate internucleoside linkages are between nucleotides 1-3 from the 5' end of the antisense strand and between nucleotides 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end of the antisense strand. In some cases, the compound comprises at least two phosphorothioate internucleoside linkages in the sense strand and three or four phosphorothioate internucleoside linkages in the antisense strand.

[0024] In some cases, the compounds disclosed herein, e.g., HSD17B13 RNAi agents, include one or more modified nucleotides and one or more modified internucleoside linkages. In some cases, the 2' modified nucleoside is linked at a modified internucleoside linkage.

[0025] [Table 1]

[0026] As used in Tables 1A, 1B, and 2 herein, the following notation is used to denote modified nucleotides, ligand groups, and bridging groups: A, C, G, and U represent adenosine, cytidine, guanosine, and uridine, respectively; a, c, g, and u represent 2'-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue (see Table 2); and (NAG37)s represents the structure shown in Table 3 below.

[0027] Unless otherwise indicated by the sequence (e.g., by a phosphorothioate linkage "s"), when present in a chain, the monomers may be linked to each other by a 5'-3'-phosphodiester bond. In some cases, the inclusion of a phosphorothioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in oligonucleotides. In some cases, the terminal nucleotide at the 3' end of a given oligonucleotide sequence generally has a hydroxyl (-OH) group at the 3' position of each of the given monomers in vitro, instead of a phosphate moiety. In some cases, as one proceeds through each chain in the 5'→3' direction, an inverted abasic residue is inserted such that the 3' position of the deoxyribose is attached at the 3' end of the preceding monomer on each chain. Furthermore, as one of skill in the art will readily appreciate and appreciate, although the chemical structures of phosphorothioates depicted herein typically show an anion on the sulfur atom, the present disclosure encompasses tautomers of phosphorothioates (e.g., those in which the sulfur atom has a double bond and the anion is on the oxygen atom). In some cases, each sense and / or antisense strand can have any of the cross-linking groups disclosed herein attached to the 5' and / or 3' ends of the sequence.

[0028] Capping Residue In some cases, the sense strands disclosed herein may include one or more capping residues, which may be referred to as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more ends of a nucleotide sequence of an RNAi agent disclosed herein. Capping residues can confer certain beneficial properties to an RNAi agent, such as protection from exonuclease degradation. In some cases, an inverted abasic residue (invAb) (also referred to as an "inverted abasic site") is added as a capping residue (see Table 2). Capping residues include, for example, inverted abasic residues as well as terminal C 3 H 7 (propyl), C6 H 13 (hexyl), or C 12 H 25 (dodecyl) group. In some cases, the capping residue is present at the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the sense strand. In some cases, the 5'-end and / or the 3'-end of the sense strand may contain two or more inverted abasic deoxyribose moieties as capping residues.

[0029] In some cases, one or more inverted abasic residues (invAbs) are added to the 3' end of the sense strand. In some cases, one or more inverted abasic residues (invAbs) are added to the 5' end of the sense strand. In some cases, one or more inverted abasic residues or inverted abasic sites are inserted between the ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some cases, inclusion of one or more inverted abasic residues or inverted abasic sites at or near one or more ends of the sense strand of the RNAi agent can enhance the activity or other desired property of the RNAi agent.

[0030] In some cases, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some cases, one or more inverted abasic residues are inserted between the nucleotide sequences of the ligand and the sense strand of the RNAi agent. The inverted abasic residues can be linked via a phosphate bond, a phosphorothioate bond (e.g., shown herein as (invAb)s), or other internucleoside bond. The chemical structure of an inverted abasic deoxyribose residue is shown in Table 2 below, and the chemical structure is shown in Figures 5A-5D and 6A-6D.

[0031] [Table 2]

[0032] Bound Ligand In some examples, compounds for use herein may include an RNA molecule, such as an HSD17B13 RNAi agent, attached to one or more non-nucleotide groups, such as a ligand, that may facilitate targeting or delivery of the RNAi agent. Examples including three N-acetyl-galactosamine moieties are described in the present disclosure. The ligand may be covalently attached to the 3' and / or 5' ends of the sense and / or antisense strands. In some cases, the compound includes a ligand attached to the 3' and / or 5' ends of the sense strand. In some cases, the ligand is attached to the 5' end of the sense strand. In some cases, the ligand comprises, consists essentially of, or consists of the structure (NAG37)s and is attached to the 5' end of the sense strand. The ligand may be attached directly to the RNAi agent or indirectly via a linker / bridging group. In some cases, the ligand is attached to the RNAi agent via a labile, cleavable, or reversible bond or linker. In some cases, the ligand is attached to an inverted abasic residue at the 5' end of the sense strand.

[0033] The ligand disclosed herein can enhance the pharmacokinetics or biodistribution of the RNAi agent to which it is attached, improving the cell-specific distribution and cell-specific uptake of the RNAi agent.In some cases, the ligand enhances the endocytosis of the RNAi agent.In some cases, the ligand can have monovalent, bivalent, trivalent, tetravalent, or more valency with respect to the target to which it is directed.Exemplary ligands include, but are not limited to, compounds that have affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics that have affinity for cell surface molecules.

[0034] Properties of ligands such as galactose derivative clusters containing N-acetyl-galactosamine are described, for example, in WO 2018 / 044350 (Patent Application No. PCT / US2017 / 021147) and WO 2017 / 156012 (Patent Application No. PCT / US2017 / 021175), the contents of both of which are incorporated herein by reference in their entireties.

[0035] For example, a ligand attached to an HSD17B13 RNAi agent described in Tables 1A and 1B may have the chemical structure of (NAG37)s as shown in Table 3.

[0036] [Table 3]

[0037] Pharmaceutical Compositions Compounds suitable for use herein, for example, HSD17B13RNAi agents, can be formulated as pharmaceutical compositions for administration to human subjects.The pharmaceutical compositions can be used to treat or prevent subjects with diseases or disorders that benefit from reducing the expression of HSD17B13 mRNA or the level of HSD17B13 protein, such as human subjects with non-alcoholic fatty liver disease.In some cases, one or more pharmacologic acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition that comprises HSD17B13 RNAi agents, thereby forming pharmaceutical formulations suitable for in vivo delivery to human subjects.

[0038] As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or simply an "effective amount" refers to an amount of an active agent that produces a pharmacological, therapeutic, or prophylactic result. In some cases, a therapeutically or prophylactically effective amount of one or more pharmaceutical compositions is administered to a subject in need thereof to reduce the number, severity, and / or frequency of symptoms of a disease in the subject.

[0039] As used herein, a pharmaceutical composition or medicament comprises a pharmacologically effective amount of at least one compound disclosed herein and one or more pharma- ceutically acceptable excipients. An excipient is a substance other than an active pharmaceutical ingredient (API, therapeutic product, e.g., HSD17B13 RNAi drug) that is intentionally included in a drug delivery system. An excipient does not exert or is not intended to exert a therapeutic effect at the intended dose. An excipient may function to a) aid in the processing of the drug delivery system during manufacture, b) protect, support or enhance the stability, bioavailability or patient acceptability of the API, c) aid in the identity of the product, and / or d) enhance the overall safety, efficacy or other attributes of the delivery of the API during storage or use.

[0040] Excipients include, but are not limited to, absorption enhancers, anti-caking agents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavorings, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, viscosity enhancers, tonicity agents, vehicles, water repellents, and wetting agents.

[0041] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if soluble in water). For subcutaneous or intravenous or intramuscular administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, NJ) or phosphate buffer. The pharmaceutical composition must be stable under the conditions of manufacture and storage and must be preserved against the influence of contaminating microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0042] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. In some cases, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0043] The pharmaceutical composition may contain other additional ingredients commonly found in pharmaceutical compositions, such as active agents such as antipruritics, astringents, local anesthetics, anti-inflammatory agents, or antihistamines.

[0044] The pharma- ceutically acceptable formulations described herein can be packaged in a kit, container, pack, or dispenser.The pharmaceutical compositions described herein can be packaged in pre-filled syringes or vials.

[0045] Treatment method As used herein, the term "treatment" can include prevention, management, prophylactic treatment, reduction, and / or inhibition of the number, severity, and / or frequency of a disease and / or one or more symptoms of a disease in a subject. In some examples, the term "treatment" refers to the management, reduction, and / or inhibition of the number, severity, and / or frequency of a disease and / or one or more symptoms of a disease in a subject. Unless otherwise specified, treatment can also include alleviation or reduction of the underlying condition and / or symptoms of the disease.

[0046] Embodiment 1: A method for treating non-alcoholic fatty liver disease in a human subject in need thereof, comprising administering to the human subject a dose of about 25 to about 200 mg of a compound or a pharma- ceutical acceptable salt thereof, wherein the dose is calculated based on the compound in free acid form, the compound comprising a double-stranded oligonucleotide bound to a ligand, the double-stranded oligonucleotide reducing expression of HSD17β13 in the human subject, and the ligand [ka] The method comprises the chemical structure of, its stereoisomer, or a salt thereof (a pharma- ceutically acceptable salt).

[0047] Embodiment 2: The method of embodiment 1, wherein said administration is a single dose.

[0048] Embodiment 3: The method according to embodiment 1, wherein the administration is repeated, for example, once every about 28 days (i.e. 4 weeks), about 12 weeks, or about 3 months, for example, about 100 mg every 4 weeks, or about 200 mg every 12 weeks, optionally for about 52 weeks or about 1 year. In some cases, the administration is repeated every 4 weeks, for example, at a dose of 100 mg. In some cases, the administration is repeated every 12 weeks, for example, at a dose of 200 mg.

[0049] Embodiment 4: The method according to any one of embodiments 1 to 3, wherein the dose is from about 50 mg to about 100 mg, for example about 50 mg.

[0050] Embodiment 5: The method according to any one of embodiments 1 to 3, wherein the dose is from about 100 mg to about 200 mg, for example about 100 mg.

[0051] Embodiment 6: The method according to any one of embodiments 1 to 3, wherein the dose is from about 50 mg to about 200 mg, for example about 200 mg.

[0052] Embodiment 7: The method of any one of embodiments 1-6, wherein the compound is in sodium salt form.

[0053] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the compound is administered by injection, for example at a concentration of 200 mg / ml.

[0054] Embodiment 9: The method of embodiment 8, wherein the injection is a subcutaneous injection.

[0055] Embodiment 10: The method of any one of embodiments 1 to 9, wherein the compound is present in a unit dosage form, including, for example, a single (unit) dosage form.

[0056] Embodiment 11: The method of any one of embodiments 1 to 10, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH), such as pre-cirrhotic non-alcoholic steatohepatitis, NASH-F3, NASH with bridging fibrosis, or biopsy-proven NASH.

[0057] Embodiment 12: The method according to any one of embodiments 1 to 11, wherein the human subject has HSD17β13 rs72613567 mutation, PNPLA3 rs738409 (I148M) mutation, or a combination thereof. In some cases, the human subject has HSD17β13 rs72613567 mutation. In some cases, the human subject has PNPLA3 rs738409 (I148M) mutation.

[0058] Embodiment 13: The double-stranded oligonucleotide has the following sequence (5'→3'): usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO:1); usCfsasUfcUfaUfcAfgAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO:5); usGfsasUfcCfaAfaAfaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO:6); or usGfsasUfcCfaaaaaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 7) where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond. 13. The method of any one of embodiments 1 to 12, comprising an antisense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following:

[0059] Embodiment 14: The double-stranded oligonucleotide has the following sequence (5'→3'): cguaagaaGfuCfuGfauagauga (nucleotide sequence SEQ ID NO:3); cguaagaaGfUfCfugauagauga (nucleotide sequence SEQ ID NO:8); gccuaggaCfAfUfuuuugiauca (nucleotide sequence SEQ ID NO:9); or gccuaggaCfaUfuUfuugiauca (nucleotide sequence SEQ ID NO: 10) where a, c, g, i, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, 2'-O-methyl inosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond. 14. The method of any one of the preceding claims, comprising a sense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following:

[0060] Embodiment 15: The double-stranded oligonucleotide has the sequence: usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1) and an antisense strand comprising the sequence: (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb) (nucleotide sequence SEQ ID NO: 11) and a sense strand comprising where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue, The method according to any one of embodiments 1 to 14.

[0061] Embodiment 16: The method of any one of embodiments 1 to 15, wherein the expression level of HSD17β13 mRNA in a human subject is reduced by at least about 50% (e.g., at least about 62%, at least about 79%, at least about 84%, at least about 90%, or at least about 96%) compared to before administration, as measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample from the human subject, for example, on about day 71 or day 113 after the first administration.

[0062] Embodiment 17: The method of any one of embodiments 1 to 16, wherein the expression of HSD17β13 protein in a human subject is reduced by at least about 30% (e.g., at least about 50%, at least about 60%, at least about 83%, at least about 92%, or at least about 97%) compared to before administration, for example, on about day 71 or day 113 after the first administration, as measured by Western blot using a liver biopsy sample from the human subject.

[0063] Embodiment 18: The method of any one of embodiments 1 to 17, wherein the ALT (alanine aminotransferase) level in a human subject is reduced by at least about 7.7% (e.g., at least about 14%, at least about 26%, at least about 36%, or at least about 39%), or at least about 40% (e.g., at least about 51%, at least about 53%, or at least about 60%), as measured in the serum of the human subject, e.g., at about 71 days or 113 days after the first administration, compared to before administration.

[0064] Embodiment 19: The method of any one of embodiments 1 to 18, wherein AST (aspartate aminotransferase) levels in a human subject are reduced by at least about 7.5% (e.g., at least about 15%), or at least about 20% (e.g., at least about 24%, or at least about 28%), as measured in the serum of the human subject, e.g., at about day 71 or day 113 after the first administration, compared to before administration.

[0065] Embodiment 20: The method of any one of embodiments 1 to 19, wherein the increase in liver fat percentage in a human subject is reduced or slowed (e.g., a reduction of at least 4-41% or at least 7-8%) compared to before administration, e.g., at about day 71 or day 113 after the first administration, as measured by magnetic resonance imaging.

[0066] Embodiment 21: The method of any one of embodiments 1 to 20, wherein the increase in liver stiffness in a human subject is reduced or slowed (e.g., by at least a 4-37% reduction) relative to pre-administration, e.g., at about day 71 or day 113 after the first administration, as measured by transient elastography.

[0067] Embodiment 22: The chemical structure of the ligand is [ka] 22. The method of any one of embodiments 1 to 21, comprising:

[0068] Embodiment 23: The method of embodiment 22, wherein the compound is in free acid form having the chemical structure shown in Figures 5A-5D.

[0069] Embodiment 24: The chemical structure of the ligand is [ka] 22. The method of any one of embodiments 1 to 21, comprising:

[0070] Embodiment 25: The method of embodiment 24, wherein the compound is in the form of a sodium salt having the chemical structure shown in Figures 6A-6D.

[0071] Embodiment 26: The method of any one of embodiments 1 to 25, wherein the GGT (gamma-glutamyltransferase) level in the human subject is reduced by at least about 1.4%, or at least 8.3%, or at least 9% compared to before administration, as measured in the serum of the human subject, for example at about day 71 or day 113 after the first administration.

[0072] Embodiment 27: A compound or a pharma- ceutical acceptable salt thereof for use in the treatment of non-alcoholic fatty liver disease in a human subject, said use comprising administering to said human subject a dose of about 25 to about 200 mg of said compound or a pharma-ceutical acceptable salt thereof, said dose being calculated based on the compound in free acid form, said compound comprising a double-stranded oligonucleotide bound to a ligand, said double-stranded oligonucleotide reducing expression of HSD17β13 in the human subject, said ligand being [ka] or a salt thereof (e.g., a pharma- ceutically acceptable salt), comprising the chemical structure of:

[0073] Embodiment 28: The compound or a pharma- ceutically acceptable salt thereof for use according to embodiment 27, wherein said administration is a single dose.

[0074] Embodiment 29: The compound or a pharma- ceutically acceptable salt thereof for use according to embodiment 28, wherein the administration is, for example, once every about 28 days (i.e. 4 weeks), about 12 weeks, or about 3 months, for example, about 100 mg every 4 weeks, or about 200 mg every 12 weeks, optionally repeated for about 52 weeks or about 1 year.Embodiment 29a: The compound or a pharma- ceutically acceptable salt thereof for use according to embodiment 28, wherein the administration is about 100 mg every 4 weeks for about 52 weeks.Embodiment 29b: The compound or a pharma- ceutically acceptable salt thereof for use according to embodiment 28, wherein the administration is about 200 mg every 12 weeks for about 52 weeks.

[0075] Embodiment 30: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 29, wherein the dosage is from about 50 mg to about 100 mg, for example about 50 mg.

[0076] Embodiment 31: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 29, wherein the dosage is from about 100 mg to about 200 mg, for example about 100 mg.

[0077] Embodiment 32: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 29, wherein the dosage is from about 50 mg to about 200 mg, for example about 200 mg.

[0078] Embodiment 33: The compound for use according to any one of embodiments 27 to 32, or a pharma- ceutically acceptable salt thereof, in sodium salt form.

[0079] Embodiment 34: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 33, wherein the compound is administered by injection, for example in a concentration of 200 mg / ml.

[0080] Embodiment 35: The compound or a pharma- ceutically acceptable salt thereof for use according to embodiment 34, wherein the injection is subcutaneous.

[0081] Embodiment 36: A compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 35, administered in unit dosage form, e.g. present in a single unit dosage form.

[0082] Embodiment 37: The compound or a pharmacologic acceptable salt thereof for use according to any one of embodiments 27 to 36, wherein said non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH), such as pre-cirrhotic non-alcoholic steatohepatitis, NASH-F3, NASH with bridging fibrosis, or biopsy-proven NASH.

[0083] Embodiment 38: A compound or a pharma- ceutical acceptable salt thereof for use according to any one of embodiments 27 to 37, wherein the human subject has an HSD17β13 rs72613567 mutation, a PNPLA3 rs738409 (I148M) mutation, or a combination thereof.

[0084] Embodiment 39: The double-stranded oligonucleotide has the following sequence (5'→3'): usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO:1); usCfsasUfcUfaUfcAfgAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO:5); usGfsasUfcCfaAfaAfaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO:6); or usGfsasUfcCfaaaaaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 7) where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond. The compound for use according to any one of embodiments 27 to 38, or a pharma- ceutically acceptable salt thereof, comprising an antisense strand comprising a modified nucleotide sequence differing by 0 or 1 nucleotide from one of the following:

[0085] Embodiment 40: The double-stranded oligonucleotide has the following sequence (5'→3'): cguaagaaGfuCfuGfauagauga (nucleotide sequence SEQ ID NO:3); cguaagaaGfUfCfugauagauga (nucleotide sequence SEQ ID NO:8); gccuaggaCfAfUfuuuugiauca (nucleotide sequence SEQ ID NO:9); or gccuaggaCfaUfuUfuugiauca (nucleotide sequence SEQ ID NO: 10) where a, c, g, i, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, 2'-O-methyl inosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond. The compound for use according to any one of embodiments 27 to 39, or a pharma- ceutically acceptable salt thereof, comprising a sense strand comprising a modified nucleotide sequence differing by 0 or 1 nucleotide from one of the following:

[0086] Embodiment 41: The double-stranded oligonucleotide has the sequence: usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1) and an antisense strand comprising the sequence: (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb) (nucleotide sequence SEQ ID NO: 11) and a sense strand comprising a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue; 41. A compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 40.

[0087] Embodiment 42: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 41, wherein the use reduces the expression of HSD17β13 mRNA in a human subject by at least about 50% (e.g., at least about 62%, at least about 79%, at least about 84%, at least about 90%, or at least about 96%) compared to before administration, as measured, for example, by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample from the human subject on about 71 or 113 days after the first administration.

[0088] Embodiment 43: A compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 42, wherein the use reduces the expression of HSD17β13 protein in a human subject by at least about 30% (e.g., at least about 50%, at least about 60%, at least about 83%, at least about 92%, or at least about 97%) compared to before administration, as measured by Western blot using a liver biopsy sample from the human subject, for example, on day 71 or day 113 after the first administration.

[0089] Embodiment 44: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 43, wherein the use reduces ALT (alanine aminotransferase) levels in a human subject by at least about 7.7% (e.g., at least about 14%, at least about 26%, at least about 36%, or at least about 39%), or at least about 40% (e.g., at least about 51%, at least about 53%, or at least about 60%), as measured in the serum of the human subject, for example, at about 71 days or 113 days after the first administration, compared to before administration.

[0090] Embodiment 45: A compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 44, wherein the use reduces AST (aspartate aminotransferase) levels in a human subject by at least about 7.5% (e.g., at least about 15%), or at least about 20% (e.g., at least about 24%, or at least about 28%) compared to before administration, as measured in the serum of the human subject, for example, on day 71 or day 113 after the first administration.

[0091] Embodiment 46: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 45, wherein the use reduces or slows the increase in liver fat percentage in a human subject, e.g., by at least 4-41% or at least 7-8%, as measured by magnetic resonance imaging, e.g., about 71 days or 113 days after the first administration, compared to before administration.

[0092] Embodiment 47: A compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 46, wherein said use reduces or slows the increase in liver stiffness in a human subject, e.g., by at least a 4 to 37% reduction, e.g., at about 71 days or 113 days after the first administration, compared to before administration, as measured by transient elastography.

[0093] Embodiment 48: The chemical structure of the ligand is [ka] or a pharma- ceutically acceptable salt thereof.

[0094] Embodiment 49: The compound for use according to embodiment 48, wherein said compound is in free acid form having the chemical structure shown in Figures 5A-5D, or a pharma- ceutically acceptable salt thereof.

[0095] Embodiment 50: The chemical structure of the ligand is [ka] or a pharma- ceutically acceptable salt thereof.

[0096] Embodiment 51: The compound for use according to embodiment 50, or a pharma- ceutically acceptable salt thereof, wherein said compound is in sodium salt form having the chemical structure shown in Figures 6A-6D.

[0097] Embodiment 52: The compound or a pharma- ceutically acceptable salt thereof for use according to any one of embodiments 27 to 51, wherein said use reduces GGT (gamma-glutamyltransferase) levels in a human subject by at least about 1.4%, or at least 8.3%, or at least 9% compared to before administration, as measured in the serum of the human subject, for example at about day 71 or day 113 after the first administration.

[0098] Embodiment 53: Use of a compound or a pharma- ceutical acceptable salt thereof for the manufacture of a medicament for the treatment of non-alcoholic fatty liver disease, wherein the compound or a pharma-ceutical acceptable salt thereof is administered in a dose of about 25 to about 200 mg, the dose being calculated based on the compound in free acid form, the compound comprising a double-stranded oligonucleotide bound to a ligand, the double-stranded oligonucleotide reducing expression of HSD17β13, and the ligand [ka] or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

[0099] Embodiment 54: The use according to embodiment 53, wherein said dose is a single dose.

[0100] Embodiment 55: The use according to embodiment 53, wherein the dosage is, for example, once every about 28 days (i.e. 4 weeks), about 12 weeks, or about 3 months, for example, about 100 mg every 4 weeks, or about 200 mg every 12 weeks, optionally repeated for about 52 weeks or about 1 year.Embodiment 55a: The use according to embodiment 53, wherein the dosage is about 100 mg every 4 weeks for 52 weeks.Embodiment 55b: The use according to embodiment 53, wherein the dosage is about 200 mg every 12 weeks for 52 weeks.

[0101] Embodiment 56: The use according to any one of embodiments 53 to 55, wherein the dosage is from about 50 mg to about 100 mg, for example about 50 mg.

[0102] Embodiment 57: The use according to any one of embodiments 53 to 55, wherein the dosage is from about 100 mg to about 200 mg, for example about 100 mg.

[0103] Embodiment 58: The use according to any one of embodiments 53 to 55, wherein the dosage is from about 50 mg to about 200 mg, for example about 200 mg.

[0104] Embodiment 59: The use of any one of embodiments 53 to 58, wherein the compound is in sodium salt form.

[0105] Embodiment 60: The use according to any one of embodiments 53 to 59, wherein the compound is administered by injection, for example in a concentration of 200 mg / ml.

[0106] Embodiment 61: The use according to embodiment 60, wherein said injection is subcutaneous.

[0107] Embodiment 62: The use of any one of embodiments 53 to 61, wherein the compound is in a unit dosage form.

[0108] Embodiment 63: The use according to any one of embodiments 53 to 62, wherein said non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH), such as pre-cirrhotic non-alcoholic steatohepatitis, NASH-F3, NASH with bridging fibrosis, or biopsy-proven NASH.

[0109] Embodiment 64: The use according to any one of embodiments 53 to 63, wherein the treatment is for a subject having the HSD17β13 rs72613567 mutation, the PNPLA3 rs738409 (I148M) mutation, or a combination thereof.

[0110] Embodiment 65: The double-stranded oligonucleotide has the following sequence (5'→3'): usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO:1); usCfsasUfcUfaUfcAfgAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO:5); usGfsasUfcCfaAfaAfaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO:6); or usGfsasUfcCfaaaaaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 7) where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond. The use according to any one of embodiments 53 to 64, comprising an antisense strand comprising a modified nucleotide sequence which differs by 0 or 1 nucleotide from one of the following:

[0111] Embodiment 66: The double-stranded oligonucleotide has the following sequence (5'→3'): cguaagaaGfuCfuGfauagauga (nucleotide sequence SEQ ID NO:3); cguaagaaGfUfCfugauagauga (nucleotide sequence SEQ ID NO:8); gccuaggaCfAfUfuuuugiauca (nucleotide sequence SEQ ID NO:9); or gccuaggaCfaUfuUfuugiauca (nucleotide sequence SEQ ID NO: 10) where a, c, g, i, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, 2'-O-methyl inosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond. The use according to any one of embodiments 53 to 65, comprising a sense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following:

[0112] Embodiment 67: The double-stranded oligonucleotide has the sequence: usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1) and an antisense strand comprising the sequence: (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb) (nucleotide sequence SEQ ID NO: 11) and a sense strand comprising a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue; Use according to any one of embodiments 53 to 66.

[0113] Embodiment 68: The use according to any one of embodiments 53 to 67, wherein the use reduces expression of HSD17β13 mRNA by at least about 50% (e.g., at least about 62%, at least about 79%, at least about 84%, at least about 90%, or at least about 96%) compared to before use, for example, on day 71 or day 113 after the first use, as measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample.

[0114] Embodiment 69: The use described in any one of embodiments 53 to 68, wherein the use reduces expression of HSD17β13 protein by at least about 30% (e.g., at least about 50%, at least about 60%, at least about 83%, at least about 92%, or at least about 97%) compared to before use, for example, on about day 71 or day 113 after first use, as measured by Western blot using a liver biopsy sample.

[0115] Embodiment 70: The use according to any one of embodiments 53 to 69, wherein the use reduces ALT (alanine aminotransferase) levels by at least about 7.7% (e.g., at least about 14%, at least about 26%, at least about 36%, or at least about 39%), or at least about 40% (e.g., at least about 51%, at least about 53%, or at least about 60%), as measured in serum, for example, on about day 71 or day 113 after first use, compared to before use.

[0116] Embodiment 71: The use according to any one of embodiments 53 to 70, wherein the use reduces AST (aspartate aminotransferase) levels, measured in serum, for example on day 71 or day 113 after the first administration, by at least about 7.5% (e.g., at least about 15%), or at least about 20% (e.g., at least about 24%, or at least about 28%) compared to before use.

[0117] Embodiment 72: The use according to any one of embodiments 53 to 71, wherein the use reduces or slows the increase in liver fat percentage (e.g., a reduction of at least 4 to 41% or at least 7 to 8%), e.g., on about day 71 or day 113 after the first use, compared to before the use, as measured by magnetic resonance imaging.

[0118] Embodiment 73: The use according to any one of embodiments 53 to 72, wherein the use reduces or slows the increase in liver stiffness (e.g., at least a 4 to 37% reduction), e.g., at about day 71 or day 113 after the first administration, compared to before the use, as measured by transient elastography.

[0119] Embodiment 74: The chemical structure of the ligand is [ka] Use according to any one of embodiments 53 to 73, comprising:

[0120] Embodiment 75: The use of embodiment 74, wherein the compound is in free acid form having the chemical structure shown in Figures 5A-5D.

[0121] Embodiment 76: The chemical structure of the ligand is [ka] Use according to any one of embodiments 53 to 73, comprising:

[0122] Embodiment 77: The use of embodiment 76, wherein the compound is in sodium salt form having the chemical structure shown in Figures 6A-6D.

[0123] Embodiment 78: The use according to any one of embodiments 53 to 77, wherein said use reduces GGT (gamma-glutamyltransferase) levels by at least about 1.4%, or at least 8.3%, or at least 9%, as measured in serum, for example on about day 71 or day 113 after first use, compared to before use.

[0124] In some examples, one or more human subjects treated with the methods described herein achieve an improvement of ≥ 1 stage (i.e., ≥ 1 stage) of histological fibrosis without NASH worsening, for example, at 52 weeks after initiation of treatment. In some examples, the improvement of histological fibrosis is assessed by Clinical Research Network (CRN) scoring. In some cases, the absence of NASH worsening is defined as no increase in NAFLD activity score (NAS) for fat deposition, ballooning, or inflammation.

[0125] In some examples, one or more human subjects treated with the methods described herein achieve NASH resolution without worsening of fibrosis, for example at 52 weeks after initiation of treatment. In some cases, NASH resolution is defined as a ballooning score of 0 and an inflammation score of 0 to 1. In some cases, no worsening of fibrosis is defined as no increase in CRN fibrosis score.

[0126] In some cases, one or more human subjects treated with the methods described herein achieve a ≧30% relative reduction in liver fat from baseline at 24 weeks and 52 weeks after initiation of treatment, e.g., as measured by MRI-PDFF. EXAMPLES

[0127] Example 1. Synthesis and formulation of HSD17B13 RNAi agents RNAi agent compounds suitable for use herein can be synthesized using standard phosphoramidite technology in solid-phase oligonucleotide synthesis as known in the art. Commercially available oligonucleotide synthesizers (e.g., MerMade96E® (Bioautomation) or MerMade12® (Bioautomation)) can be used. Synthesis can be performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, Prime Synthesis, Aston, PA, USA). The monomers located at the 3' end of each strand can be attached to the solid support as the starting point for synthesis. All 2'-modified RNA phosphoramidites, and inverted abasic phosphoramidites, can be purchased commercially. Ligand-containing phosphoramidites suitable for addition to the 5' end of the sense strand can be synthesized. Standard cleavage, deprotection, purification, and annealing steps can be utilized as known in the art. Further description related to the synthesis of HSD17B13 RNAi agents can be found, for example, in International Publication No. 2020 / 061177 (Patent Application No. PCT / US2019 / 051707) and International Publication No. 2018 / 044350 (PCT / US2017 / 021147), each of which is incorporated herein by reference in its entirety. The HSD17B13 RNAi agent can then be formulated by dissolving in a suitable pharma- ceutically acceptable excipient.

[0128] Example 2. Phase 1 / 2a study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamic effects of HSD17B13 RNAi agents in normal healthy volunteers and patients with NASH or NAFLD The test compounds exemplified herein reduce the expression of HSD17β13 messenger RNA (mRNA) in hepatocytes, the sequence of which is shown in Table 4, and the physical properties of which are shown in Table 5.

[0129] [Table 4]

[0130] [Table 5]

[0131] Full chemical structure representations are shown in Figures 5A-5D (free acid form) and 6A-6D (sodium salt form).

[0132] The clinical trial evaluated the safety, tolerability, and pharmacodynamics of the test substance in 32 healthy volunteers (NHVs) and 18 patients with or suspected of having NASH (e.g., nonalcoholic fatty liver disease [NAFLD] with elevated alanine aminotransferase [ALT]). The double-blind NHV cohort received a single subcutaneous (SC) dose of 25 mg, 50 mg, 100 mg, or 200 mg of the test compound or placebo on day 1. The open-label patient cohort received SC doses of 25 mg, 100 mg, or 200 mg of the test compound on days 1 and 29. Patients underwent liver biopsies prior to dosing and on day 71 to assess HSD17β13 mRNA and protein expression.

[0133] Treatment with the test compound was well tolerated in both NHVs and patients, with no treatment-related serious adverse events (SAEs) or discontinuations during the study. The most frequently reported treatment-emergent adverse events (TEAEs) were mild injection site reactions, which were mild in severity and short in duration. Reductions in hepatic HSD17β13 mRNA were observed after 25 mg (-57%), 100 mg (-86%), and 200 mg (-93%) on day 71 of dosing. Hepatic HSD17β13 protein levels were similarly reduced at all dose levels.

[0134] The mean maximum reductions in ALT and AST in patients were -42% to -44% and -24% to -28%, respectively, at doses of 100 mg or more.

[0135] The test compound was well tolerated at doses up to 200 mg on day 1 (NHVs) or days 1 and 29 (NASH or NAFLD patients), and there were no treatment-related SAEs or discontinuations in the study. The incidence of TEAEs was similar between the test compound and placebo groups in NHVs and consistent across patient populations. This proof-of-concept study demonstrated that the test compound reduced hepatic HSD17β13 mRNA and protein, accompanied by reductions in ALT and AST.

[0136] method Participants and study design This was a multicenter Phase 1 / 2a clinical trial to evaluate the safety, tolerability, pharmacokinetic, and pharmacodynamic effects of injections of the test compound in normal healthy volunteers (NHVs) and patients with or suspected of NASH (e.g., NAFLD with elevated alanine aminotransferase [ALT]). Criteria for NAFLD were based on magnetic resonance imaging proton density fat fraction (MRI-PDFF) >8% and ALT > upper limit of normal (ULN) (ULN = 30 U / L for men and 19 U / L for women). The study included patients with a body mass index (BMI) <35 kg / m 2 (NHV) aged 18-55 years or BMI ≤ 40.0 kg / m 2 The study was conducted on adult men and women aged 19 to 65 years (patients with NASH or NAFLD).

[0137] Genotyping was performed at screening of patients with NASH or NAFLD to determine the mutation status of HSD17β13 rs72613567 and patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 (I148M) variants. Individuals were divided into T / T (homozygous wild type, or no mutation) or T / TA (heterozygous mutation) groups according to the HSD17β13 rs726131567 genotype analyzed. Individuals homozygous for rs726131567:TA were excluded from the study. Similarly, individuals were divided into C / C (homozygous wild type, or no mutation), C / G (heterozygous mutation), or G / G (homozygous mutation) groups according to the PNPLA3 genotype analyzed.

[0138] Subjects were enrolled in the study in a double-blind (NHV cohorts 1 [25 mg], 2 [50 mg], 3 [100 mg], and 4 [200 mg]) or open-label (patient cohorts 1b [25 mg], 3b [100 mg], and 4b [200 mg]) fashion (Table 6). NHV subjects were randomized in a 4:4 (active drug:placebo) ratio to receive a single subcutaneous (SC) injection of the test compound (25, 50, 100, or 200 mg) or placebo (cohorts 1–4) on day 1. Patients with NASH or NAFLD received the test compound (25, 100, or 200 mg for cohorts 1b, 3b, and 4b, respectively) in an open-label fashion on days 1 and 29. The study design is summarized in Figure 4 and Table 6.

[0139] [Table 6]

[0140] In cohorts 1-4, the test compound or placebo was administered to two sentinel subjects (one test compound, one placebo). After the day 3 evaluation of these subjects, the remaining subjects in the cohort were dosed at least 30 minutes apart to ensure that no two subjects were dosed at the same time. Cohorts 1-4 were enrolled sequentially, and enrollment of patient cohorts began in a rolling manner. No sentinel dosing was required in any patient cohort. A summary of dosing by cohort is shown in Table 6. NHVs stayed at the clinical site for 3 days to be dosed on day 1 (cohorts 1, 2, 3, and 4). NHVs returned to the clinical site for an outpatient visit. Patients in cohorts 1b, 3b, and 4b stayed at the clinical site for approximately 4 hours (2 hours pre-dose and 2 hours post-dose) on dosing days (days 1 and 29).

[0141] Study treatment Fifty subjects (32 NHVs and 18 patients) were enrolled. Of the 58 NHVs screened, 8 could not be screened (mostly because they did not meet the inclusion or exclusion criteria) and 18 were not assigned (because the 45-day screening period had elapsed due to the COVID-19 pandemic). In NHV cohorts 1–4, 8 subjects (4 active drug: 4 placebo) were enrolled in each double-blind cohort. All NHV cohorts received a single dose of test compound or placebo on day 1 at dose levels of 25 mg (cohort 1), 50 mg (cohort 2), 100 mg (cohort 3), and 200 mg (cohort 4).

[0142] Of the 34 NASH or NAFLD patients screened, 16 were screened out (due to not meeting inclusion or exclusion criteria) (Table 6). Of the 18 patients who entered open-label cohorts 1b, 3b, and 4b, six were enrolled per cohort. All patient cohorts received the test compound at dose levels of 25 mg (cohort 1b), 100 mg (cohort 3b), or 200 mg (cohort 4b) on days 1 and 29. Patients were followed through day 113 (week 16).

[0143] Test Assessment and Procedures In patients with NASH or NAFLD, the pharmacodynamic effects of test compounds were measured by hepatic HSD17β13 mRNA (qRT-PCR) and protein (western blot) expression from paired liver biopsies taken at screening and day 71 to measure the activity of test compounds against HSD17β13. For gene expression measurements by qRT-PCR, HSD17B13 and endogenous control β-actin (ACTB) and mRNA were measured in biopsies both at day 71 and screening. Relative expression was calculated using the 2(-ΔC[T]) normalization method. Changes in HSD17B13 protein expression were assessed as the ratio of HSD17B13 / vinculin protein at day 71 relative to pre-dose baseline levels. Liver injury markers such as ALT and AST were measured from screening to day 113. Liver imaging was performed to assess changes in hepatic fat percentage using MRI-PDFF at screening and day 71, and liver stiffness using transient elastography (FibroScan, kPa). Single nucleotide polymorphisms (SNPs) of HSD17B13 rs72613567 and patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 were detected in whole blood by real-time PCR, and the results were confirmed by Sanger sequencing at screening.

[0144] Safety assessments included determining the incidence of adverse events (AEs) / serious adverse events (SAEs) in both the NHV and patient cohorts, performing physical examinations and recording vital sign measurements (blood pressure, heart rate, temperature, and respiratory rate), electrocardiograms (ECGs), laboratory tests (blood and urine), concomitant medications / therapies, and recording reasons for treatment discontinuation.

[0145] statistical analysis All safety outcomes, demographic and baseline characteristic data, and pharmacodynamic results were compiled based on data collected from all enrolled subjects who received at least one dose of active drug or placebo. Treatment-emergent adverse events were coded using the Medical Dictionary for Regulatory Activities (MedDRA, version 24.0).

[0146] For the analysis of pharmacodynamic parameters, descriptive summary statistics and derived knockdown data from baseline were provided for HSD17β13 mRNA and protein. A nonparametric Wilcoxon signed rank test was performed to test the significance of the change from baseline at day 71. Descriptive summary statistics were also provided for liver enzyme parameters such as ALT and AST, along with derived change from baseline and percent change from baseline summary. A nonparametric Wilcoxon signed rank test was also performed to test the significance of the change from baseline at days 29, 71, and 113 for ALT and AST. Descriptive summary statistics and derived percent change from baseline were provided for liver imaging (MRI-PDFF and FibroScan). Baseline values ​​were defined as the last nonmissing observation (pre-dose value closest to the first dose) before study drug administration (i.e., start of injection on day 1) for each subject. Data were analyzed using SAS software (version 9.4), and figures were generated using GraphPad Prism software (version 8.3.0).

[0147] Exam Supervisor The ethical committees of each participating institution approved the protocol. The study was conducted in accordance with the principles of the Declaration of Helsinki, the International Conference on Harmonisation Good Clinical Practice guidelines, and applicable regulatory requirements. All subjects gave written informed consent before enrolment.

[0148] result Patient characteristics and disposition The demographic characteristics of the healthy volunteers are shown in Table 7. All enrolled NHVs completed all planned assessments.

[0149] [Table 7]

[0150] Patients with NASH or NAFLD: Mean (SD) age was 44.5 (9.9) years. Four subjects (22.2%) were female and 14 subjects (77.8%) were male. Thirteen (72.2%) subjects were Asian, three subjects (16.7%) were Caucasian, one subject (5.6%) was Black or African American, and one subject (5.6%) was other. At baseline, MRI-PDFF rank was 10.34%-30.73% and ALT rank was 28 U / L (female subjects)-144 U / L. Suspected NASH (e.g., NAFLD with elevated ALT) was defined by MRI-PDFF>8% and ALT>ULN (male ULN=30 U / L and female 19 U / L). Alternatively, biopsy-confirmed NASH within 1 year of screening visit could be included in the registry. These patients showed typical NAFLD complications, with 50% (n = 9), 44.4% (n = 8), and 44.4% (n = 8) having hyperlipidemia, type 2 diabetes, and hypertension, respectively.

[0151] A summary of the genotype results of NAFLD patients by cohort is shown in Table 8. For the HSD17β13 rs72613567 variant, 12 subjects (67%) had no detectable mutations (T / T), and 6 subjects (33%) were heterozygous (T / TA). For the PNPLA3 rs738409 variant, 11 subjects (61%) had no detectable mutations (C / C), 4 subjects (22%) were heterozygous (C / G), and 3 subjects (17%) were homozygous (G / G). At baseline, concomitant medications taken by NASH or NAFLD patients in clinical trials included statins (HMG COA reductase inhibitors) (39% of subjects), biguanides (metformin) (33% of subjects), ACE inhibitors (28% of subjects), angiotensin II receptor blockers (22% of subjects), calcium channel blockers (dihydropyridine derivatives) (22% of subjects), glucagon-like peptide-1 (GLP-1) analogs (5.6% of subjects), and sodium-glucose cotransporter 2 (SGLT2) inhibitors (5.6% of subjects). All subjects completed the study without early termination.

[0152] [Table 8]

[0153] Pharmacodynamics Hepatic HSD17B13 protein and mRNA At day 71, the test compound reduced hepatic mRNA encoding HSD17β13 protein by 56.9% (50.7%-60.5%), 85.5% (61.6%-96.1%), and 93.4% (90.8%-98.6%) mean (min-max) values ​​from pretreatment baseline in the 25 mg, 100 mg, and 200 mg dose cohorts, respectively (Figure 1). The pooled HSD17β13 mRNA reduction rate for all subjects was 78.6% (50.7%-98.6%, p-value < 0.0001). In the 200 mg dose cohort, all six subjects showed a greater than 90% reduction in hepatic expression of HSD17β13 mRNA. Hepatic HSD17β13 protein levels were similarly reduced by an average of >34%, 86%, and 83% in the 25 mg, 100 mg, and 200 mg dose cohorts, respectively, with the mean percent change from baseline in HSD17β13 protein for the pooled cohort being 63% (range -97.8% to 53.8%, p-value: 0.0017), with multiple measurements (three subjects at baseline and six subjects at day 71) below the quantification level of the assay (Table 9).

[0154] [Table 9]

[0155] The data observed for reduction of HSD17B13 mRNA suggested that a robust on-target pharmacological response was achieved in the liver, with a trend towards dose-dependence at the 100mg and 200mg dose levels, as well as a clear dose-dependence at the 25mg and 100mg dose levels. The observed pharmacodynamic effects were not affected by HSD17β13 (rs72613567, T>TA) and / or PNPLA3 (rs738409, C>G) mutations. At each dose level, there was a similar reduction in HSD17β13 mRNA in patients without mutations compared to patients who were heterozygous for the rs72613567 splice variant (Figure 3A). Meanwhile, at each dose level, there was a similar reduction in HSD17β13 mRNA in patients without mutations compared to patients who were heterozygous or homozygous for the PNPLA3 rs738409 polymorphism (Figure 3B). Thus, knockdown of HSD17β13 was consistent in this study regardless of genotype or mutation status.

[0156] ALT, AST, and GGT In patients with NASH or NAFLD, serum biomarkers of liver damage were reduced after treatment with the test compound, with a reduction in ALT between the 25 mg and 100 mg dose levels. The 100 mg and 200 mg dose levels showed similar reductions in ALT. The mean percent change from baseline in ALT at day 71 (concurrent with biopsy) ranged from -7.7% (25 mg dose group) to -39.3% (100 mg) and -42.2% (200 mg) (P<0.001 for pooled cohort) (Figure 2A). The mean percent change from baseline in ALT at day 113, i.e., end of study, was -14%, -36%, and -39% at the 25 mg, 100 mg, and 200 mg dose levels, respectively (P<0.01 for pooled cohort) (Figure 2A). Similar to ALT, AST was also reduced between the 25mg and 100mg dose levels, with a similar reduction seen between the 100mg and 200mg doses. The mean percent change from baseline in AST at day 71 was 4%, -24%, and -28% at the 25mg, 100mg, and 200mg dose levels, respectively (P<0.01 for the pooled cohort) (Figure 2B). The mean percent change from baseline in AST at day 113 was -7.5%, -14.7%, and -19.7% at the 25mg, 100mg, and 200mg dose levels, respectively (Figure 2B). Two doses of the test compound, 25mg, 100mg, and 200mg, were administered to patients on days 1 and 29, and safety and pharmacodynamic parameters were measured until the end of the study, i.e., day 113. The ALT reduction period was sustained until day 85, i.e., 8 weeks after the last dose, at the 100mg and 200mg dose levels. The duration of AST reduction was sustained at the 100 mg and 200 mg dose levels up to day 71, 6 weeks after dosing. *** P < .001; ** P < .01, *<.01 is the p-value for the pooled cohort (25, 100, and 200 mg dose levels at the indicated time points using nonparametric tests (Wilcoxon signed rank test)). Mean body weight remained stable throughout the study in each dose group. Two subjects lost weight during the study and demonstrated the greatest percentage reduction in ALT from baseline. These subjects had weights of 4.6 kg (25 mg dose group) and 2.2 kg (100 mg dose group) by day 71, corresponding to a 60% and 51% reduction in ALT, respectively. All other subjects maintained stable weight throughout the study, so the reductions in all other liver enzymes were seen independently of changes in body weight. GGT levels, as measured in serum, were reduced by approximately 1.4%, 8.3%, and 9% compared to pre-dose in the 25 mg, 100 mg, and 250 mg patient cohorts, respectively.

[0157] Liver fat percentage and stiffness The mean relative changes from baseline to day 71 in hepatic fat percentage by MRI-PDFF were 14.4%, -7.6%, and -7.3% for the 25 mg, 100 mg, and 200 mg doses, respectively (Table 10).

[0158] [Table 10]

[0159] The mean percent change from baseline in liver stiffness (kPa) at day 71 was +16.7%, +2.2%, and +4.2% for the 25 mg, 100 mg, and 200 mg doses, respectively (Table 10).

[0160] Both the 100 mg and 200 mg dose levels showed a less significant increase in liver stiffness and a reduction in liver fat percentage when measured as mean change from baseline at day 71 compared to the 25 mg dose level.

[0161] safety The test compound was well tolerated in both NHVs and NASH or NAFLD patients (Tables 11 and 12), with no study or drug discontinuations, dose-limiting toxicities, or deaths related to AEs. There was no recurrent pattern of adverse laboratory findings indicative of end-organ toxicity. The most frequently reported TEAEs were injection site reactions (injection site bruising, injection site erythema, and injection site swelling), which were mild in severity and short in duration. These events occurred in 5 of 16 NHVs who received the test compound, and none of the patients with NASH or NAFLD. The remaining TEAEs were those commonly seen in clinical trials (Table 12). One treatment-emergent SAE of soft tissue injury requiring hospitalization was reported in the 200 mg cohort. The subject was hospitalized for a right arm soft tissue injury following an accident (unrelated to time and site of injection) that occurred at work and was discharged 2 days later. The subject recovered from the accident and continued on the study. This SAE was considered unrelated to the study drug.

[0162] conclusion The test compound was well tolerated at doses up to 200 mg administered once (NHV) or twice (patients with NASH or NAFLD) with no treatment-related serious adverse events, no patterns of drug-related adverse laboratory findings, and no drug discontinuations during the study. The incidence of TEAEs was similar in the test compound and placebo groups in NHV and was consistent across patient cohorts. There was no mean increase in liver fat and no adverse changes in biomarkers of liver injury at all dose levels. Reductions in hepatic HSD17β13 mRNA and protein were observed, consistent with reductions in ALT and AST, signaling clinically meaningful reductions in liver injury. AST concentrations were reduced by a mean of 7.5%, 24.5%, and 28.3% at the 25 mg, 100 mg, and 200 mg doses, respectively, and sustained for 12 weeks at the 100 mg and 200 mg doses. Similarly, ALT levels were reduced by 14.1%, 43.6%, and 42.3% at the 25mg, 100mg, and 200mg doses, respectively, on days 1 and 29, which were sustained for 12 weeks at the 100mg and 200mg doses. Reductions in hepatic HSD17B13 mRNA and HSD17B13 protein indicate a robust on-target pharmacological response in the liver with the 100mg and 200mg doses. Thus, the data support a dosing regimen of at least 100mg every 4 weeks (Q4W).

[0163] Pharmacokinetic / pharmacodynamic (PK / PD) modeling of HSD17β13 protein knockdown and ALT predicts that the 200 mg dose will maintain reductions in HSD17β13 protein and ALT for 90 days, supporting the study dosing schedule of 200 mg every 3 months or 12 weeks. As shown in Figure 7, the 100 mg and 200 mg doses maintain a >80% reduction from baseline in HSD17β13 protein for >3 months. 1 / 2 Since the time required for the induction of HSD17β13 protein is 10.6 days, it is estimated that a dose of 200 mg induces complete knockdown of HSD17β13 protein, assuming, for example, a reduction of about 87% with complete knockdown in 30 days.

[0164] [Table 11] [Table 12]

[0165] Example 3. Minimizing HSD17B13 for the Treatment of NASH: A Double-Blind, Placebo-Controlled Phase 2b Study Evaluating the Efficacy and Safety of an HSD17B13 RNAi Agent in Adults with Precirrhotic Nonalcoholic Steatohepatitis This is a double-blind, randomized, placebo-controlled, Phase 2b, multicenter study designed to evaluate the efficacy and safety of test compounds (Table 4) in adult patients with NASH and bridging (F3) fibrosis.

[0166] The study included three treatment arms, including two arms receiving the test compound at two different dosing regimens and one placebo group. After a screening period of up to 70 days, eligible participants were randomized into one of the three treatment arms. 1) 100 mg of the test compound once every 4 weeks (Q4W) for 52 weeks. 2) 200 mg of the test compound once every 12 weeks (Q12W) for 52 weeks, or 3) Placebo once every 4 weeks (Q4W) for 52 weeks

[0167] The study is expected to enroll approximately 246 participants. Dose cohorts will be randomly assigned in a 1:1:1 ratio with approximately 82 participants enrolled in parallel in each treatment arm.

[0168] Selection criteria include, but are not limited to, the following: - Body mass index (BMI) at screening of 23 kilograms per square meter (kg / m 2 ) and above, except for Asian participants who were eligible for the study. 2 Above (all ethnic groups) - Patients had features of metabolic syndrome, with NAFLD being the most likely cause of liver disease, in the opinion of the investigator Baseline liver biopsy with NAFLD Activity Score (NAS) ≥ 4, with at least 1 each of steatosis, inflammation, and ballooning, and a fibrosis CRN score of 3 Able and willing to comply with all study evaluations, including liver biopsy at week 52

[0169] Exclusion criteria include, but are not limited to, the following: - Liver cirrhosis (confirmed by screening biopsy or previous biopsy) Current alcohol consumption is 14 or more alcoholic beverages (24 units or 196g of ethanol) per week for women and 21 or more alcoholic beverages (37 units or 294g of ethanol) per week for men Weight loss surgery (including gastric banding and intragastric balloon insertion) within 2 years of Screening 1 History of cancer within the past 2 years from Screening 1 (excluding adequately excised non-melanoma skin cancer)

[0170] [Table 13]

[0171] Secondary endpoints include, at a minimum, the percentage of participants achieving a 30% or greater relative reduction in liver fat from baseline using magnetic resonance imaging such as MRI-PDFF at weeks 24 and 52, change from baseline in liver stiffness measurement (LSM) by transient elastography such as vibration-controlled transient elastography (VCTE), and change from baseline in alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) (units / liter) [time frames: baseline (day 1), weeks 24 and 52].

Claims

1. A pharmaceutical composition for treating non-alcoholic fatty liver disease in human subjects, comprising a dose of 25 to 200 mg of a compound or a pharmaceutically acceptable salt thereof, wherein the dose is calculated based on the compound in free acid form, the compound comprising a ligand-bound double-stranded oligonucleotide, the double-stranded oligonucleotide reducing the expression of HSD17β13 in the human subject, and the ligand, 【Chemistry 1】 A pharmaceutical composition comprising the chemical structure of, its stereoisomer, or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, which is intended to be administered in a single dose.

3. The pharmaceutical composition according to claim 1, wherein the administration is to be repeated at intervals of 28 days, 12 weeks, or 3 months, and in some cases for 52 weeks or 1 year.

4. The pharmaceutical composition according to claim 1, wherein the dose is 50 mg to 100 mg.

5. The pharmaceutical composition according to claim 1, wherein the aforementioned dose is 100 mg to 200 mg.

6. The pharmaceutical composition according to claim 1, wherein the dose is 50 mg to 200 mg.

7. The pharmaceutical composition according to claim 1, wherein the compound is in the form of a sodium salt.

8. The pharmaceutical composition according to claim 1, which is to be administered by injection.

9. The pharmaceutical composition according to claim 8, wherein the injection is a subcutaneous injection.

10. The pharmaceutical composition according to claim 1, which exists in a single unit dosage form.

11. The pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

12. The pharmaceutical composition according to claim 1, wherein the human subject has the HSD17β13 rs72613567 mutation, the PNPLA3 rs738409 (I148M) mutation, or a combination thereof.

13. The aforementioned double-stranded oligonucleotide has the following sequence (5'→3'): usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence, Sequence ID 1); usCfsasUfcUfaUfcAfgAfcUfuCfuUfaCfsg (nucleotide sequence, SEQ ID NO: 5); usGfsasUfcCfaAfaAfaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 6); or usGfsasUfcCfaaaaaUfgUfcCfuAfgGfsc (nucleotide sequence, SEQ ID NO: 7) Here, a, c, g, and u represent 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, and 2'-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, and 2'-fluorouridine, respectively; and s represents a phosphorothioate bond. The pharmaceutical composition according to claim 1, comprising an antisense chain having one to zero or one nucleotide in a different modified nucleotide sequence.

14. The aforementioned double-stranded oligonucleotide has the following sequence (5'→3'): cguaagaaGfuCfuGfauagaugaa (nucleotide sequence, SEQ ID NO: 3); cguaagaaGfUfCfugaugaugauga (nucleotide sequence, SEQ ID NO: 8); gccuaggaCfAfUfuuuuugiauca (nucleotide sequence SEQ ID NO: 9); or gccuaggaCfaUfuUfuugiauca (nucleotide sequence, SEQ ID NO: 10) Here, a, c, g, i, and u represent 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylinosine, and 2'-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, and 2'-fluorouridine, respectively; and s represents a phosphorothioate bond. The pharmaceutical composition according to claim 1, comprising a sense chain having one to zero or one nucleotide differently in its modified nucleotide sequence.

15. The aforementioned double-stranded oligonucleotide has the sequence (5'→3'): usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence, SEQ ID NO: 1) An antisense chain comprising the following elements and sequence (5'→3'): (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb) (nucleotide sequence SEQ ID NO: 11) It includes a sense chain which includes, a, c, g, and u represent 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, and 2'-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, and 2'-fluorouridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted debasic deoxyribose residue. The pharmaceutical composition according to claim 1.

16. The pharmaceutical composition according to claim 1, for reducing the expression of HSD17β13 mRNA in a human subject by at least 50% compared to before administration, as measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample from the human subject.

17. The pharmaceutical composition according to claim 1, which reduces the expression of HSD17β13 protein in a human subject by at least 30% compared to before administration, as measured by Western blotting using a liver biopsy sample from the human subject.

18. The pharmaceutical composition according to claim 1, for reducing the ALT (alanine aminotransferase) level in a human subject by at least 40% compared to before administration, as measured in the serum of a human subject.

19. The pharmaceutical composition according to claim 1, for reducing the AST (aspartate aminotransferase) level in a human subject by at least 20% compared to before administration, as measured in the serum of a human subject.

20. The pharmaceutical composition according to claim 1, for reducing or slowing the increase in liver fat percentage in human subjects compared to before administration, as measured by magnetic resonance imaging.

21. The pharmaceutical composition according to claim 1, for reducing or slowing the increase in liver stiffness in human subjects compared to before administration, as measured by transient elastography.

22. The chemical structure of the ligand is 【Chemistry 2】 The pharmaceutical composition according to claim 1, comprising the following:

23. The chemical structure of the aforementioned compound is shown in Figures 5A to 5D below: 【Transformation 3】 【change】 【change】 【change】 The pharmaceutical composition according to claim 22, wherein the free acid form is having the same properties.

24. The chemical structure of the ligand is 【Chemistry 4】 The pharmaceutical composition according to claim 1, comprising the following:

25. The chemical structure of the aforementioned compound is shown in Figures 6A to 6D below: 【Transformation 5】 【change】 【change】 【change】 The pharmaceutical composition according to claim 24, wherein it is a sodium salt form having the properties of the sodium salt.

26. The pharmaceutical composition according to any one of claims 1 to 3 and 7 to 25, wherein the dose is 50 mg.

27. The pharmaceutical composition according to any one of claims 1 to 3 and 7 to 25, wherein the dose is 100 mg.

28. The pharmaceutical composition according to any one of claims 1 to 3 and 7 to 25, wherein the dose is 200 mg.

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