Treatment of non-alcoholic fatty liver disease

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 329,081, filed on April 8, 2022, and U.S. Provisional Application No. 63 / 401,858, filed on August 29, 2022, the entire contents of each of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in XML format, and the entire contents of which are hereby incorporated by reference herein. The XML copy is named 70162WO01_SL.XML, is approximately 115,715 bytes in size, and was created on March 7, 2023. Technical Field

[0005] Disclosed herein are methods for treating non-alcoholic fatty liver disease (NAFLD), which can reduce (e.g., inhibit or silence) the gene expression of the hepatic lipid droplet protein 17β-hydroxysteroid dehydrogenase type 13 (also known as HSD17B13, 17β-HSD13, HSD17β13, 17beta-HSD13, 17beta-HSD type 13 or 17β-HSD13) and its protein expression. Summary of the invention

[0006] In some aspects, the present disclosure provides a method for treating non-alcoholic fatty liver disease in a human subject, comprising administering a compound or a pharmaceutically acceptable salt thereof to the human subject at a dose of about 25 to about 200 mg, the dose being calculated based on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide linked to a ligand, wherein the double-stranded oligonucleotide reduces expression of HSD17β13 in the human subject, and wherein the ligand comprises the following chemical structure:

[0007]

[0008] (NAG37)s, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shown is the effect of the test compound on HSD17β13 mRNA expression relative to baseline in NASH (non-alcoholic steatohepatitis) or NAFLD patients at day 71. Boxes show Q1, median and Q3. Crosses show mean values, while whiskers extend to minimum and maximum values.

[0010] Figure 2A and Figure 2BShown are the effects of the test compounds on the liver enzymes ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in patients with NASH or NAFLD, respectively.

[0011] Figure 3A and Figure 3B The results show that the test compound has a positive correlation with HSD17β13rs72613567 ( Figure 3A , boxes show Q1, median, and Q3, crosses show mean, and whiskers extend to minimum and maximum values) and PNPLA3 rs738409 ( Figure 3B Effect of messenger RNA knockdown relative to baseline by genotype in patients with NASH or NAFLD (with boxes showing Q1, median, and Q3, diamonds showing means, and whiskers extending to minimum and maximum values).

[0012] Figure 4 The clinical study design and dosing regimen for the test compounds are shown.

[0013] Figures 5A to 5D The chemical structures of the test compounds described in Example 2 in free acid form are shown. The numbers in circles indicate the connection points of the parts of the structure that are divided into four pages due to their length.

[0014] Figures 6A to 6D The chemical structure of the test compound described in Example 2 in the form of a sodium salt is shown. The numbers in the circles indicate the connection points of the parts of the structure that are divided into four pages due to their length.

[0015] Figure 7 Target engagement simulations based on the clinical results in Example 2 are shown to further support a dose of 200 mg every 12 weeks or 3 months. DETAILED DESCRIPTION

[0016] In some cases, the treatment disclosed herein can reduce the hepatocyte production of HSD17β13 and provide treatment and / or protection in human subjects with or suspected NAFLD and potential other chronic liver diseases. In some cases of human patients with NASH or suspected NASH (e.g., NAFLD with high ALT), based on a biopsy on the 71st day after administration, treatment can cause a decrease in HSD17β13 mRNA expression at a dose level of about 25 to about 200 mg, such as a near maximum decrease (e.g., at least a decrease of about 90%). For the doses disclosed herein, such as 100 and 200 mg, treatment inhibits HSD17β13 in subjects carrying PNPLA3 rs738409CG or GG genotypes, which are associated with an increased risk of NASH cirrhosis, and this inhibitory effect can be substantially the same compared to subjects lacking PNPLA3 mutations. In some cases, reduced expression of HSD17β13 can result in or be accompanied by a decrease in ALT and AST, which are surrogate markers of liver inflammation in NASH.

[0017] As used herein, unless otherwise indicated, the term "about" means plus or minus 5% of the referenced figure.

[0018] Unless otherwise specified, the terms "a," "an," and "the" include singular and plural references.

[0019] Compounds used

[0020] The compounds disclosed herein for use in treating human subjects may include, for example, RNA interference (RNAi) agents "HSD17B13 RNAi agents" that reduce HSD17B13 expression. In some cases, the compound comprises a double-stranded oligonucleotide targeting HSD17β13, which is linked to a galactose-containing ligand and can be preferentially taken up by hepatocytes to promote liver specificity, after which the compound will engage the RNAi machinery of the cell to target HSD17β13 mRNA for degradation, thereby reducing the amount of free HSD17β13 mRNA available for translation. These factors can minimize off-target effects or extrahepatic inhibition of the compound and contribute to an acceptable safety profile of the compound. In some cases, the compound targeting herein is associated with knockdown of HSD17β13, mimicking LOF (loss of function) mutations in NASH.

[0021] As used herein, when referring to the expression of HSD17B13, the terms "reduced", "silenced", "inhibited", "down-regulated" or "knockdown" may mean that in a cell, cell group, tissue, organ or subject in which the gene is transcribed, when the cell, cell group, tissue, organ or subject is treated with an RNAi agent, the expression of the gene is reduced compared to a second cell, cell group, tissue, organ or subject that has not been treated in this way, 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. The reduction of HSD17B13 can be measured by any suitable assay or method known in the art. Non-limiting example assays include microarrays, Affymetrix gene chips, and qRT-PCR (quantitative reverse transcriptase polymerase chain reaction) for HSD17B13 gene quantification, and gel electrophoresis (such as Western blot) and mass spectrometry for HSD17B13 protein measurement, and those described in International Patent Application Publication No. WO 2020 / 061177 (Patent Application No. PCT / US2019 / 051707), the entire contents of which are incorporated herein by reference. The reference HSD17B13 mRNA gene transcript for a normal person may 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 treatment disclosed herein with the level of HSD17B13 mRNA or HSD17B13 protein after administration of the compound.

[0022] Compounds disclosed herein may contain one or more asymmetric centers (also referred to as chiral centers), and therefore may exist as single enantiomers, diastereomers or other forms of stereoisomers or as mixtures thereof.Chiral centers, such as chiral carbon atoms, may also be present in substituents such as alkyl groups. In the absence of specific descriptions of the stereochemistry of the chiral centers in any chemical structure shown herein, the structure is expected to encompass any stereoisomer and all mixtures thereof. In some cases, compounds disclosed herein containing one or more chiral centers may be used as racemic modifications, including racemic mixtures and racemates, mixtures enriched with enantiomers, or as enantiomerically pure single stereoisomers. In some cases, compounds herein are provided in the form of salts, mixed salts or free acids.

[0023] 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 (1) by forming diastereomeric salts, complexes or other derivatives; (2) by selective reaction with stereoisomer-specific reagents, such as by enzymatic oxidation or reduction; or (3) by gas-liquid or liquid chromatography in a chiral environment, such as on a chiral support such as silica with a bound chiral ligand or in the presence of a chiral solvent. It will be understood that in the case where the desired stereoisomer is converted into another chemical entity by one of the above separation procedures, further steps are required to release the desired form. Alternatively, a specific stereoisomer can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into another by asymmetric transformation. In some cases, the compounds used herein include a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand) annealed to form a double strand.

[0024] In some cases, the compound comprises a double-stranded oligonucleotide, for example, comprising RNA, chemically modified RNA, DNA or chemically modified DNA oligonucleotide molecules, any of which can have two complementary sequences. In some cases, the double-stranded oligonucleotide can degrade or inhibit the translation of the messenger RNA (mRNA) transcript of HSD17B13 mRNA in a sequence-specific manner. In some cases, the compounds disclosed herein can be operated by RNA interference mechanisms (i.e., by interacting with the RNA interference pathway mechanisms (RNA induced silencing complex or RISC) of mammalian cells) or by any alternative (one or more) mechanisms or approaches. Although it is believed that HSD17B13 RNAi agents (as used herein) are mainly operated by RNA interference mechanisms, the disclosed RNAi agents are not subject to the constraints or limitations of any particular pathway or mechanism of action. In some cases, the compound comprises short or small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) or dicer (dicer) substrates. In some cases, the compounds used herein include an antisense strand having a region complementary to at least a portion of HSD17B13 mRNA.

[0025] In some cases, the compounds used herein have a sense strand of 15 to 49 nucleotides in length and an antisense strand of 18 to 49 nucleotides in length. In some cases, the length of the sense strand and the antisense strand is independently 18 to 26 nucleotides. In some cases, the length of the sense strand and the antisense strand is independently 21 to 26 nucleotides. In some cases, the length of the sense strand and the antisense strand is independently 21 to 24 nucleotides. In some cases, the length of the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some cases, the length of the antisense strand is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some cases, the length of the sense strand and the antisense strand is 21 nucleotides. The sense strand and the antisense strand may be of the same length or of different lengths. The sense strand and the antisense strand may also form overhanging nucleotides at one or both ends of the nucleotide chain.

[0026] Compounds suitable for use herein can be covalently linked to a ligand comprising one or more N-acetylgalactosamine moieties. The N-acetylgalactosamine moieties can promote the targeting of HSD17B13 RNAi agents to the asialoglycoprotein receptor (ASGPR) readily present on the surface of hepatocytes, which results in the internalization of the HSD17B13-RNAi agent by endocytosis or other means.

[0027] Modified nucleotides and modified internucleoside linkages

[0028] Compounds disclosed herein, such as HSD17B13 RNAi agents, can include modified nucleotides, which can maintain the activity of RNAi agents while improving serum stability and minimizing the possibility of activating interferon activity in humans. As used herein, "modified nucleotides" are nucleotides other than ribonucleotides (2'-hydroxy 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 nucleotides are modified nucleotides. As used herein, modified nucleotides include but are not limited to deoxyribonucleotides, nucleotide mimics, 2'-modified nucleotides, reverse nucleotides, nucleotides comprising modified nucleobases, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3'-open ring nucleotide mimics (unlocked nucleobase analogs), locked nucleotides, 3'-O-methoxy (2'-internucleoside connected) nucleotides, 2'-F-arabinonucleotides, 5'-Me, 2'-fluoronucleotides, morpholino nucleotides, nucleotides containing vinyl phosphonate and nucleotides containing cyclopropyl phosphonate. In some cases, the modified nucleotides of HSD17B13 RNAi agents are 2' modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of a five-membered sugar ring). 2'-modified nucleotides include but are not limited to 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides (usually referred to as 2'-fluoro nucleotides), 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-amino nucleotides and 2'-alkyl nucleotides. It may not be necessary to uniformly modify all nucleotides in a given RNAi agent. In some cases, more than one modification can be incorporated into a single HSD17B13 RNAi agent, or even in a single nucleotide thereof. The sense strand and antisense strand of the HSD17B13 RNAi agent can be synthesized and / or modified by methods known in the art. The modification on one nucleotide can be independent of the modification of another nucleotide.

[0029] In some cases, the nucleobases disclosed herein (often referred to simply as "bases") may be modified. Natural nucleobases may include the basic purine bases adenine and guanine, and the basic pyrimidine bases cytosine, thymine, and uracil. Nucleobases may 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 containing modified nucleobases) is known in the art.

[0030] 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) derivatives of adenine and guanine, and other alkyl derivatives, 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-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (such as 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0031] In some cases, all or substantially all nucleotides of compounds disclosed herein (e.g., HSD17B13 RNAi agents) are modified nucleotides. As used herein, the RNAi agent wherein substantially all nucleotides are modified nucleotides is the RNAi agent having four or less (i.e., 0, 1, 2, 3 or 4) nucleotides as ribonucleotides (i.e., unmodified) in both sense strand and antisense strand. As used herein, the sense strand wherein substantially all nucleotides are modified nucleotides is the sense strand having two or less (i.e., 0, 1 or 2) nucleotides as ribonucleotides in the sense strand. As used herein, the antisense strand wherein substantially all nucleotides are modified nucleotides is the antisense strand having two or less (i.e., 0, 1 or 2) nucleotides as ribonucleic acids in the sense strand.

[0032] In some cases, one or more nucleotides of a compound disclosed herein (e.g., HSD17B13 RNAi agent) are connected by a non-standard bond or a backbone (i.e., a modified internucleoside bond or a modified backbone). The modified internucleoside bond or a backbone include, but are not limited to, a thiophosphate (phosphorothioate) group, a chiral thiophosphate, a thiophosphate (thiophosphate), a dithiophosphate, a phosphotriester, an aminoalkyl phosphotriester, an alkylphosphonate (e.g., a methylphosphonate or a 3'-alkylenephosphonate), a chiral phosphonate, a phosphinate, a phosphoramidate (e.g., a 3'-aminophosphoramidate, an aminoalkylphosphoramidate or a thiophosphoramidate), a thioalkylphosphonate, a thioalkylphosphotriester, a morpholino bond, a borane phosphate with a normal 3'-5' bond, a borane phosphate analog connected with a 2'-5' bond, or a borane phosphate with an inverted polarity, wherein adjacent nucleoside units are connected as 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some cases, the internucleoside bond or the skeleton of modification lack phosphorus atom.The nucleoside bond of the modification lacking phosphorus atom includes but is not limited to the bond between short chain alkyl or cycloalkyl sugar, the bond between mixed heteroatoms and alkyl or cycloalkyl sugar, or the bond between one or more short chain heteroatoms or heterocyclic sugar.In some cases, the internucleoside skeleton of modification includes but is not limited to siloxane skeleton, sulfide skeleton, sulfoxide skeleton, sulfone skeleton, methyl acetyl and thiomethyl acetyl skeleton, methylene methyl acetyl and thiomethyl acetyl skeleton, containing olefin skeleton, sulfamic acid salt skeleton, methylene imino and methylene hydrazine skeleton, sulfonate and sulfonamide skeleton, amide skeleton and having mixed N, O, S and CH 2 Other skeletons of components.

[0033] In some cases, the sense strand of the compounds disclosed herein (e.g., HSD17B13 RNAi agents) may contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds, the antisense strand of the HSD17B13-RNAi agent may contain 1, 2, 3, 4, 5, 6 thiosulfate bonds, or both the sense strand and the antisense strand may independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds. In some cases, the sense strand of the HSD17B13 RNAi agent may contain 1, 2, 3, or 4 phosphorothioate bonds, the antisense strand of the HSD17B13 RNAi agent may contain 1, 2, 3, or 4 phosphorothioate bonds, or both the sense strand and the antisense strand may independently contain 1, 2, 3, or 4 phosphorothioate bonds.

[0034] In some cases, the sense strand of a compound disclosed herein (e.g., HSD17B13 RNAi agent) contains at least two thiophosphate nucleoside bonds. In some cases, at least two thiophosphate nucleoside bonds are located between the nucleotides at positions 1-3 of the sense strand 3' end. In some cases, at least two thiophosphate nucleoside bonds are located between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 of the sense strand 5' end. In some cases, the thiophosphate nucleoside bonds are used to connect the terminal nucleotides in the sense strand to the capping residues of the 5' end, 3' end, or 5' and 3' end of the nucleotide sequence. In some cases, the thiophosphate nucleoside bonds are used to connect the ligand to the sense strand.

[0035] In some cases, the antisense strand of a compound disclosed herein (e.g., HSD17B13 RNAi agent) contains three or four phosphorothioate nucleoside interkeys. In some cases, the antisense strand comprises three phosphorothioate nucleoside interkeys. In some cases, three phosphorothioate nucleoside interkeys are located between the nucleotides at positions 1-3 of the 5' end of the antisense strand, and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25 ​​or 24-26 of the 5' end. In some cases, the compound contains at least two phosphorothioate nucleoside interkeys in the sense strand, and contains three or four phosphorothioate nucleoside interkeys in the antisense strand.

[0036] In some cases, the compounds disclosed herein (eg, HSD17B13 RNAi agents) contain one or more modified nucleotides and one or more modified internucleoside bonds. In some cases, the 2' modified nucleoside is bound to the modified internucleoside bond.

[0037] Table 1A. Exemplary antisense strand sequences

[0038]

[0039] Table 1B. Exemplary sense strand nucleotide sequences (shown as modified versions without inverted abasic residues or NAG ligands)

[0040]

[0041] As used in Tables 1A, 1B and 2 herein, the following symbols are used to indicate modified nucleotides, ligand groups and linker groups: A, C, G and U represent adenosine, cytidine, guanosine and uridine, respectively; a, c, g and u represent 2'-O-methyladenosine, cytidine, guanosine and uridine, respectively; Af, Cf, Gf and Uf represent 2'-fluoroadenosine, 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.

[0042] Unless the sequence indicates otherwise (e.g., by a phosphorothioate bond "s"), when a monomer is present in a chain, the monomer can be interconnected by a 5'-3'-phosphodiester bond. In some cases, the phosphorothioate bond shown in the modified nucleotide sequence disclosed herein replaces the phosphodiester bond commonly present in an oligonucleotide. In some cases, the terminal nucleotide at the 3' end of a given oligonucleotide sequence will generally have a hydroxyl group (-OH) at the corresponding 3' position of a given monomer, rather than an isolated phosphate moiety. In some cases, when 5'→3' observes the corresponding chain, a reverse abasic residue is inserted so that the 3' position of deoxyribose is connected to the 3' end of the previous monomer on the corresponding chain. In addition, those of ordinary skill in the art will readily understand and appreciate that, although the phosphorothioate chemical structure described herein generally shows an anion on a sulfur atom, the present disclosure includes phosphorothioate tautomers (e.g., wherein the sulfur atom has a double bond, and an anion is on an oxygen atom). In some cases, each sense strand and / or antisense strand can have any linking group disclosed herein, connected to the 5' and / or 3' end of the sequence.

[0043] Capping residues

[0044] In some cases, the sense strand disclosed herein may include one or more capping residues, sometimes referred to as "caps", "end caps" or "capping residues". As used herein, "capping residues" are non-nucleotide compounds or other moieties that can be incorporated into one or more ends of the nucleotide sequence of the RNAi agent disclosed herein. Capping residues can provide RNAi agents with certain beneficial properties, such as preventing exonuclease degradation. In some cases, inverted abasic residues (invAb) (also referred to as "inverted abasic sites") are added as capping residues (see Table 2). Capping residues include, for example, inverted abasic residues and carbon chains such as terminal C 3 H 7 (propyl), C 6 H 13 (Hexyl) or C 12 H 25 In some cases, the capping residue is present at the 5' end, the 3' end, or both the 5' and 3' ends of the sense strand. In some cases, the 5' end and / or the 3' end of the sense strand may contain more than one inverted abasic deoxyribose moiety as a capping residue.

[0045] In some cases, one or more reverse abasic residues (invAb) are added to the 3' end of the sense strand. In some cases, one or more reverse abasic residues (invAb) are added to the 5' end of the sense strand. In some cases, one or more reverse abasic residues or reverse abasic sites are inserted between the nucleotide sequence of the sense strand of the part and the RNAi agent. In some cases, one or more reverse abasic residues or reverse abasic sites are included near one or both ends of the sense strand of the RNAi agent or near the end to allow the RNAi agent to enhance activity or other desired characteristics.

[0046] In some cases, one or more reverse abasic residues (invAb) are added to the 5' end of the sense strand. In some cases, one or more reverse abasic residues can be inserted between the nucleotide sequence of the ligand and the RNAi agent sense strand. Reverse abasic residues can be connected via phosphate, thiophosphate (e.g., shown herein as (invAb)s) or other internucleoside bonds. The chemical structures of reverse abasic deoxyribose residues are shown in Table 2 below and Figures 5A to 5D and Figures 6A to 6D The chemical structure shown.

[0047] Table 2. Reverse abasic (deoxyribose) chemical structures

[0048]

[0049]

[0050] Ligand

[0051] In some cases, the compound used herein may include an RNA molecule, such as an HSD17B13 RNAi agent, which is connected to one or more non-nucleotide groups, such as a ligand that can enhance RNAi agent targeting or delivery. Examples include three N-acetylgalactosamine moieties described in the present disclosure. The ligand can be covalently linked to the 3' and / or 5' ends of the sense strand and / or antisense strand. In some cases, the compound contains a ligand connected to the 3' and / or 5' ends of the sense strand. In some cases, the ligand is connected to the 5' end of the sense strand. In some cases, the ligand comprises structure (NAG37) s, is essentially composed of it or is composed of it, and is connected to the 5' end of the sense strand. The ligand can be directly or indirectly connected to the RNAi agent via a joint / linking group. In some cases, the ligand is connected to the RNAi agent via an unstable, cleavable or reversible bond or joint. In some cases, the ligand is connected to the reverse abasic residue at the 5' end of the sense strand.

[0052] The ligands disclosed herein can enhance the pharmacokinetics or biodistribution properties of the RNAi agents to which they are attached, to improve the cell-specific distribution and cell-specific uptake of the RNAi agents. In some cases, the ligands enhance the endocytosis of the RNAi agents. In some cases, the ligands can be monovalent, divalent, trivalent, tetravalent, or have a higher valence state for the target to which it is directed. Representative ligands include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetics with affinity for cell surface molecules.

[0053] The preparation of ligands, such as clusters of galactose derivatives including N-acetylgalactosamine, is described in, for example, International Patent Application Publication No. WO 2018 / 044350 (Patent Application No. PCT / US2017 / 021147) and International Patent Application Publication No. WO2017 / 156012 (Patent Application No. PCT / US2017 / 021175), the entire contents of both of which are incorporated herein by reference.

[0054] For example, a ligand linked to the HSD17B13 RNAi agent described in Tables 1A and 1B can have a chemical structure of (NAG37)s, as shown in Table 3.

[0055] Table 3. Chemical structures of (NAG37)s.

[0056]

[0057]

[0058] Drug ingredients

[0059] Compounds suitable for use herein, such as HSD17B13 RNAi agents, can be formulated as pharmaceutical compositions for administration to human subjects. Pharmaceutical compositions can be used to treat or prevent subjects with diseases or conditions that would benefit from inhibiting the expression of HSD17B13 mRNA or reducing the level of HSD17B13 protein, such as human subjects with non-alcoholic fatty liver disease. In some cases, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents and / or delivery polymers) are added to a pharmaceutical composition comprising an HSD17B13 RNAi agent to form a pharmaceutical formulation suitable for in vivo delivery to a human subject.

[0060] As used herein, "pharmacologically effective amount", "therapeutically effective amount" or simply "effective amount" refers to the amount of an active agent that produces a pharmacological, therapeutic or preventive result. In some cases, a therapeutically or preventively 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 subject's disease.

[0061] As used herein, pharmaceutical compositions or medicines include at least one compound disclosed herein and one or more pharmaceutically acceptable excipients in a pharmacologically effective amount. Excipients are substances other than active pharmaceutical ingredients (API, therapeutic products, such as HSD17B13 RNAi agents), which are intentionally included in drug delivery systems. Excipients do not or are not expected to have therapeutic effects at the expected dose. Excipients can play the following roles: a) assist in the processing of drug delivery systems during manufacturing, b) protect, support or enhance the stability, bioavailability or patient acceptability of API, c) assist in product identification, and / or d) enhance the overall safety and effectiveness of API delivery during storage or use Any other attributes.

[0062] Excipients may include, but are not limited to, absorption enhancers, antiadherents, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, dextran, glucose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release bases, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, and wetting agents.

[0063] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble). For subcutaneous, intravenous or intramuscular administration, suitable carriers may include physiological saline, bacteriostatic water, ELTM (BASF, Parsippany, NJ) or phosphate buffer. It should be stable under the conditions of manufacture and storage and should prevent the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof.

[0064] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or more of the above ingredients, as required, followed by filtered sterilization. In some cases, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those described above.

[0065] The pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. Such additional components are selected from active agents such as antipruritic agents, hemostatic agents, local anesthetics, anti-inflammatory agents or antihistamines.

[0066] The pharmaceutically acceptable formulation may be packaged into a kit, container, pack or dispenser.The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.

[0067] Treatment

[0068] As used herein, the terms "treat", "treatment", and "treating", etc., may include prevention, management, preventive treatment, reduction, and / or suppression of the number, severity, and / or frequency of one or more symptoms of a disease and / or a disease in a subject. In some cases, the terms "treat", "treatment", and "treating", etc., mean management, reduction, and / or suppression of the number, severity, and / or frequency of one or more symptoms of a disease and / or a disease in a subject. Unless otherwise expressly stated, treatment may also include alleviation or reduction of the underlying conditions and / or symptoms of a disease.

[0069] Embodiment 1: A method of treating non-alcoholic fatty liver disease in a human subject in need thereof, comprising administering a compound or a pharmaceutically acceptable salt thereof to the subject at a dose of about 25 to about 200 mg, wherein the dose is calculated based on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide linked to a ligand, wherein the double-stranded oligonucleotide reduces the expression of HSD17β13 in the human subject, and wherein the ligand comprises the following chemical structure:

[0070]

[0071] (NAG37)s, a stereoisomer thereof, or a salt thereof (eg, a pharmaceutically acceptable salt).

[0072] Embodiment 2: The method of Embodiment 1, wherein the administration is a single dose.

[0073] Embodiment 3: The method of embodiment 1, wherein the administration is repeated once, for example with an interval of about 28 days (i.e., 4 weeks), about 12 weeks, or about 3 months between doses, 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 once every 4 weeks, for example at a dose of 100 mg. In some cases, the administration is repeated once every 12 weeks, for example at a dose of 200 mg.

[0074] Embodiment 4: The method of any one of embodiments 1-3, wherein the dose is from about 50 mg to about 100 mg, such as about 50 mg.

[0075] Embodiment 5: The method of any one of embodiments 1-3, wherein the dose is from about 100 mg to about 200 mg, such as about 100 mg.

[0076] Embodiment 6: The method of any one of embodiments 1-3, wherein the dose is from about 50 mg to about 200 mg, such as about 200 mg.

[0077] Embodiment 7: The method of any one of Embodiments 1-6, wherein the compound is in the form of a sodium salt.

[0078] Embodiment 8: The method of any one of Embodiments 1-7, wherein the compound is administered by injection, for example at a concentration of 200 mg / ml.

[0079] Embodiment 9: The method according to embodiment 8, wherein the injection is subcutaneous injection.

[0080] Embodiment 10: The method of any one of Embodiments 1-9, wherein the compound is present in unit dosage form, eg, comprising a single (unit) dosage form.

[0081] Embodiment 11: The method of any one of Embodiments 1-10, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH), such as pre-cirrhotic non-alcoholic hepatitis, NASH-F3, NASH with bridging fibrosis, or biopsy-proven NASH.

[0082] Embodiment 12: The method of any one of Embodiments 1-11, wherein the human subject has a HSD17β13rs72613567 mutation, a PNPLA3rs738409(I148M) mutation, or a combination thereof. In some cases, the human subject has a HSD17β13rs72613567 mutation. In some cases, the human subject has a PNPLA3rs738409(I148M) mutation.

[0083] Embodiment 13: The method of any one of Embodiments 1-12, wherein the double-stranded oligonucleotide comprises an antisense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (5'→3'):

[0084] usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1);

[0085] usCfsasUfcUfaUfcAfgAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 5);

[0086] usGfsasUfcCfaAfaAfaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 6); or

[0087] usGfsasUfcCfaaaaaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 7);

[0088] And wherein 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.

[0089] Embodiment 14: The method of any one of embodiments 1-13, wherein the double-stranded oligonucleotide comprises a sense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (5'→3'):

[0090] cguaagaaGfuCfuGfauagauga (nucleotide sequence SEQ ID NO: 3);

[0091] cguaagaaGfUfCfugauagauga (nucleotide sequence SEQ ID NO: 8);

[0092] gccuaggaCfAfUfuuuugiauca (nucleotide sequence SEQ ID NO: 9); or

[0093] gccuaggaCfaUfuUfuugiauca (nucleotide sequence SEQ ID NO: 10);

[0094] And wherein 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.

[0095] Embodiment 15: The method of any one of embodiments 1-14, wherein the double-stranded oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprises the sequence: usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1), and the sense strand comprises the sequence:

[0096] (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb)(nucleotide sequence

[0097] SEQ ID NO: 11),

[0098] And wherein 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; and (invAb) represents an inverted abasic deoxyribose residue.

[0099] Embodiment 16: The method of any one of Embodiments 1-15, wherein the method reduces the expression level 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, e.g., at about day 71 or 113 from the first administration, as measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample from a human subject.

[0100] Embodiment 17: The method of any one of Embodiments 1-16, wherein the method 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, e.g., at about day 71 or 113 from the first administration, as measured by Western blot using a liver biopsy sample from the human subject.

[0101] Embodiment 18: The method of any one of Embodiments 1-17, wherein the method reduces the ALT (alanine aminotransferase) level of the 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, e.g., at about day 71 or 113 from the first administration, compared to before administration.

[0102] Embodiment 19: The method of any one of Embodiments 1-18, wherein the method reduces the AST (aspartate aminotransferase) level in the 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%), as measured in the serum of the human subject, e.g., at about day 71 or 113 from the first administration, compared to before administration.

[0103] Embodiment 20: The method of any one of embodiments 1-19, wherein the method reduces or slows the increase in liver fat fraction in the human subject (e.g., by at least 4-41% or at least 7-12% or less) at, for example, about day 71 or 113 from the first administration, compared to before administration.

[0104] 8%), as measured by magnetic resonance imaging.

[0105] Embodiment 21: The method of any one of Embodiments 1-20, wherein the method reduces or slows the increase in liver stiffness in a human subject (e.g., by at least 4-37%), compared to before administration, as measured by transient elastography, e.g., at about day 71 or 113 from the first administration.

[0106] Embodiment 22: The method according to any one of embodiments 1-21, wherein the chemical structure of the ligand comprises:

[0107]

[0108] Embodiment 23: A method according to Embodiment 22, wherein the compound is in the form of Figures 5A to 5D The chemical structures shown are of the free acid form.

[0109] Embodiment 24: The method according to any one of embodiments 1-21, wherein the chemical structure of the ligand comprises:

[0110]

[0111] Embodiment 25: The method of Embodiment 24, wherein the compound is in the form of Figures 6A to 6D The chemical structure shown is the sodium salt form.

[0112] Embodiment 26: The method of any one of Embodiments 1-25, wherein the method reduces the GGT (gamma-glutamyl transferase) level in the human subject by at least about 1.4% or at least 8.3% or at least 9% as measured in the serum of the human subject, e.g., at about day 71 or 113 from the first use, compared to before administration.

[0113] Embodiment 27: A compound or a pharmaceutically acceptable salt thereof for use in treating non-alcoholic fatty liver disease in a human subject, wherein the use comprises administering the compound or a pharmaceutically acceptable salt thereof to the subject at a dose of about 25 to about 200 mg, wherein the dose is calculated based on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide linked to a ligand, wherein the double-stranded oligonucleotide reduces the expression of HSD17β13 in the human subject, and wherein the ligand comprises the following chemical structure:

[0114]

[0115] (NAG37)s, a stereoisomer thereof, or a salt thereof (eg, a pharmaceutically acceptable salt).

[0116] Embodiment 28: A compound for use according to Embodiment 27 or a pharmaceutically acceptable salt thereof, wherein the administration is a single dose.

[0117] Embodiment 29: A compound or a pharmaceutically acceptable salt thereof for use according to Embodiment 28, wherein the administration is repeated once, for example with an interval of about 28 days (i.e., 4 weeks), about 12 weeks, or about 3 months between doses, for example about 100 mg every 4 weeks, or about 200 mg every 12 weeks, optionally for about 52 weeks or about 1 year. Embodiment 29a: A compound or a pharmaceutically acceptable salt thereof for use according to Embodiment 28, wherein about 100 mg is administered every 4 weeks for about 52 weeks. Embodiment 29b: A compound or a pharmaceutically acceptable salt thereof for use according to Embodiment 28, wherein about 200 mg is administered every 12 weeks for about 52 weeks.

[0118] Embodiment 30: A compound for use according to any one of Embodiments 27-29, or a pharmaceutically acceptable salt thereof, wherein the dose is from about 50 mg to about 100 mg, such as about 50 mg.

[0119] Embodiment 31: A compound for use according to any one of Embodiments 27-29, or a pharmaceutically acceptable salt thereof, wherein the dose is from about 100 mg to about 200 mg, such as about 100 mg.

[0120] Embodiment 32: A compound for use according to any one of Embodiments 27-29, or a pharmaceutically acceptable salt thereof, wherein the dose is from about 50 mg to about 200 mg, such as about 200 mg.

[0121] Embodiment 33: A compound for use according to any one of Embodiments 27-32, or a pharmaceutically acceptable salt thereof, wherein the compound is in the form of a sodium salt.

[0122] Embodiment 34: A compound for use according to any one of Embodiments 27-33, or a pharmaceutically acceptable salt thereof, wherein the compound is administered by injection, for example at a concentration of 200 mg / ml.

[0123] Embodiment 35: A compound for use according to Embodiment 34 or a pharmaceutically acceptable salt thereof, wherein the injection is subcutaneous.

[0124] Embodiment 36: A compound for use according to any one of Embodiments 27-35, or a pharmaceutically acceptable salt thereof, wherein the compound is administered in unit dosage form, eg, in a single unit dosage form.

[0125] Embodiment 37: A compound for use according to any one of Embodiments 27-36 or a pharmaceutically acceptable salt thereof, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH), such as pre-cirrhotic non-alcoholic hepatitis, NASH-F3, NASH with bridging fibrosis, or biopsy-proven NASH.

[0126] Embodiment 38: A compound for use according to any one of Embodiments 27-37, or a pharmaceutically acceptable salt thereof, wherein the human subject has the HSD17β13rs72613567 mutation, the PNPLA3 rs738409(I148M) mutation, or a combination thereof.

[0127] Embodiment 39: A compound for use according to any one of Embodiments 27-38, or a pharmaceutically acceptable salt thereof, wherein the double-stranded oligonucleotide comprises an antisense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (5'→3'):

[0128] usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1);

[0129] usCfsasUfcUfaUfcAfgAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 5);

[0130] usGfsasUfcCfaAfaAfaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 6); or

[0131] usGfsasUfcCfaaaaaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 7);

[0132] And wherein 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.

[0133] Embodiment 40: A compound for use according to any one of Embodiments 27 to 39, or a pharmaceutically acceptable salt thereof, wherein the double-stranded oligonucleotide comprises a sense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (5'→3'):

[0134] cguaagaaGfuCfuGfauagauga (nucleotide sequence SEQ ID NO: 3);

[0135] cguaagaaGfUfCfugauagauga (nucleotide sequence SEQ ID NO: 8);

[0136] gccuaggaCfAfUfuuuugiauca (nucleotide sequence SEQ ID NO: 9); or

[0137] gccuaggaCfaUfuUfuugiauca (nucleotide sequence SEQ ID NO: 10);

[0138] And wherein 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.

[0139] Embodiment 41: A compound or a pharmaceutically acceptable salt thereof for use according to any one of Embodiments 27-40, wherein the double-stranded oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprises the sequence: usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1), and the sense strand comprises the sequence:

[0140] (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb)(nucleotide sequence

[0141] SEQ ID NO: 11),

[0142] And wherein 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; and (invAb) represents an inverted abasic deoxyribose residue.

[0143] Embodiment 42: A compound for use according to any one of Embodiments 27-41, or a pharmaceutically acceptable salt thereof, wherein the use reduces the expression level 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%), e.g., at about day 71 or 113 from the first administration, compared to before administration, as measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample from a human subject.

[0144] Embodiment 43: A compound for use according to any one of Embodiments 27-42, or a pharmaceutically acceptable salt thereof, 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, e.g., at about day 71 or 113 from the first administration, as measured by Western blotting using a liver biopsy sample from a human subject.

[0145] Embodiment 44: A compound for use according to any one of Embodiments 27-43, or a pharmaceutically acceptable salt thereof, 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, e.g., at about day 71 or 113 from the first administration, compared to before administration.

[0146] Embodiment 45: A compound for use according to any one of Embodiments 27-44, or a pharmaceutically acceptable salt thereof, 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, e.g., at about day 71 or 113 from the first administration.

[0147] Embodiment 46: A compound for use according to any one of Embodiments 27-45, or a pharmaceutically acceptable salt thereof, wherein the use reduces or slows the increase in liver fat fraction in a human subject (e.g., by at least 4-41% or at least about 7-8%) compared to before administration, as measured by magnetic resonance imaging, e.g., at about day 71 or 113 from the first administration.

[0148] Embodiment 47: A compound for use according to any one of Embodiments 27-46, or a pharmaceutically acceptable salt thereof, wherein the use reduces or slows the increase in liver stiffness in a human subject (e.g., by at least 4-37%) compared to before administration, as measured by transient elastography, e.g., at about day 71 or 113 from the first administration.

[0149] Embodiment 48: A compound for use according to any one of Embodiments 27-47 or a pharmaceutically acceptable salt thereof, wherein the chemical structure of the ligand comprises:

[0150]

[0151] Embodiment 49: The compound for use according to Embodiment 48 or a pharmaceutically acceptable salt thereof, wherein the compound has Figures 5A to 5D The chemical structures shown are of the free acid form.

[0152] Embodiment 50: A compound for use according to any one of Embodiments 27-47 or a pharmaceutically acceptable salt thereof, wherein the chemical structure of the ligand comprises:

[0153]

[0154] Embodiment 51: The compound for use according to Embodiment 50 or a pharmaceutically acceptable salt thereof, wherein the compound has Figures 6A to 6D The chemical structure shown is the sodium salt form.

[0155] Embodiment 52: A compound for use according to any one of Embodiments 27-51, or a pharmaceutically acceptable salt thereof, wherein the use reduces GGT (gamma-glutamyl transferase) 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, e.g., at about day 71 or 113 from the first use.

[0156] Embodiment 53: Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating non-alcoholic fatty liver disease, wherein the compound or a pharmaceutically acceptable salt thereof is used at a dose of about 25 to about 200 mg, wherein the dose is calculated based on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide linked to a ligand, wherein the double-stranded oligonucleotide reduces the expression of HSD17β13, and wherein the ligand comprises the following chemical structure:

[0157]

[0158] (NAG37)s, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0159] Embodiment 54: The use of Embodiment 53, wherein the dose is a single dose.

[0160] Embodiment 55: The use of embodiment 53, wherein the administration is repeated once, for example with an interval of about 28 days (i.e. 4 weeks), about 12 weeks or about 3 months between doses, for example about 100 mg every 4 weeks, or about 200 mg every 12 weeks, optionally for about 52 weeks or about 1 year. Embodiment 55a: The use of embodiment 53, wherein about 100 mg is administered every 4 weeks for 52 weeks. Embodiment 55b: The use of embodiment 53, wherein about 200 mg is administered every 12 weeks for 52 weeks.

[0161] Embodiment 56: The use of any one of Embodiments 53-55, wherein the dosage is from about 50 mg to about 100 mg, such as about 50 mg.

[0162] Embodiment 57: The use of any one of Embodiments 53-55, wherein the dosage is from about 100 mg to about 200 mg, such as about 100 mg.

[0163] Embodiment 58: The use of any one of Embodiments 53-55, wherein the dosage is from about 50 mg to about 200 mg, such as about 200 mg.

[0164] Embodiment 59: The use of any one of Embodiments 53-58, wherein the compound is in the form of a sodium salt.

[0165] Embodiment 60: The use according to any one of Embodiments 53-59, wherein the compound is administered by injection, for example at a concentration of 200 mg / ml.

[0166] Embodiment 61: The use of Embodiment 60, wherein the injection is subcutaneous.

[0167] Embodiment 62: The use of any one of Embodiments 53-61, wherein the compound is in unit dosage form.

[0168] Embodiment 63: The use of any one of Embodiments 53-62, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH), such as pre-cirrhotic non-alcoholic hepatitis, NASH-F3, NASH with bridging fibrosis, or biopsy-proven NASH.

[0169] Embodiment 64: The use of any one of Embodiments 53-63, wherein the treatment is for a subject having the HSD17β13rs72613567 mutation, the PNPLA3 rs738409(I148M) mutation, or a combination thereof.

[0170] Embodiment 65: The use of any one of embodiments 53-64, wherein the double-stranded oligonucleotide comprises an antisense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (5'→3'):

[0171] usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1);

[0172] usCfsasUfcUfaUfcAfgAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 5);

[0173] usGfsasUfcCfaAfaAfaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 6); or

[0174] usGfsasUfcCfaaaaaUfgUfcCfuAfgGfsc (nucleotide sequence SEQ ID NO: 7);

[0175] And wherein 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.

[0176] Embodiment 66: The use of any one of embodiments 53-65, wherein the double-stranded oligonucleotide comprises a sense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (5'→3'):

[0177] cguaagaaGfuCfuGfauagauga (nucleotide sequence SEQ ID NO: 3);

[0178] cguaagaaGfUfCfugauagauga (nucleotide sequence SEQ ID NO: 8);

[0179] gccuaggaCfAfUfuuuugiauca (nucleotide sequence SEQ ID NO: 9); or

[0180] gccuaggaCfaUfuUfuugiauca (nucleotide sequence SEQ ID NO: 10);

[0181] And wherein 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.

[0182] Embodiment 67: The use of any one of embodiments 53-66, wherein the double-stranded oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprises the sequence: usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1), and the sense strand comprises the sequence:

[0183] (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb)(nucleotide sequence

[0184] SEQ ID NO: 11),

[0185] And wherein 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; and (invAb) represents an inverted abasic deoxyribose residue.

[0186] Embodiment 68: The use of any one of embodiments 53-67, wherein the use reduces the expression level 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, e.g., at about day 71 or 113 from the first use, as measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample.

[0187] Embodiment 69: The use of any one of Embodiments 53-68, wherein the use reduces the 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, e.g., at about day 71 or 113 from the first use, as measured by Western blot using a liver biopsy sample.

[0188] Embodiment 70: The use of any one of Embodiments 53-69, 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, e.g., at about day 71 or 113 from the first use, compared to before use.

[0189] Embodiment 71: The use of any one of Embodiments 53-70, wherein the use reduces AST (aspartate aminotransferase) levels 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 serum, e.g., at about day 71 or 113 from the first use, compared to before use.

[0190] Embodiment 72: The use of any one of Embodiments 53-71, wherein the use reduces or slows the increase in liver fat fraction (e.g., by at least 4-41% or at least 7-8%) compared to before use, as measured by magnetic resonance imaging, e.g., at about day 71 or 113 from the first use.

[0191] Embodiment 73: The use of any one of Embodiments 53-72, wherein the use reduces or slows the increase in liver stiffness (e.g., by at least 4-37%), compared to before use, as measured by transient elastography, e.g., at about day 71 or 113 from the first use.

[0192] Embodiment 74: The use of any one of Embodiments 53-73, wherein the chemical structure of the ligand comprises:

[0193]

[0194] Embodiment 75: The use of Embodiment 74, wherein the compound is Figures 5A to 5D The chemical structures shown are of the free acid form.

[0195] Embodiment 76: The use of any one of Embodiments 53-73, wherein the chemical structure of the ligand comprises:

[0196]

[0197] Embodiment 77: The use of Embodiment 76, wherein the compound is Figures 6A-6D The chemical structure shown is the sodium salt form.

[0198] Embodiment 78: The use of any one of Embodiments 53-77, wherein the use reduces GGT (gamma-glutamyl transferase) levels by at least about 1.4% or at least 8.3% or at least 9% as measured in serum, e.g., on day 71 or 113 from the first use, compared to before use.

[0199] In some cases, for example, at the 52nd week after treatment begins, one or more human subjects treated with the methods described herein achieve an improvement of => 1 stage (i.e., equal to or greater than 1 stage) in tissue fibrosis without NASH exacerbation. In some cases, the improvement in tissue fibrosis is assessed with a Clinical Research Network (CRN) score. In some cases, no NASH exacerbation is defined as no increase in the NAFLD activity score (NAS) for fatty degeneration, ballooning, or inflammation.

[0200] In some cases, for example, at week 52 after initiation of treatment, one or more human subjects treated with the methods described herein achieve NASH regression without worsening fibrosis. In some cases, NASH regression is defined as a ballooning score of 0 and an inflammation score of 0-1. In some cases, no worsening of fibrosis is defined as no increase in the CRN fibrosis score.

[0201] In some cases, one or more human subjects treated with the methods described herein achieve a ≥30% relative reduction from baseline in liver fat at week 24 and week 52 following initiation of treatment, e.g., as measured by MRI-PDFF.

[0202] Example

[0203] Example 1. Synthesis and formulation of HSD17B13 RNAi agent

[0204] Compounds suitable for use as RNAi agents herein can be synthesized on solid phase oligonucleotide synthesis using standard phosphoramidite techniques known in the art. Commercially available oligonucleotide synthesizers (e.g. (Bioautomation) or (Bioautomation)). It can be made of controlled pore glass (CPG, or The synthesis is carried out on a solid support made from Prime Synthesis, Aston, PA, USA. The monomer at the 3' end of the corresponding chain can be attached to the solid support as the starting point of the synthesis. All 2'-modified RNA phosphoramidites and reverse abasic phosphoramidites are commercially available. Ligand-containing phosphoramidites suitable for addition to the 5' end of the sense strand can be synthesized. Standard cleavage, deprotection, purification and annealing steps as known in the art can be used. Further descriptions related to the synthesis of HSD17B13 RNAi agents can be found, for example, in International Patent Application Publication Nos. WO 2020 / 061177 (Patent Application No. PCT / US2019 / 051707) and WO

[0205] 2018 / 044350 (PCT / US2017 / 021147), the entire contents of each of which are incorporated herein by reference. The HSD17B13 RNAi agent can then be formulated by dissolving in a suitable pharmaceutically acceptable excipient.

[0206] Example 2. Phase 1 / 2a study evaluating the safety, tolerability, pharmacokinetic and pharmacodynamic effects of HSD17B13 RNAi agents in normal healthy volunteers and patients with NASH or NAFLD.

[0207] The test compounds exemplified herein reduce the expression of HSD17β13 messenger RNA (mRNA) in hepatocytes. Their sequences are shown in Table 4, and their physical properties are listed in Table 5:

[0208] Table 4. Test compounds

[0209]

[0210] Table 5. Properties of the test compounds

[0211] Chemical formula: <![CDATA[C 493 H 610 F 11 N 163 Na 43 O 311 P 43 S 7 (Na + form)]]> <![CDATA[C 493 H 653 F 11 N 163 O 311 P 43 S 7 (H + Form)]]> Molecular Weight: <![CDATA[16548.9Da(Na + Form)]]> <![CDATA[15309.7Da(H + Form)]]> Physical Appearance: White to off-white powder

[0212] The complete chemical structure is shown in Figures 5A to 5D (free acid form) and Figures 6A to 6D (sodium salt form).

[0213] This clinical study evaluated the safety, tolerability, and pharmacodynamics of the test compound in 32 normal healthy volunteers (NHV) and 18 patients with NASH or suspected NASH (e.g., nonalcoholic fatty liver disease [NAFLD] with elevated alanine aminotransferase [ALT]). The double-blind NHV cohort received a single dose of 25 mg, 50 mg, 100 mg, or 200 mg of the test compound or placebo subcutaneously (SC) on day 1. The open-label patient cohort received 25 mg, 100 mg, or 200 mg SC of the test compound on days 1 and 29. Liver biopsies were performed on patients before dosing and on day 71 to assess the expression levels of HSD17β13 mRNA and protein.

[0214] Treatment with the test compound was well tolerated in both NHVs and patients, with no treatment-related serious adverse events (SAEs) or drug discontinuations in the study. The most commonly reported treatment-emergent adverse events (TEAEs) were mild injection site reactions that were mild in severity and short in duration. Reductions in liver HSD17β13 mRNA were observed after doses of 25 mg (-57%), 100 mg (-86%), and 200 mg (-93%) on day 71. Liver HSD17β13 protein levels were also reduced at all dose levels. In patients, the mean maximum decreases in ALT and AST ranged from -42% to -44% and -24% to -28%, respectively, at doses ≥100 mg.

[0215] The test compound was well tolerated at doses up to 200 mg on day 1 (NHV) or day 1 and day 29 (NASH or NAFLD patients), with no treatment-related SAEs or drug discontinuations in the study. In NHV, the incidence of TEAEs was similar between the test compound and placebo groups and was generally consistent across patient groups. This proof-of-concept study showed that the test compound reduced HSD17β13 mRNA and protein in the liver, accompanied by reductions in ALT and AST.

[0216] method

[0217] Participants and study design

[0218] This is a multicenter Phase 1 / 2a clinical study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the test compound injection in normal healthy volunteers (NHV) and patients with NASH or suspected NASH (e.g., NAFLD with elevated alanine aminotransferase [ALT]). NAFLD criteria are 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 ULN = 19 U / L for women). The study included patients aged 18 to 55 years with a body mass index (BMI) < 35 kg / m2 The study was conducted in adult men and women aged 19 to 65 years with a BMI ≤ 40.0 kg / m2 (NHV) or NASH or NAFLD.

[0219] Genotyping was performed in the screening of patients with NASH or NAFLD to determine the mutation status of HSD17β13rs72613567 and patatin-like phospholipase domain-containing 3 (PNPLA3)rs738409(I148M) variant. Individuals were divided into T / T (homozygous wild type or no mutation) or T / TA (heterozygous mutation) groups according to HSD17β13rs726131567 genotype. Individuals who were 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 PNPLA3 genotype.

[0220] Subjects were enrolled in the study in a double-blind (NHV cohorts 1 [25 mg], 2 [50 mg], 3 [100 mg], 4 [200 mg]) or open-label (patient cohorts 1b [25 mg], 3b [100 mg], 4b [200 mg]) fashion (Table 6). NHV subjects were randomized in a 4:4 ratio (active:placebo) on day 1 to receive a single subcutaneous (SC) injection of test compound (25, 50, 100, or 200 mg) or placebo (cohorts 1 to 4). Patients with NASH or NAFLD received open-label test compound on days 1 and 29 (25, 100, or 200 mg for cohorts 1b, 3b, and 4b, respectively). The study designs are summarized in Figure 4 and Table 6.

[0221] Table 6: Queue summary

[0222]

[0223] NASH=nonalcoholic steatohepatitis; NHV=normal healthy volunteers.

[0224] For cohorts 1 to 4, test compounds or placebos were administered to 2 sentinel subjects (1 test compound, 1 placebo). After these subjects were evaluated on day 3, the remaining subjects in the cohort were treated, at least 30 minutes apart, so that no 2 subjects were administered at the same time. Cohorts 1 to 4 were included in sequence, and patient cohorts were open for inclusion in a step-by-step manner. Sentinel dosing was not required in patient cohorts. Table 6 summarizes the dose levels divided by cohort. NHVs stayed in the clinical institution for 3 days and dosed on day 1 (cohorts 1, 2, 3, and 4). NHVs returned to the clinical institution for outpatient visits. On dosing days (days 1 and 29), patients in cohorts 1b, 3b, and 4b stayed in the clinical institution for approximately 4 hours (2 hours before and 2 hours after dosing).

[0225] Study Treatment

[0226] Fifty subjects were enrolled (32 NHVs and 18 patients). Of the 58 NHVs screened, 8 failed screening (primarily due to non-compliance with inclusion or exclusion criteria) and 18 were not allocated (screening was delayed 45 days due to the COVID-19 global pandemic). For NHV Cohorts 1 to 4, 8 subjects (4 active: 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).

[0227] Of the 34 NASH or NAFLD patients screened, 16 failed screening (due to not meeting inclusion or exclusion criteria) (Table 6). For the 18 patients enrolled in open-label cohorts 1b, 3b, and 4b, 6 ​​patients were enrolled in each 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 up until day 113 (week 16).

[0228] Research Assessment and Procedures

[0229] In patients with NASH or NAFLD, the efficacy of the test compounds was measured by hepatic HSD17β13 mRNA (qRT-PCR) and protein (Western blot) expression extracted from paired liver biopsies at screening and day 71 to measure the activity of the test compounds on HSD17β3. For gene expression measurements by qRT-PCR, HSD17B13 and endogenous controls, β-actin (ACTB) and mRNA were measured at day 71 and in screening biopsies. Relative expression was calculated using the 2(-delta C[T]) normalization method. Changes in HSD17B13 protein expression were assessed as the ratio of HSD17B13 / vinculin at day 71 to pre-dose baseline levels. Markers of liver injury, such as ALT and AST, were measured from screening to day 113. Liver imaging was performed using MRI-PDFF at screening and day 71 to assess changes in liver fat fraction, and liver stiffness was assessed using transient elastography (FibroScan, kPa). HSD17B13 rs72613567 and patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 single nucleotide polymorphisms (SNPs) were detected in whole blood by real-time PCR, and the results were confirmed by Sanger sequencing at the time of screening.

[0230] Safety assessments included determining the incidence of adverse events (AEs) / serious adverse events (SAEs), performing physical examinations, and recording vital sign measurements (blood pressure, heart rate, temperature, and respiratory rate), electrocardiograms (ECGs), clinical laboratory tests (blood and urine), concomitant medications / treatments, and recording reasons for treatment discontinuations in both the NHV and patient cohorts.

[0231] Statistical analysis

[0232] All safety results, demographic and baseline characteristics data, and pharmacodynamic results were summarized 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).

[0233] In order to analyze the pharmacodynamic parameters, descriptive statistical summaries of HSD1713 mRNA and protein and knockdown data derived from baseline are provided. Nonparametric Wilcoxon signed rank test was performed to test the significance of the change from baseline on day 71. In addition, for liver enzyme parameters such as ALT and AST, descriptive statistical summaries, as well as changes derived from baseline and percentage changes with baseline summary are provided. Nonparametric Wilcoxon signed rank test was also performed to test the significance of ALT and AST changes from baseline on days 29, 71 and 113. For liver imaging (MRI-PDFF and FibroScan), descriptive statistical summaries and percentage changes derived from baseline are provided. Baseline values ​​are defined as the last non-missing observation (pre-dose value closest to the first dose) of each subject before the study drug is given (i.e., injection starts on day 1). Data were analyzed using SAS software version 9.4, and data were generated using GraphPad Prism software version 8.3.0.

[0234] Study Supervision

[0235] The ethics committee of each participating center approved the protocol. The study was conducted in accordance with the Declaration of Helsinki, the International Conference on Harmonisation Good Clinical Practice guidelines, and applicable regulatory requirements. All subjects provided written informed consent before inclusion.

[0236] result

[0237] Patient Characteristics and Disposition

[0238] The demographic characteristics of the healthy volunteers are shown in Table 7. All included NHVs completed all planned assessments.

[0239] Table 7: Summary of demographic data – healthy volunteer cohort

[0240]

[0241] BMI = body mass index.

[0242] Patients with NASH or NAFLD: The mean (SD) age was 44.5 (9.9) years. Four subjects (22.2%) were female, while 14 (77.8%) were male. Thirteen (72.2%) subjects were Asian, three (16.7%) subjects were white, one (5.6%) was African American, and one subject was other (5.6%). At baseline, MRI-PDFF ranged from 10.34% to 30.73%, while ALT ranged from 28 U / L (for female subjects) to 144 U / L. Suspected NASH (e.g., NAFLD with elevated ALT) was defined as MRI-PDFF>8% and ALT>ULN (ULN=30 U / L for men and 19 U / L for women. Alternatively, biopsy-proven NASH within 1 year after the screening visit could also be included.) These patients presented with typical NAFLD comorbidities, with 50% (n=9), 44.4% (n=8), and 44.4% (n=9).

[0243] (n=8) suffered from hyperlipidemia, type 2 diabetes and hypertension.

[0244] Table 8 provides a summary of the genotyping results of NAFLD patients divided by cohort. For the HSD17β13rs72613567 variant, 12 subjects (67%) had no detected mutation (T / T), while 6 subjects (33%) were heterozygous (T / TA). For the PNPLA3 rs738409 variant, 11 subjects (61%) had no detected mutation (C / C), 4 subjects (22%) were heterozygous (C / G), and 3 subjects (17%) were homozygous (G / G). In the clinical study, baseline concomitant medications taken by patients with NASH or NAFLD 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 premature termination.

[0245] Table 8: Summary of demographics and medical history of patients with NASH or NAFLD

[0246]

[0247]

[0248] ALT = alanine aminotransferase; AST = aspartate aminotransferase; BMI = body mass index; C / C = no mutation; C / G = heterozygous; G / G = homozygous; max = maximum; min = minimum; NASH = nonalcoholic steatohepatitis; SD = standard deviation; T / T = no mutation; T / TA = heterozygous.

[0249] a Codes were coded using the Medical Dictionary for Regulatory Activities version 23.0.

[0250] Pharmacodynamics

[0251] Hepatic HSD17B13 protein and mRNA

[0252] On Day 71, the test compound reduced liver mRNA encoding HSD17β13 protein by a mean (min-max) value of 56.9% (50.7%-60.5%), 85.5% (61.6%-96.1%), and 93.4% (90.8%-98.6%) from pre-treatment baseline in the 25 mg, 100 mg, and 200 mg dose cohorts, respectively. Figure 1 ). The overall pooled subject HSD17β13 mRNA decreased by 78.6% (50.7%-98.6%, p value <0.0001). In the 200 mg dose cohort, liver HSD17β13 mRNA expression in all 6 subjects decreased by greater than 90%. In the 25 mg, 100 mg, and 200 mg dose cohorts, liver HSD17β13 protein levels similarly decreased by an average of >34%, >86%, and 83%, respectively, while the average change from baseline in the combined cohort HSD17β3 protein was 63% (range -97.8% to 53.8%, p = 0.0017), and multiple measurements (3 patients at baseline and 6 patients on day 71) were all below the quantitative level of the assay (Table 9).

[0253] Table 9: Summary of HSD17β13 mRNA and protein knockdown by tested compounds in patients with NASH or NAFLD

[0254]

[0255] LLOQ = lower limit of quantitation; mRNA = messenger RNA; NASH = non-alcoholic steatohepatitis.

[0256] a Several patients had HSD17β13 protein levels below the LLOQ on day 71, in which case the LLOQ was used to calculate the mean.

[0257] b n=2 (3 samples had baseline HSD17β13 below LLOQ and 1 sample failed the assay acceptance criteria).

[0258] The observed data on HSD17B13 mRNA reductions indicated that a robust on-target pharmacological response was achieved in the liver, with a dose-dependent trend at the 100 and 200 mg dose levels and a clear dose-dependency at the 25 and 100 mg dose levels. The observed pharmacodynamic effects were not affected by HSD17β13 (rs72613567, T>TA) and / or PNPLA3 (rs738409, C>G) mutations. At each individual dose level, similar reductions in HSD17β13 mRNA were observed in patients without mutations compared to patients heterozygous for the rs72613567 splice variant ( Figure 3A At the same time, at each dose level, similar reductions in HSD17β13 mRNA were observed in patients without the mutation compared with patients who were heterozygous or homozygous for the PNPLA3rs738409 polymorphism ( Figure 3B ). Therefore, knockdown of HSD17β13 was consistent in this study regardless of genotype or mutation status.

[0259] ALT, AST, and GGT

[0260] In patients with NASH or NAFLD, serum biomarkers of liver injury decreased after treatment with the test compound, as indicated by a decrease in ALT between the 25 and 100 mg dose levels. The 100 and 200 mg dose levels also showed similar decreases in ALT. The mean percentage 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 the combined cohort) ( Figure 2A At day 113 or end of study, mean percent changes from baseline in ALT were -14%, -36%, and -39% for the 25, 100, and 200 mg dose levels, respectively (P < 0.01 for the combined cohort) ( Figure 2A ). Similar to ALT, AST decreased between the 25 to 100 mg dose levels, and similar decreases were observed between the 100 and 200 mg doses. The mean percentage changes from baseline in AST at day 71 were 4%, -24%, and -28% for the 25, 100, and 200 mg dose levels, respectively (P < 0.01 for the combined cohort) ( Figure 2B At Day 113, the mean percentage changes from baseline in AST were -7.5%, -14.7%, and -19.7% for the 25, 100, and 200 mg dose levels, respectively ( Figure 2B). Patients were given two doses of 25, 100, or 200 mg of the test compound on days 1 and 29, and safety and pharmacodynamic parameters were measured until the end of the study or day 113. For the 100 and 200 mg dose levels, the duration of ALT reduction continued until day 85, or 8 weeks after the last dose. For the 100 and 200 mg dose levels, the duration of AST reduction continued until day 71, or 6 weeks after dosing. ***P<0.001; **P<.01, *<.01 are p values ​​for the combined cohort (25, 100, and 200 mg dose levels at the specified time points using a nonparametric test (Wilcoxon signed-rank test)). The mean body weight in each dose group remained stable during the study. Two subjects lost weight during the study, showing the largest percentage decrease in ALT from baseline in their dose groups. By day 71, these subjects had lost 4.6 kg (25 mg dose group) and 2.2 kg (100 mg group) in body weight, respectively, with corresponding ALT reductions of 60% and 51%, respectively. All other subjects maintained a stable body weight throughout the study; therefore, all other decreases in liver enzymes were observed to be independent of changes in body weight. GGT levels in the 25 mg, 100 mg, and 250 mg patient cohorts were reduced by approximately 1.4%, 8.3%, and 9%, respectively, as measured in serum, compared to before administration.

[0261] Liver fat fraction and liver stiffness

[0262] The mean relative changes from baseline in liver fat fraction MRI-PDFF by day 71 were 14.4%, -7.6%, and -7.3% at the 25, 100, and 200 mg doses, respectively (Table 10).

[0263] Table 10: Summary of liver fat fraction (MRI-PDFF) and liver stiffness (FibroScan) in patients with NASH or NAFLD

[0264]

[0265] kPa = kilopascal; MRI-PDFF = magnetic resonance imaging proton density fat fraction

[0266] The mean percent change from baseline in liver stiffness (in kPa) on Day 71 was +16.7%, +2.2%, and +4.2% at the 25, 100, and 200 mg doses, respectively (Table 10).

[0267] Increases in liver stiffness were less pronounced and liver fat fraction decreased at both the 100 mg and 200 mg dose levels compared to the 25 mg dose level, as shown by mean changes from baseline at Day 71.

[0268] Security

[0269] The test compounds were well tolerated by NHV and NASH or NAFLD patients (Tables 11 and 12), with no AE-related study or drug interruptions, no dose-limiting poisonings or deaths. There was no recurring pattern of adverse experimental findings suggesting end-organ toxicity. The most commonly 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 the 16 NHVs who received the test compound, and there were no NASH or NAFLD patients. The remaining TEAEs were common TEAEs in clinical studies (Table 12). One treatment-emergent SAE of soft tissue injury was reported in the 200mg cohort, requiring hospitalization. The subject was hospitalized for soft tissue injury in the right arm after an accident at work (unrelated to the time and place of injection) and was discharged 2 days later. The subject recovered from the event and continued on the study. The SAE was considered unrelated to the study drug.

[0270] in conclusion

[0271] The test compound was well tolerated at doses up to 200 mg administered once (NHV) or twice (NASH or NAFLD patients), with no treatment-related serious adverse events, no drug-related pattern of adverse laboratory findings, and no drug discontinuations during the study. In NHV, the incidence of TEAEs was similar between the test compound and placebo groups and was generally consistent across patient cohorts. There was no mean increase in liver fat at all dose levels, and there were no adverse changes in biomarkers of liver damage. Reductions in liver HSD17β13 mRNA and protein were observed, corresponding to reductions in ALT and AST, a clinically meaningful signal of reduced liver damage. AST concentrations decreased by an average of 7.5%, 24.5%, and 28.3% at 25, 100, and 200 mg doses, respectively, and the 100 and 200 mg doses lasted for 12 weeks. Similarly, ALT levels were also reduced by 14.1%, 43.6%, and 42.3% at 25, 100, and 200 mg doses, respectively, and were sustained for 12 weeks in the groups administered 100 and 200 mg doses on days 1 and 29. The reductions in liver HSD17B13 mRNA and HSD17B13 protein indicate that the hit pharmacological response in the liver was robust at 100 and 200 mg doses. Therefore, the data at least supports a dosing regimen of 100 mg every 4 weeks (Q4W).

[0272] Pharmacokinetic / pharmacodynamic (PK / PD) modeling of HSD17β13 protein knockdown and ALT predicted that the 200 mg dose maintained reductions in HSD17β3 protein and ALT for 90 days, further supporting the testing of a 200 mg dosing regimen every 3 months or 12 weeks. Figure 7As shown in Figure 2, doses of 100 mg and 200 mg resulted in a >80% decrease in HSD17β13 protein from baseline for 3 months or longer. 1 / 2 It is 10.6 days, assuming complete knockdown within 30 days, for example, a reduction of about 87%, it is inferred that a dose of 200 mg can induce complete knockdown of HSD17β13 protein.

[0273] Table 11: Overall summary of adverse events (single-dose healthy volunteer cohort)

[0274]

[0275]

[0276] AE = adverse event, NA = not applicable; NASH = nonalcoholic steatohepatitis; TEAE = treatment-emergent adverse event, LISR = local injection site reaction, SAE = serious adverse event; TESAE = treatment-emergent serious adverse event.

[0277] Note: Categories are not mutually exclusive. n(%)# represents the number of subjects, percentage of subjects, and number of events in each category, respectively.

[0278] Table 12: Summary of TEAEs reported by preferred term in 2 or more subjects (NHV and NASH or NAFLD) in the cohort

[0279]

[0280]

[0281] NASH = nonalcoholic steatohepatitis; NHV = normal healthy volunteers; TEAE = treatment-emergent adverse event.

[0282] a All reported TEAEs were single-event terms for different system organ classes.

[0283] Note: Coding was performed using the Medical Dictionary for Regulatory Activities version 23.0. Subjects were counted once within each preferred term. Events were counted by the number of times they occurred. n(%) # represents the number of subjects, percentage of subjects, and number of events in each category, respectively.

[0284] Example 3. HSD17B13 minimization 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

[0285] This is a double-blind, randomized, placebo-controlled, Phase 2b multicenter study designed to evaluate the efficacy and safety of the test compound in adult patients with NASH and bridging (F3) fibrosis (Table 4).

[0286] The study includes 3 treatment arms, including 2 treatment arms that receive 2 different dosing schedules of the test compound and one placebo arm. After a screening period of up to 70 days, eligible participants are randomized to one of the 3 treatment arms:

[0287] 100 mg test compound once every 4 weeks (Q4W) for 52 weeks

[0288] 200 mg of test compound once every 12 weeks (Q12W) for 52 weeks, or

[0289] Placebo was administered every 4 weeks (Q4W) for 52 weeks.

[0290] Approximately 246 participants are planned to participate in the study. Approximately 82 participants in each treatment group will be enrolled in parallel in dose cohorts and randomized in a 1:1:1 ratio.

[0291] Inclusion criteria include but are not limited to

[0292] Body mass index (BMI) ≥ 25 kg / m 2 ) (all ethnic origins), except for Asian participants who had a BMI ≥ 23 kg / m at screening 2 Comply with research.

[0293] The researchers believe that NAFLD is the most likely cause of the liver disease, with features of metabolic syndrome.

[0294] ●Baseline liver biopsy showing a NAFLD Activity Score (NAS) >= 4, with at least 1 point each for steatosis, inflammation, and ballooning, and a CRN score for fibrosis of 3.

[0295] Able and willing to comply with all study assessments, including liver biopsy at Week 52.

[0296] Exclusion criteria include but are not limited to:

[0297] Cirrhosis (based on screening biopsy or historical biopsy showing definite cirrhosis).

[0298] Women currently drink ≥14 standard drinks (24 units, 196 g ethanol) per week, and men currently drink ≥21 standard drinks (37 units, 294 g ethanol) per week.

[0299] Bariatric surgery (including gastric banding and intragastric balloon insertion) within 2 years of screening1.

[0300] History of cancer within 2 years prior to screening1, excluding adequately resected non-melanoma skin cancer.

[0301] Table 13: Main outcome measures

[0302]

[0303] Secondary outcome measures included at least the percentage of participants achieving a ≥30% relative reduction from baseline in liver fat at Week 24 and Week 52 using magnetic resonance imaging such as MRI-PDFF, change from baseline in liver stiffness measurement (LSM) using 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 / L) [time frame: baseline (Day 1) and Weeks 24 and 52].

Claims

1. A method of treating non-alcoholic fatty liver disease in a human subject in need thereof, comprising administering a compound or a pharmaceutically acceptable salt thereof to the subject at a dose of about 25 to about 200 mg, wherein the dose is calculated based on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide linked to a ligand, wherein the double-stranded oligonucleotide reduces the expression of HSD17β13 in the human subject, and wherein the ligand comprises the following chemical structure: (NAG37)s, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the administration is a single dose.

3. The method of claim 1, wherein the administration is repeated with an interval of about 28 days, about 12 weeks, or about 3 months between doses, optionally for about 52 weeks or about 1 year.

4. The method of any one of claims 1-3, wherein the dose is from about 50 mg to about 100 mg.

5. The method of any one of claims 1-3, wherein the dose is about 100 mg to about 200 mg.

6. The method of any one of claims 1-3, wherein the dose is from about 50 mg to about 200 mg.

7. The method of any one of claims 1-6, wherein the compound is in the form of a sodium salt.

8. The method according to any one of claims 1-7, wherein the compound is administered by injection.

9. The method of claim 8, wherein the injection is subcutaneous.

10. The method of any one of claims 1-9, wherein the compound is present in a single unit dosage form.

11. The method according to any one of claims 1-10, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis (NASH).

12. The method of any one of claims 1-11, wherein the human subject has a HSD17β13rs72613567 mutation, a PNPLA3 rs738409(I148M) mutation, or a combination thereof.

13. The method according to any one of claims 1 to 12, wherein the double-stranded oligonucleotide comprises an antisense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (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); And wherein 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.

14. The method according to any one of claims 1 to 13, wherein the double-stranded oligonucleotide comprises a sense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the following sequences (5'v3'): 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); And wherein 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.

15. The method according to any one of claims 1 to 14, wherein the double-stranded oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprising the sequence (5'→3'): usCfsasUfcUfaucagAfcUfuCfuUfaCfsg (nucleotide sequence SEQ ID NO: 1), and the sense strand comprises the sequence (5'→3'): (NAG37)s(invAb)scguaagaaGfuCfuGfauagaugas(invAb) (nucleotide sequence SEQ ID NO:11), And wherein 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; and (invAb) represents an inverted abasic deoxyribose residue.

16. The method of any one of claims 1-15, wherein the method reduces the expression of HSD17β13 mRNA in the human subject by at least about 50% compared to before the administration, as measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using a liver biopsy sample from the human subject.

17. The method of any one of claims 1-16, wherein the method reduces expression of HSD17β13 protein in the human subject by at least about 30% compared to before the administration, as measured by Western blot using a liver biopsy sample from the human subject.

18. The method of any one of claims 1-17, wherein said method reduces ALT (alanine aminotransferase) levels of said human subject by at least about 40% compared to before said administration, as measured in the serum of said human subject.

19. The method of any one of claims 1-18, wherein said method reduces AST (aspartate aminotransferase) levels in said human subject by at least about 20% compared to before said administration, as measured in the serum of said human subject.

20. The method of any one of claims 1-19, wherein the method reduces or slows an increase in liver fat fraction in the human subject compared to before the administration, as measured by magnetic resonance imaging.

21. The method of any one of claims 1-20, wherein the method reduces or slows an increase in liver stiffness in the human subject compared to before the administration, as measured by transient elastography.

22. The method according to any one of claims 1-21, wherein the chemical structure of the ligand comprises:

23. The method of claim 22, wherein the compound is in the free acid form having the chemical structure as shown in Figures 5A to 5D.

24. The method according to any one of claims 1-21, wherein the chemical structure of the ligand comprises:

25. The method of claim 24, wherein the compound is in the form of a sodium salt having the chemical structure as shown in Figures 6A to 6D.

26. The method of any one of claims 1-3 and 7-25, wherein the dose is about 50 mg.

27. The method of any one of claims 1-3 and 7-25, wherein the dose is about 100 mg.

28. The method of any one of claims 1-3 and 7-25, wherein the dose is about 200 mg.

29. A compound or a pharmaceutically acceptable salt thereof for use in treating non-alcoholic fatty liver disease in a human subject, wherein the use comprises administering the compound or a pharmaceutically acceptable salt thereof to the subject at a dose of about 25 to about 200 mg, wherein the dose is calculated based on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide linked to a ligand, wherein the double-stranded oligonucleotide reduces the expression of HSD17β13 in the human subject, and wherein the ligand comprises the following chemical structure: (NAG37)s, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

30. The compound for use according to claim 29 or a pharmaceutically acceptable salt thereof, wherein the compound is in the form of a free acid having a chemical structure as shown in Figures 5A to 5D, or in the form of a sodium salt having a chemical structure as shown in Figures 6A to 6D.

31. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating non-alcoholic fatty liver disease, wherein the compound or a pharmaceutically acceptable salt thereof is used at a dose of about 25 to about 200 mg, wherein the dose is calculated based on the free acid form of the compound, wherein the compound comprises a double-stranded oligonucleotide linked to a ligand, wherein the double-stranded oligonucleotide reduces the expression of HSD17β13 in the human subject, and wherein the ligand comprises the following chemical structure: (NAG37)s, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

32. The use according to claim 31, wherein the compound is in the form of a free acid having a chemical structure as shown in Figures 5A to 5D, or in the form of a sodium salt having a chemical structure as shown in Figures 6A to 6D.

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