Combination Therapy for the Treatment of Liver Disease - Patent application

Abstract

Methods of treating liver disease in a subject are provided, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor. Medicaments and kits comprising i) an inhibitor of PNPLA3 expression and ii) an agonist of the glucagon receptor and / or the GLP-1 receptor are also provided.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on Jun. 4, 2021, is named 0098-0076PR1_SL.txt, and is 60,642 bytes in size.

[0002] The present disclosure provides a method of treating liver disease in a subject, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor. Also provided are medicaments and kits comprising i) an inhibitor of PNPLA3 expression and ii) an agonist of the glucagon receptor and / or the GLP-1 receptor. [Background technology]

[0003] The incidence of liver disease is increasing worldwide, especially in Western countries. The most common is nonalcoholic fatty liver disease (NAFLD). NAFLD encompasses a variety of liver diseases ranging from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is defined as the accumulation of more than 5% fat by weight in the liver in the absence of significant alcohol intake, steatogenic medication, or genetic disorders (Non-Patent Document 1).

[0004] Nonalcoholic steatohepatitis (NASH) is a NAFLD with signs of inflammation and liver damage. NASH is histologically defined by macrovesicular steatosis, ballooning of hepatocytes, and lobular inflammatory infiltration (Non-Patent Document 2). NASH is estimated to affect 2-3% of the general population. In the presence of other pathologies such as obesity or diabetes, the estimated prevalence increases to 7% and 62%, respectively (Non-Patent Document 3).

[0005] PNPLA3 is a 481 amino acid member of the patatin-like phospholipase domain-containing family expressed in the ER and lipid droplets. PNPLA3 is highly expressed in the liver in humans, but its expression in adipose tissue is five times lower (Non-Patent Document 4).

[0006] Glucagon and glucagon-like peptide-1 (GLP-1) are derived from a 158 amino acid precursor polypeptide, preproglucagon, which is proteolytically processed in various tissues to form several different proglucagon-derived peptides, such as glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM), which are involved in diverse physiological functions such as glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the control of food intake. Glucagon is a 29 amino acid peptide corresponding to amino acids 33-61 of proglucagon (53-81 of preproglucagon), whereas GLP-1 is generated as a 37 amino acid peptide corresponding to amino acids 72-108 of proglucagon (92-128 of preproglucagon). GLP-1(7-36)amide or GLP-1(7-37)acid are biologically active forms of GLP-1 that exhibit essentially equivalent activity at the GLP-1 receptor.

[0007] Glucagon is produced by pancreatic islets and activates the glucagon receptor ("GCGR"). Glucagon acts in the liver to increase blood glucose through gluconeogenesis and glycogenolysis. When blood glucose begins to fall, glucagon signals the liver to break down glycogen to release glucose, and stimulates glucose production, raising blood glucose levels to normal levels. Glucagon is also known to increase energy expenditure, increase the production of ketone bodies, inhibit lipogenesis, promote the oxidation of fatty acids, delay gastric emptying, and suppress appetite (Non-Patent Document 5) (Non-Patent Document 6).

[0008] GLP-1 has different biological activities compared to glucagon. GLP-1 is secreted from gut L cells and binds to the GLP-1 receptor. The activities of GLP-1 include enhancing insulin secretion through the incretin effect, inhibiting glucagon secretion, and inhibiting food intake. Both glucagon and GLP-1 act as agonists at their respective receptors and have been shown to be effective in weight loss. Certain GLP-1 analogs are marketed or in development for the treatment of obesity, including liraglutide (VICTOZA®, Novo Nordisk) and exenatide (Byetta®, AstraZeneca AB). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Kotronen et al,Arterioscler Thromb.Vasc.Biol.2008,28:27-38 [Non-Patent Document 2] Sanyal,Hepatol.Res.2011.41:670-4 [Non-Patent Document 3] Hashimoto et al,J.Gastroenterol.2011.46(1):63-69 [Non-Patent Document 4] Huang et al,Proc.Natl.Acad.Sci.USA 2010.107:7892-7 [Non-Patent Document 5] Mueller et al,Proc.Intl.Journal of Molecular Sciences 2020.21(2):383 [Non-Patent Document 6] Boland et al., Nat Metab., 2020.2(5):413-431 Summary of the Invention [Means for solving the problem]

[0010] The present disclosure is directed to a method of treating liver disease in a subject, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor.

[0011] In some embodiments, the inhibitor of PNPLA3 expression is an antisense oligonucleotide complementary to a region of a nucleic acid encoding PNPLA3. In some embodiments, the antisense oligonucleotide is complementary to a site within nucleotides 5567-5731 of a nucleic acid encoding PNPLA3. In some embodiments, the antisense oligonucleotide is complementary to a site within nucleotides 5644-5731 of a nucleic acid encoding PNPLA3. In some embodiments, the antisense oligonucleotide is complementary to a site within nucleotides 5567-5642 of a nucleic acid encoding PNPLA3. In some embodiments, the antisense oligonucleotide is complementary to a site within nucleotides 5567-5620 of a nucleic acid encoding PNPLA3. In some embodiments, the nucleic acid encoding PNPLA3 is an mRNA. In some embodiments, the antisense oligonucleotide is 12-30 nucleosides in length. In some embodiments, the antisense oligonucleotide is 16-30 nucleosides in length.

[0012] In some embodiments, the antisense oligonucleotide comprises one or more modified sugar moieties. In some embodiments, the one or more modified sugar moieties are 2'-deoxy, 2'-O-methyl, 2'-O-methoxymethyl, 2'-O-methoxyethyl, 2'-fluoro, 4'-CH(CH3)-O-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', or combinations thereof. In some embodiments, the antisense oligonucleotide comprises one or more modified bases. In some embodiments, the one or more modified bases are 5-methylcytosine. In some embodiments, all cytosines in the antisense oligonucleotide are 5'methylcytosine. In some embodiments, the antisense oligonucleotide comprises one or more non-natural internucleoside linkages. In some embodiments, one or more internucleoside linkages are phosphorothioate linkages. In some embodiments, all internucleoside linkages are phosphorothioate linkages.

[0013] In some embodiments, the antisense oligonucleotide comprises a sequence having at least 8 contiguous bases of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the antisense oligonucleotide comprises one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the antisense oligonucleotide comprises a) a gap segment of 10 linked deoxynucleosides; b) a 5' wing segment of three linked nucleosides; and c) a 3' wing segment of three linked nucleosides, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a constrained ethyl sugar, each internucleoside linkage is a phosphorothioate linkage, and each cytosine is a 5-methylcytosine.

[0014] In some embodiments, the inhibitor of PNPLA3 expression further comprises a conjugate group.

[0015] In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is a peptide. In some embodiments, the peptide has the amino acid sequence: HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30 (sequence number 25) wherein (1) X2 is S, X10 is Y, X12 is K, X13 is K, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is V, X28 is A, and X30 is G (SEQ ID NO: 14); (2) X2 is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 15); (3) X2 is S, X10 is K, X12 is K, X13 is Y, X15 is E, X16 is G, X17 is Q, X18 is A, X20 is K, X21 is E, X23 is I, X24 is A, X27 is E, X28 is K, and X30 is R (SEQ ID NO: 20); (4) X2 is S, X10 is K, X12 is S, X13 is Y, X15 is D, X16 is S, X17 is R, X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 18); (5) X2 is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 33); or (6) X2 is S, X10 is K, X12 is S, X13 is Y, X15 is D, X16 is S, X17 is R, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (sequence number 19).

[0016] In some embodiments, the peptide comprises the amino acid sequence HSQGTFTSDKSEYLDSERARDFVAWLEAGG (SEQ ID NO:33).

[0017] In some embodiments, the peptide further comprises a modification to an amino acid in the amino acid sequence. In some embodiments, the modification is the addition of an acyl moiety. In some embodiments, the modification is a palmitoyl moiety on the N(ε) group of a lysine residue. In some embodiments, the palmitoyl group is attached to the lysine via a linker. In some embodiments, the linker is gamma glutamic acid. In at least one embodiment, the peptide is HSQGTFTSDKSEYLDSERARDFVAWLEAGG (SEQ ID NO: 33), where the lysine is modified with a palmitoyl moiety via a glutamic acid linker.

[0018] In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered simultaneously. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 1 hour of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 24 hours of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 72 hours of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 1 week of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 2 weeks of each other. In some embodiments, the inhibitor of PNPLA3 expression is administered parenterally. In some embodiments, the inhibitor of PNPLA3 expression is administered once a day, twice a day, or three times a day. In some embodiments, the inhibitor of PNPLA3 expression is administered once a week, twice a week, or three times a week. In some embodiments, the inhibitor of PNPLA3 expression is administered once a month, twice a month, or three times a month.

[0019] In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered parenterally. In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered once a day, twice a day, or three times a day. In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered once a week, twice a week, or three times a week. In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered once a month, twice a month, or three times a month.

[0020] In some embodiments, the subject is obese and / or has type 2 diabetes. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the liver disease is non-alcoholic steatohepatitis (NASH). In some embodiments, the liver disease is liver fibrosis and / or cirrhosis.

[0021] In some embodiments, the present disclosure is directed to a method of reducing hepatic steatosis in the liver of a subject having liver disease, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor.

[0022] In some embodiments, the total fatty liver of the subject is reduced compared to the total fatty liver of the subject when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone. In some embodiments, the total fatty liver of the subject is reduced by at least 30% compared to the total fatty liver of the subject when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone. In some embodiments, the total fatty liver of the subject is reduced by at least 30% compared to the total fatty liver of the subject when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the liver disease is non-alcoholic steatohepatitis. In some embodiments, the liver disease is liver fibrosis.

[0023] In some embodiments, the present disclosure provides a method for reducing inflammation in the liver of a subject with non-alcoholic fatty liver disease (NAFLD), comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of glucagon receptor and / or glucagon-like peptide-1 (GLP-1) receptor. In some embodiments, the inflammation in the liver is reduced by at least 50% compared to the inflammation in the liver when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone.

[0024] In some embodiments, the present disclosure provides a method for reducing hepatic collagen in a subject with liver disease, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of glucagon receptor and / or glucagon-like peptide-1 (GLP-1) receptor. In some embodiments, the hepatic collagen in the subject is reduced by at least 25% compared to the hepatic collagen when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone.

[0025] In some embodiments, the present disclosure provides a pharma- ceutically acceptable composition comprising: i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression; ii) an agonist of glucagon receptor and / or glucagon-like peptide-1 (GLP-1) receptor; and iii) at least one pharma- ceutically acceptable excipient. In some embodiments, the composition is formulated for parenteral administration.

[0026] In some embodiments, the present disclosure provides a kit comprising i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor.

[0027] The following drawings form part of the present specification and are included to further demonstrate illustrative embodiments of certain aspects of the present disclosure. [Brief description of the drawings]

[0028] [Figure 1A-B] Figure 1A shows a plot of the percentage body weight change for homozygous Pnpla3148M / M knock-in mice fed a NASH-inducing diet for 36 weeks and treated for 14 weeks with either 1) control ASO + saline, 2) Pnpla3 ASO + saline, 3) control ASO + Cotadutide, or 4) Pnpla3 ASO + Cotadutide, as described in Example 1. Figure 1B shows a plot of liver mPnpla3 mRNA concentrations for the same mice, measured as described in Example 1. [Figure 2A-D] Figure 2 shows plots of total hepatic steatosis (Figure 2A), macrodroplet steatosis (Figure 2B), and microdroplet steatosis (Figure 2C) measured from stained liver sections taken from the mice described above with respect to Figure 1A and Example 1. Figure 2D shows images of the stained sections along with the percentage of total lipid droplets per area for each section. [Figure 3A-B] Figure 3A shows a plot of the percentage of liver macrophages measured on liver sections taken from the mice described above with reference to Figure 1A and Example 1. Figure 3B shows a plot of liver inflammation scores obtained from the mice described above with reference to Figure 1A and Example 1. [Figure 4] 1A and 1B show plots of NAFLD Activity Score (NAS), calculated as described in Example 1, for the mice described above in relation to Example 1. [Diagram 5] 1 shows a plot of the percentage of hepatic collagen A1A in liver sections taken from the mice described above with respect to FIG. 1A and Example 1. Liver collagen is measured as described in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present disclosure provides a method for treating liver disease (e.g., NASH and / or NAFLD).In some embodiments, the present disclosure provides a method for treating liver disease in a subject, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of glucagon receptor and / or glucagon-like peptide-1 (GLP-1) receptor.

[0030] definition Unless otherwise indicated, the following terms have the following meanings.

[0031] Throughout this specification, the term "about" is used to indicate that a value includes the variation of error inherent in the method / device used to determine the value or includes the variation that exists between test subjects. Typically, the term "about" means to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% variability, or greater, depending on the context. In embodiments, a person skilled in the art will understand the level of variation indicated by the term "about" due to the context in which it is used herein. It should also be understood that the use of the term "about" includes the specifically recited value.

[0032] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or unless the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0033] As used herein, "to" refers to a range that includes both ends of the range. For example, a number from x to y explicitly includes the numbers x and y, as well as any numbers that fall within the range between x and y.

[0034] "2'-deoxynucleoside" means a nucleoside containing a 2'-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, 2'-deoxynucleosides may contain modified nucleobases or may contain RNA nucleobases (uracil).

[0035] "2'-O-Methoxyethyl" (also referred to as 2'-MOE) refers to a 2'-O(CH2)2--OCH3) in place of the 2'--OH group on the ribosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0036] "2'-MOE nucleoside" (also referred to as 2'-O-methoxyethyl nucleoside) means a nucleoside that includes a 2'-MOE modified sugar moiety.

[0037] "2'-substituted nucleoside" or "2-modified nucleoside" means a nucleoside that includes a 2'-substituted or 2'-modified sugar moiety. As used herein, "2'-substituted" or "2-modified" with respect to a sugar moiety means a sugar moiety that includes at least one 2'-substituent group other than H or OH.

[0038] "3' target site" refers to the nucleotide of a target nucleic acid that is complementary to the 3'-most nucleotide of a particular compound.

[0039] "5' target site" refers to the nucleotide of a target nucleic acid that is complementary to the 5'-most nucleotide of a particular compound.

[0040] "5-methylcytosine" means a cytosine with a methyl group attached to the 5 position.

[0041] "Administration" or "administering" refers to a route by which a compound or composition provided herein is introduced into an individual to perform its intended function. One example of an administration route that may be used includes, but is not limited to, parenteral administration (e.g., subcutaneous, intravenous, or intramuscular injection or infusion).

[0042] "Concurrent administration" or "co-administration" means administering two or more compounds in any manner that produces the pharmacological effects of both in a patient. Concurrent administration does not require that both compounds be administered in a single pharmaceutical composition, in the same dosage form, by the same route of administration, or at the same time. The effects of both compounds need not be simultaneous. The effects need only overlap over a period of time, not necessarily over the same time. Concurrent administration or co-administration includes parallel or sequential administration.

[0043] "Amelioration" refers to the improvement or alleviation of at least one indicator, symptom, or symptom of the associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression or severity of one or more indicators of a condition or disease. The progression or severity of the indicator may be determined by objective or subjective measures known to those of skill in the art.

[0044] "Animal" refers to humans or non-human animals (such as, but not limited to, mice, rats, rabbits, dogs, cats, pigs), and non-human primates (such as, but not limited to, monkeys and chimpanzees).

[0045] "Antisense compound" refers to a compound that includes an oligonucleotide and, optionally, one or more additional mechanisms (e.g., a conjugate group or a terminal group). Examples of antisense compounds include single-stranded and double-stranded compounds, such as oligonucleotides, ribozymes, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

[0046] "Antisense oligonucleotide" means an oligonucleotide having a nucleobase sequence complementary to a target nucleic acid, or a region or segment thereof. In certain embodiments, an antisense oligonucleotide is capable of specifically hybridizing to a target nucleic acid, or a region or segment thereof.

[0047] "cEt" or "constrained ethyl" means a ribosyl bicyclic sugar moiety where the second ring of the bicyclic sugar is formed via a bridge connecting the 4'-carbon and the 2'-carbon, the bridge having the formula: 4'-CH(CH3)-O-2', and the methyl group of the bridge is in the S configuration.

[0048] "cEt nucleoside" means a nucleoside that includes a cEt modified sugar moiety.

[0049] A "chemical modification" in a compound describes the replacement or change by chemical reaction of any such unit compared to the original state of such unit in the compound. A "modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and / or a modified nucleobase. A "modified oligonucleotide" means an oligonucleotide containing at least one modified internucleoside linkage, modified sugar, and / or modified nucleobase.

[0050] A "chemically distinct region" refers to a region of a compound that is in some way chemically different from another region of the same compound. For example, a region having 2'-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without the 2'-O-methoxyethyl modification.

[0051] "Chimeric antisense compound" means an antisense compound having at least two chemically distinct regions, where each position has multiple subunits.

[0052] "Conjugate group" means a group of atoms attached to an oligonucleotide. A conjugate group includes a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

[0053] "Conjugate linker" means a group of atoms containing at least one bond that connects a conjugate moiety to an oligonucleotide.

[0054] "Conjugate moiety" means a group of atoms attached to an oligonucleotide via a conjugate linker.

[0055] "Contiguous" with respect to oligonucleotides refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to one another. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to one another in the sequence.

[0056] "Dose" refers to a specific amount of a compound or pharmaceutical agent provided in a single administration or provided over a specific period of time. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose may require a volume that is not easily accommodated in a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reactions in an individual. In other embodiments, the compound or pharmaceutical agent is administered by infusion over an extended period of time or continuously. A dose may be given as the amount of pharmaceutical agent per hour, day, week, or month.

[0057] A "dosage regimen" is a combination of doses designed to achieve one or more desired effects.

[0058] "Effective amount" means an amount of a compound sufficient to achieve a desired physiological result in an individual in need of the compound. The effective amount may vary from individual to individual, depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, an evaluation of the individual's medical condition, and other relevant factors.

[0059] "Gapmer" means an oligonucleotide comprising an internal region having multiple nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings."

[0060] "Directly adjacent" means that there are no intervening elements between elements of the same type that are directly adjacent (eg, there are no intervening nucleobases between directly adjacent nucleobases).

[0061] "Internucleoside linkage" means a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. "Modified internucleoside linkage" means any internucleoside linkage other than the naturally occurring phosphate internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages.

[0062] "Linker nucleoside" refers to a nucleoside that connects an oligonucleotide to a conjugate moiety. The linker nucleoside is located within the conjugate linker of the compound. The linker nucleoside is not considered part of the oligonucleotide portion of the compound, even if it is contiguous to the oligonucleotide.

[0063] "Mismatch" or "non-complementary" refers to the nucleobase of the first oligonucleotide that is not complementary to the corresponding nucleobase of the second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned.For example, a nucleobase (such as, but not limited to, universal nucleobase, inosine, and hypoxanthine) can hybridize with at least one nucleobase, but is still mismatched or non-complementary to the nucleobase with which it is hybridized.As another example, the nucleobase of the first oligonucleotide that cannot hybridize to the corresponding nucleobase of the second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned is a mismatched nucleobase or a non-complementary nucleobase.

[0064] "Modulating" refers to changing or adjusting the mechanism in a cell, tissue, organ, or organism. For example, modulating PNPLA3 RNA can mean increasing or decreasing the level of PNPLA3 RNA and / or PNPLA3 protein in a cell, tissue, organ, or organism. A "modulator" is something that brings about a change in a cell, tissue, organ, or organism. For example, a PNPLA3 compound can be a modulator that reduces the amount of PNPLA3 RNA and / or PNPLA3 protein in a cell, tissue, organ, or organism.

[0065] "MOE" means methoxyethyl.

[0066] By "non-bicyclic modified sugar" or "non-bicyclic modified sugar moiety" is meant a modified sugar moiety that includes modifications such as substituents that do not form a bridge between two atoms of the sugar to form a second ring.

[0067] "Oligomeric compound" means a compound comprising a single oligonucleotide and, optionally, one or more additional features (eg, a conjugate group or a terminal group).

[0068] "Oligonucleotide" means a polymer of linked nucleosides, each of which may be modified or unmodified, independently of the others. Unless otherwise indicated, an oligonucleotide consists of 8 to 80 linked nucleosides. "Modified oligonucleotide" means an oligonucleotide in which at least one sugar, nucleobase, or internucleoside linkage is modified. "Unmodified oligonucleotide" means an oligonucleotide that does not contain any sugar modification, nucleobase modification, or internucleoside modification.

[0069] "Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.

[0070] "Portion" refers to a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound.

[0071] A "RefSeq number" is a unique combination of letters and numbers assigned to a sequence to indicate that the sequence is for a particular target transcript (e.g., a target gene). Such sequences and information (collectively, a gene record) for a target gene can be found in gene sequence databases, including the NCBI Reference Sequence database, GenBank, the European Nucleotide Archive, and the Japan DNA Data Bank (the latter three form the International Nucleotide Sequence Database Collaboration, or INSDC).

[0072] "RNAi compound" means an antisense compound that acts at least in part through RISC or Ago2, but not through RNase H, to modulate a target nucleic acid and / or a protein encoded by the target nucleic acid. RNAi compounds include, but are not limited to, double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.

[0073] "Sugar moiety" refers to an unmodified sugar moiety or a modified sugar moiety. "Unmodified sugar moiety" or "unmodified sugar" refers to a 2'-OH(H) ribosyl moiety as found in RNA (an "unmodified RNA sugar moiety"), or a 2'-H(H) moiety as found in DNA (an "unmodified DNA sugar moiety"). "Modified sugar moiety" or "modified sugar" refers to a modified furanosyl sugar moiety or sugar surrogate. "Modified furanosyl sugar moiety" refers to a furanosyl sugar that contains a non-hydrogen substituent in place of at least one hydrogen or hydroxyl group of the unmodified sugar moiety. In certain embodiments, the modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic and non-bicyclic sugars.

[0074] "Sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group within an oligonucleotide (e.g., an internucleoside linkage, a conjugate group, or a terminal group). Modified nucleosides containing sugar surrogates can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides can hybridize to complementary compounds or nucleic acids.

[0075] "Therapeutically effective amount" means an amount of a compound, pharmaceutical agent, or composition that confers a therapeutic benefit to an individual.

[0076] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" and multiple "polypeptides" and to include any single or multiple chains of two or more amino acids. Thus, as used herein, a "peptide", a "peptide subunit", a "protein", an "amino acid chain", an "amino acid sequence", or any other term used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide", even though each of these terms may have a more specific meaning. The term "polypeptide" may be used in place of or interchangeably with any of these terms. The term further includes polypeptides that have undergone post-translational or post-synthetic modifications (e.g., glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids).

[0077] The term "sequence identity" as used herein refers to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. If a position in one sequence has the same nucleic acid base or amino acid as the corresponding position in the comparison sequence, the sequences are said to be "identical" at that position. The percentage of "sequence identity" is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid is present to obtain the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window, and this value is multiplied by 100 to obtain the percentage of "sequence identity". The percentage of "sequence identity" is determined by comparing two optimally aligned sequences over the comparison window. To optimally align sequences for comparison, portions of the polynucleotide or polypeptide sequences in the comparison window may contain additions or deletions, called gaps, while the reference sequence is kept constant. An optimal alignment is one that maximizes the number of "identical" positions between the reference and comparison sequences as much as possible, even if there are gaps. The percentage of "sequence identity" between two sequences may be determined using the version of the program "BLAST 2 Sequences" available from the National Center for Biotechnology Information (as of September 1, 2004), which includes the programs BLASTN (for comparison of nucleotide sequences) and BLASTP (for comparison of polypeptide sequences), which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST 2 Sequences", parameters that were the default parameters as of September 1, 2004, may be used for word length (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expectation value (10), and any other required parameters (such as, but not limited to, matrix options).

[0078] PNPLA3 In some embodiments, the disclosure provides a method of treating liver disease by administering a PNPLA3 inhibitor. PNPLA3 is a 481 amino acid member of the patatin-like phospholipase domain-containing family expressed in the ER and lipid droplets. PNPLA3 is highly expressed in the liver in humans, but is 5-fold less expressed in adipose tissue (Huang et al, Proc. Natl. Acad. Sci. USA 2010.107:7892-7). In some embodiments, PNPLA3 refers to SEQ ID NO: 1. "PNPLA3" refers to any nucleic acid or protein of PNPLA3. "PNPLA3 nucleic acid" refers to any nucleic acid encoding PNPLA3. For example, in certain embodiments, PNPLA3 nucleic acid includes DNA sequences encoding PNPLA3, RNA sequences transcribed from DNA encoding PNPLA3 (e.g., genomic DNA including introns and exons), and mRNA sequences encoding PNPLA3. "PNPLA3 mRNA" refers to mRNA encoding PNPLA3 protein. Targets may be shown in either upper or lower case.

[0079] In some embodiments, the present disclosure provides methods, compounds, and compositions for inhibiting PNPLA3 (PNPLA3) expression in combination with agonists of glucagon receptor and / or GLP-1 receptor for the treatment of liver disease. Certain embodiments provided herein relate to a method of treating liver disease by administering an inhibitor of PNPLA3. "PNPLA3 specific inhibitor" can refer to any agent that can specifically inhibit the expression or activity of PNPLA3 RNA and / or PNPLA3 protein at the molecular level. For example, PNPLA3 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents that can inhibit the expression of PNPLA3 RNA and / or PNPLA3 protein.

[0080] The terms "inhibitor of PNPLA3", "PNPLA3 inhibitor" and "inhibitor of PNPLA3 expression" are used interchangeably herein. Inhibiting PNPLA3 expression may be useful for treating, preventing or ameliorating PNPLA3-related diseases in individuals by administering a compound that targets PNPLA3. In certain embodiments, the PNPLA3 inhibitor may be a PNPLA3-specific inhibitor. In certain embodiments, the PNPLA3 inhibitor may be an antisense compound, an oligomeric compound, or an oligonucleotide that targets PNPLA3. In some embodiments, PNPLA3 is an antisense oligonucleotide. In some embodiments, the oligonucleotide is an siRNA, a microRNA-targeting oligonucleotide, or a single-stranded RNAi compound, such as a small hairpin RNA (shRNA), a single-stranded siRNA (ssRNA), and a microRNA mimic.

[0081] In some embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide targeting the PNPLA3 nucleic acid. In certain embodiments, the PNPLA3 nucleic acid has a sequence described in U.S. Pat. No. 10,774,333 (incorporated by reference), such as RefSeq or GENBANK Accession No. NM_025225.2; NC_000022.11 (SEQ ID NO: 2) with nucleotides 43921001-43,954,500 truncated; AK123806.1; BQ686328.1; BF762711.1; DA290491.1; and the sequences listed as "SEQ ID NOs: 7, 8, 9, and 10" in U.S. Pat. No. 10,774,333. In certain embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide or an oligomeric compound. In certain embodiments, the PNPLA3 inhibitor is single-stranded. In certain embodiments, the PNPLA3 inhibitor is double-stranded.

[0082] In certain embodiments, the PNPLA3 inhibitor comprises a modified oligonucleotide 16 linked nucleosides in length. In certain embodiments, the PNPLA3 inhibitor is an antisense compound or an oligomeric compound.

[0083] In some embodiments, the PNPLA3 inhibitor is a modified oligonucleotide having a length of 12-30 linked nucleosides and a nucleobase sequence including any of the nucleobase sequences described in U.S. Pat. No. 10,774,333 (herein incorporated by reference) (e.g., any one of SEQ ID NOs: 17-2169 in U.S. Pat. No. 10,774,333). In certain embodiments, the PNPLA3 inhibitor is an antisense compound or an oligomeric compound. In certain embodiments, the PNPLA3 inhibitor is single-stranded. In certain embodiments, the PNPLA3 inhibitor is double-stranded. In certain embodiments, the PNPLA3 inhibitor is a modified oligonucleotide having a length of 16-30 linked nucleosides.

[0084] In some embodiments, the PNPLA3 inhibitor comprises a modified oligonucleotide consisting of any of the nucleobase sequences found in U.S. Patent No. 10,774,333 (herein incorporated by reference) (e.g., any one of SEQ ID NOs: 17-2169 in U.S. Patent No. 10,774,333). In certain embodiments, the PNPLA3 inhibitor is an antisense compound or an oligomeric compound. In certain embodiments, the PNPLA3 inhibitor is single-stranded. In certain embodiments, the PNPLA3 inhibitor is double-stranded.

[0085] In some embodiments, the PNPLA3 inhibitor comprises a modified oligonucleotide that is 12-30 linked nucleosides in length and complementary within the nucleobases found in U.S. Patent No. 10,774,333 (hereby incorporated by reference) (e.g., nucleobases 5567-5642, 5644-5731, 5567-5731, 5567-5620, 13697-13733, 20553-20676, 20664-20824, 20553-20824, and 25844-25912 of SEQ ID NO:1), wherein the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO:1. In certain embodiments, the PNPLA3 inhibitor is an antisense compound or an oligomeric compound. In certain embodiments, the PNPLA3 inhibitor is single stranded. In certain embodiments, the PNPLA3 inhibitor is double stranded. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.

[0086] In some embodiments, the inhibitor of PNPLA3 expression is an antisense oligonucleotide complementary to a region of the nucleic acid encoding PNPLA. In certain embodiments, the PNPLA3 inhibitor targets nucleotides 5567-5620 of the PNPLA3 nucleic acid. In certain embodiments, the PNPLA3 inhibitor targets within nucleotides found within nucleotides 5567-5642, 5644-5731, 5567-5731, 5567-5620 of the PNPLA3 nucleic acid having the nucleobase sequence of SEQ ID NO:1. In certain embodiments, the PNPLA3 inhibitor is complementary to a site within 5567-5731 of the nucleic acid sequence encoding PNPLA3 (e.g., SEQ ID NO:1). In certain embodiments, the PNPLA3 inhibitor is complementary to a site within 5644-5731 of the nucleic acid sequence encoding PNPLA3 (e.g., SEQ ID NO:1). In certain embodiments, the PNPLA3 inhibitor is complementary to a site within 5567-5642 of a nucleic acid sequence encoding PNPLA3 (e.g., SEQ ID NO:1). In certain embodiments, the PNPLA3 inhibitor is complementary to a site within 5567-5620 of a nucleic acid sequence encoding PNPLA3 (e.g., SEQ ID NO:1). In certain embodiments, the compounds are antisense compounds, oligomeric compounds, or oligonucleotides. In some embodiments, the nucleic acid encoding PNPLA3 is mRNA.

[0087] In certain embodiments, the PNPLA3 inhibitor comprises a modified oligonucleotide having a length of 12-30 linked nucleosides. In certain embodiments, the PNPLA3 inhibitor comprises a modified oligonucleotide having a length of 16-30 linked nucleosides. In certain embodiments, the PNPLA3 inhibitor comprises a modified oligonucleotide having a length of 12-30 linked nucleosides and a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleobase portions of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the PNPLA3 inhibitor comprises an antisense oligonucleotide comprising at least 8 consecutive nucleobases of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the modified oligonucleotide is 16-30 linked nucleosides in length.

[0088] In certain embodiments, the PNPLA3 inhibitor comprises a modified oligonucleotide having a nucleobase sequence that is 12-30 linked nucleosides in length and includes any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the modified oligonucleotide is 16-30 linked nucleosides in length. In certain embodiments, the PNPLA3 inhibitor comprises an antisense oligonucleotide comprising a sequence having at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive bases of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the PNPLA3 inhibitor comprises an antisense oligonucleotide comprising a sequence having at least 8 consecutive bases of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0089] In certain embodiments, the PNPLA3 inhibitor comprises an antisense oligonucleotide comprising any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0090] In certain embodiments, the PNPLA3 inhibitor targeting PNPLA3 is ION 916333, 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612, which are described in U.S. Patent No. 10,774,333, which is incorporated herein by reference.

[0091] In certain embodiments, any of the aforementioned modified oligonucleotides comprises at least one modified internucleoside linkage, at least one modified sugar, and / or at least one modified nucleobase. In certain embodiments, any of the aforementioned modified oligonucleotides comprises at least one modified sugar moiety. In some embodiments, at least one modified sugar moiety is 2'-deoxy, 2'-O-methyl, 2'-O-methoxymethyl, 2'-O-methoxyethyl, 2'-fluoro, 4'-CH(CH3)-O-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', or combinations thereof. In certain embodiments, at least one modified sugar comprises a 2'-deoxy, 2'-O-methoxyethyl group. In certain embodiments, at least one modified sugar is a bicyclic sugar, such as a 4'-CH(CH3)-O-2' group, a 4'-CH2-O-2' group, or a 4'-(CH2)2-O-2' group.

[0092] In certain embodiments, any of the above modified oligonucleotides comprises one or more modified bases. In some embodiments, the modified base is 5-methylcytosine. In some embodiments, 1, 2, 3, 4, 5, 6 or more cytosines are 5-methylcytosines. In some embodiments, all cytosines in the antisense oligonucleotide are 5-methylcytosines.

[0093] In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, for example a phosphorothioate internucleoside linkage. In some embodiments, all internucleoside linkages are phosphorothioate internucleoside linkages.

[0094] In certain embodiments, any of the foregoing modified oligonucleotides comprises a gap segment comprised of linked deoxynucleosides; a 5' wing segment comprised of linked nucleosides; and a 3' wing segment comprised of linked nucleosides, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the modified oligonucleotide is 12 to 30 linked nucleosides in length and has a nucleobase sequence comprising a sequence listed in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length and has a nucleobase sequence comprising a sequence listed in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the modified oligonucleotide is 16 linked nucleosides in length having a nucleobase sequence consisting of the sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0095] In certain embodiments, the PNPLA3 inhibitor is a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of three linked nucleosides; and A 3' wing segment consisting of three linked nucleosides an antisense oligonucleotide comprising A gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a cEt sugar, each internucleoside linkage is a phosphorothioate linkage, and each cytosine is a 5-methylcytosine.

[0096] In certain embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide comprising a modified oligonucleotide of 12 to 30 linked nucleobases in length having a nucleobase sequence comprising a sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, or 10, the modified oligonucleotide comprising: a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of three linked nucleosides; and A 3' wing segment consisting of three linked nucleosides Including, A gap segment is located between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a cEt sugar, each internucleoside linkage is a phosphorothioate linkage, and each cytosine is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide consists of 16 to 30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides.

[0097] In certain embodiments, the compound comprises or consists of a modified oligonucleotide that is 16 linked nucleosides in length and consists of the sequence of SEQ ID NO:2, the modified oligonucleotide comprising: a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of three linked nucleosides; and A 3' wing segment consisting of three linked nucleosides Including, A gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a cEt sugar, each internucleoside linkage is a phosphorothioate linkage, and each cytosine is a 5-methylcytosine.

[0098] In some embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide further comprising a conjugate group. In some embodiments, the conjugate group is present at the 5' end of the antisense oligonucleotide. Thus, in certain embodiments, the compound is comprised of a modified oligonucleotide and a conjugate group, the modified oligonucleotide being 16 linked nucleosides in length and comprising the sequence of SEQ ID NO:2, the modified oligonucleotide comprising: a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of three linked nucleosides; and A 3' wing segment consisting of three linked nucleosides Including, a gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a cEt sugar, each internucleoside linkage is a phosphorothioate linkage, each cytosine is a 5-methylcytosine, and a conjugate group is positioned at the 5' terminus of the modified oligonucleotide; [ka] It is.

[0099] In some embodiments, the inhibitor of PNPLA3 expression has the following formula (SEQ ID NO:2): [ka] It is a compound of the formula:

[0100] In some embodiments, the inhibitor of PNPLA3 expression has the following formula (SEQ ID NO:2): [ka] It is a compound of the formula:

[0101] In any of the foregoing embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide, which may be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to a nucleic acid encoding PNPLA3.

[0102] In any of the above-mentioned embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide, and the antisense oligonucleotide can be single-stranded.In certain embodiments, the PNPLA3 inhibitor comprises deoxyribonucleotide.In certain embodiments, the PNPLA3 inhibitor is double-stranded.In certain embodiments, the PNPLA3 inhibitor is double-stranded and comprises ribonucleotide.

[0103] In any of the foregoing embodiments, the PNPLA3 inhibitor can be an antisense compound or an oligomeric compound.

[0104] In any of the foregoing embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide, which can be 8-80, 10-30, 12-50, 13-30, 13-50, 14-30, 14-50, 15-30, 15-50, 16-30, 16-50, 17-30, 17-50, 18-22, 18-24, 18-30, 18-50, 19-22, 19-30, 19-50, or 20-30 linked nucleosides in length. In some embodiments, the PNPLA3 inhibitor is an oligonucleotide.

[0105] In certain embodiments, the PNPLA3 inhibitor is an antisense oligonucleotide, and the antisense oligonucleotide comprises a modified oligonucleotide as described herein and a conjugate group. In certain embodiments, the conjugate group is attached to the modified oligonucleotide at the 5' end of the modified oligonucleotide. In certain embodiments, the conjugate group is attached to the modified oligonucleotide at the 3' end of the modified oligonucleotide. In certain embodiments, the conjugate group comprises at least one N-acetylgalactosamine (GalNAc), at least two N-acetylgalactosamine (GalNAc), or at least three N-acetylgalactosamine (GalNAc).

[0106] In certain embodiments, the PNPLA3 inhibitor provided herein comprises a pharma- ceutically acceptable salt of modified oligonucleotide.In certain embodiments, the salt is a sodium salt.In certain embodiments, the salt is a potassium salt.

[0107] In certain embodiments, the PNPLA3 inhibitors described herein have an in vitro IC of less than 2 μM, less than 1.5 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, less than 0.05 μM, less than 0.04 μM, less than 0.03 μM, less than 0.02 μM, or less than 0.01 μM. 50 The compound is active by having at least one of the following:

[0108] In certain embodiments, the PNPLA3 inhibitors described herein are well tolerated, as indicated by at least one of the following: an increase in alanine transaminase (ALT) or aspartate transaminase (AST) levels of 4-fold, 3-fold, or 2-fold or less compared to control animals, or an increase in liver, spleen, or kidney weight of 30%, 20%, 15%, 12%, 10%, 5%, or 2% or less compared to control animals. In certain embodiments, the PNPLA3 inhibitors described herein are well tolerated, as indicated by no increase in ALT or AST compared to control animals. In certain embodiments, the PNPLA3 inhibitors described herein are well tolerated, as indicated by no increase in liver, spleen, or kidney weight compared to control animals.

[0109] Certain embodiments provide compositions comprising any of the PNPLA3 inhibitors of the above-mentioned embodiments, or any pharma- ceutically acceptable salts thereof, and at least one of pharma- ceutically acceptable carriers or diluents.In certain embodiments, the compositions have a viscosity of less than about 40 centipoise (cP), less than about 30 centipoise (cP), less than about 20 centipoise (cP), less than about 15 centipoise (cP), or less than about 10 centipoise (cP).In certain embodiments, the compositions having any of the above-mentioned viscosities comprise the PNPLA3 inhibitors provided herein at a concentration of about 100 mg / mL, about 125 mg / mL, about 150 mg / mL, about 175 mg / mL, about 200 mg / mL, about 225 mg / mL, about 250 mg / mL, about 275 mg / mL, or about 300 mg / mL. In certain embodiments, a composition having any of the aforementioned viscosities and / or PNPLA3 inhibitor concentrations has a temperature at room temperature or about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C.

[0110] GLP-1 Peptides Glucon and glucagon-like peptide-1 (GLP-1) are derived from a 158 amino acid precursor polypeptide, preproglucagon, which is processed in various tissues to form several different proglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM), which are involved in diverse physiological functions such as glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake. Glucagon is a 29 amino acid peptide corresponding to amino acids 33-61 of proglucagon (53-81 of preproglucagon), whereas GLP-1 is generated as a 37 amino acid peptide corresponding to amino acids 72-108 of proglucagon (92-128 of preproglucagon). GLP-1(7-36)amide or GLP-1(7-37)acid are biologically active forms of GLP-1 that exhibit essentially equivalent activity at the GLP-1 receptor, see, e.g., U.S. Patent No. 9,765,130, incorporated herein by reference.

[0111] As used herein, a "GLP-1 / glucagon agonist peptide" is a chimeric peptide that exhibits at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more activity at the glucagon receptor compared to native glucagon and at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more activity at the GLP-1 receptor compared to native GLP-1 under the conditions of Assay 1.

[0112] As used herein, the term "native glucagon" refers to naturally occurring glucagon (e.g., human glucagon) that includes the sequence of SEQ ID NO: 11. The term "native GLP-1" refers to GLP-1 of natural origin (e.g., human GLP-1), and is a generic term that encompasses, for example, GLP-1(7-36)amide (SEQ ID NO: 12), GLP-1(7-37)acid (SEQ ID NO: 13), or a mixture of these two compounds. As used herein, a general reference to "glucagon" or "GLP-1" is intended to mean native human glucagon or native human GLP-1, respectively, unless otherwise specified. Unless otherwise indicated, "glucagon" refers to human glucagon and "GLP-1" refers to human GLP-1.

[0113] Glucagon can be produced by the pancreas and can interact with the glucagon receptor ("GCGR"). Glucagon can act in the liver to increase blood glucose through gluconeogenesis and glycogenolysis. When blood glucose begins to fall, glucagon can signal the liver to break down glycogen and release glucose, and blood glucose levels rise to normal levels. GLP-1 can have different biological activities compared to glucagon. GLP-1 can be secreted from L cells in the gastrointestinal tract and can bind to the GLP-1 receptor. GLP-1 activity can include stimulating insulin synthesis and secretion, inhibiting glucagon secretion, and inhibiting food intake.

[0114] Provided herein are agonists of glucagon receptor or glucagon-like peptide-1 (GLP-1). In some embodiments, the agonists of glucagon receptor or glucagon-like peptide-1 (GLP-1) include polypeptides. Peptides that bind to both glucagon receptor and GLP-1 receptor. In certain embodiments, the peptides provided herein are co-agonists of glucagon and GLP-1 activity. Such peptides are referred to herein as GLP-1 / glucagon agonist peptides. The GLP-1 / glucagon agonist peptides provided herein have GLP-1 activity and glucagon activity in a ratio that is favorable for promoting weight loss, preventing weight gain, or maintaining a desired weight, and have optimal solubility, formulation, and stability. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are active at the human GLP-1 and human glucagon receptors, and in certain embodiments, the relative activity at the GLP-1 receptor compared to the native ligand is at least about 1-fold, 2-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 25-fold greater than at the glucagon receptor.

[0115] As used herein, the term "peptide" encompasses full-length peptides, as well as fragments, variants, or derivatives thereof, including, for example, GLP-1 / glucagon agonist peptides (e.g., 29, 30, or 31 amino acids in length). The "peptides" (e.g., GLP-1 / glucagon agonist peptides) disclosed herein may be part of a fusion polypeptide that includes another component, such as, for example, an Fc domain or an albumin domain to extend half-life. The peptides described herein may also be derivatized in a number of different ways.

[0116] The terms "fragment," "analog," "derivative," or "variant," when referring to a GLP-1 / glucagon agonist peptide, include any peptide that retains at least some of the desired activity (e.g., binding to the glucagon and / or GLP-1 receptor). Fragments of the GLP-1 / glucagon agonist peptides provided herein include proteolytic fragments, deletion fragments that exhibit desirable properties upon expression, purification, and / or administration to a subject.

[0117] In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides have desirable potencies at the glucagon and GLP-1 receptors, and desirable relative potencies in promoting weight loss. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides exhibit in vitro potency at the GLP-1 receptor as shown by EC50 in cAMP Assay 1 (see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides exhibit in vitro potency at the GLP-1 receptor as demonstrated by EC50 in a cAMP assay (Assay 2, see Example 2) in 4.4% human serum albumin of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides exhibit in vitro potency at the glucagon receptor as demonstrated by EC50 in cAMP Assay 1 (see Example 2 of U.S. Pat. No. 9,765,130, incorporated herein by reference) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides exhibit in vitro potency at the glucagon receptor as demonstrated by EC50 in a cAMP assay (Assay 2, see Example 2 of U.S. Pat. No. 9,765,130, incorporated herein by reference) in 4.4% human serum albumin of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, the disclosed GLP-1 / Glucagon agonist peptides have a relative GLP1-R / GCGR potency ratio when compared to the native ligand using Assay 2 in the range of about 0.01 to 0.50, e.g., in the range of about 0.02 to 0.30, e.g., about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, or 0.30.

[0118] In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides exhibit in vitro potency in the glucose-dependent insulinotropic peptide (gastric inhibitory peptide) (GIPR) as demonstrated by EC50 in cAMP Assay 1 (see Example 2 of U.S. Pat. No. 9,765,130, incorporated herein by reference) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides exhibit in vitro potency in the GIPR as demonstrated by EC50 in a cAMP assay (Assay 2, see Example 2 of U.S. Pat. No. 9,765,130, incorporated herein by reference) in 4.4% human serum albumin of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

[0119] In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein have one or more of the following criteria: acceptable solubility, ease of formulation, plasma stability, and improved pharmacokinetic properties. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides are soluble in standard buffers over a wide pH range.

[0120] In certain embodiments, the GLP-1 / glucagon agonist peptide is soluble in common buffer solutions at concentrations of up to 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, or more, and in buffer systems ranging from 0.25 to 150 mM ionic strength, including, but not limited to, phosphate buffer, Tris buffer, glutamate buffer, acetate buffer, succinate buffer, or histidine buffer. Exemplary buffers include 100 mM glutamate pH 4.5 buffer, 100 mM acetate pH 5 buffer, 100 mM succinate pH 5 buffer, 100 mM phosphate pH 6 buffer, 100 mM histidine pH 6 buffer, 100 mM phosphate pH 6.5 buffer, 100 mM phosphate pH 7.0 buffer, 100 mM histidine pH 7.0 buffer, 100 mM phosphate pH 7.5 buffer, 100 mM Tris pH 7.5 buffer, and 100 mM Tris pH 8.0 buffer. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides are soluble in standard buffers at 0.8 mg / ml, e.g., over a pH range of pH 4.0 to pH 8.0, e.g., at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides are soluble in standard buffers at pH 4.5-8.0, 5.0-8.0, 5.5-8.0, 6.0-8.0, 6.5-8.0, 7.0-8.0, 4.5-8.5, 5.5-8.5, 5.5-8.5, 6.0-8.5, 6.5-8.5, or 7.0-8.5.

[0121] In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides can be formulated into standard pharmaceutical formulations. Representative formulations include, but are not limited to, 0.1M Tris pH 7.5, 150mM mannitol, final formulation pH=7.2; 0.05M Tris, 50mM arginine / proline, final formulation pH=8.0; or sodium phosphate buffer (pH 8) / 1.85% W / V propylene glycol, final formulation pH=7.0. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides are soluble in these or other formulations at concentrations of up to 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, or more.

[0122] In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides are acceptably stable against proteases in serum or plasma. Common degradation products of glucagon or GLP-1 include +1 products (acids) and DPP IV-degradation products. The +1 mass products can result from degradation at the amide group or C-terminus of glutamine, and degradation products result from the action of protease DPP IV in plasma. In certain embodiments, the disclosed GLP-1 / glucagon agonist peptides remain stable in plasma at levels up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after 24 hours in plasma at 37°C.

[0123] Provided herein is a GLP-1 / glucagon agonist peptide comprising the amino acid sequence: HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30 (Wherein, X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D or E, X16 is S or G, X17 is E, R, Q, or K, X18 is R, S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I, X24 is A or Q, X27 is E or V, X28 is A or K, and X30 is G or R) (SEQ ID NO: 25). In certain embodiments, an isolated peptide as shown above is provided, wherein X2 is S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D, X16 is S, X17 is E, R, Q, or K, X18 is R, S, or A, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E or V, X28 is A, and X30 is G (SEQ ID NO:26). In certain embodiments, an isolated peptide as shown above is provided, wherein if X17 is E and X18 is R, then X2 is S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D, X16 is S, or if X17 is R and X18 is S, then X20 is R, X21 is D, X23 is V, X24 is A, X27 is E or V, X28 is A, and X30 is G (SEQ ID NO:27 and SEQ ID NO:28, respectively).

[0124] In certain embodiments, there is provided an isolated peptide as shown above, wherein if X17 is E and X18 is R, then X2 is S, X10 is Y, X12 is K, X13 is K, X15 is D, X16 is S; or if X17 is R and X18 is S, then X20 is R, X21 is D, X23 is V, X24 is A, X27 is V, X28 is A, and X30 is G (SEQ ID NO:29 and SEQ ID NO:30, respectively). In certain embodiments, there is provided an isolated peptide as shown above, wherein if X12 is K, E, or R, and if X12 is K, E, R, or S, then X2 is S; if X10 is K, X17 is E, and X18 is R, and if X17 is R and X18 is S, then X13 is Y, X15 is D, X16 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO:31 and SEQ ID NO:32, respectively). In certain embodiments, there is provided an isolated peptide as shown above, wherein if X17 is E and X18 is R, then X2 is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S; or if X17 is R and X18 is S, then X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO:33 and SEQ ID NO:34, respectively). In certain embodiments, there is provided an isolated peptide as shown above, wherein if X17 is E and X18 is R, then X2 is S, X10 is K, X12 is R, X13 is Y, X15 is D, X16 is S; or if X17 is R and X18 is S, then X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO:35 and SEQ ID NO:36, respectively).

[0125] GLP-1 / glucan agonist peptides provided herein may include, but are not limited to, G730 (SEQ ID NO: 14), G797 (SEQ ID NO: 15), G849 (SEQ ID NO: 16), G933 (SEQ ID NO: 17), G865 (SEQ ID NO: 18), G796 (SEQ ID NO: 19), G812 (SEQ ID NO: 20), and G380 (SEQ ID NO: 21). These GLP-1 / glucan agonist peptides are listed in Table 1.

[0126] [Table 1]

[0127] i. Methods for Producing Agonists for the Glucagon Receptor and / or the GLP-1 Receptor GLP-1 / glucagon agonist peptide. The GLP-1 / glucagon agonist peptide provided herein can be produced by any suitable method, for example, by the method described in U.S. Patent No. 9,765,130, which is incorporated herein by reference.For example, in certain embodiments, the GLP-1 / glucagon agonist peptide provided herein is chemically synthesized by methods known to those skilled in the art, for example, by the solid-phase method described by Merrifield (1963, J.Am.Chem.Soc.85:2149-2154).For example, solid-phase peptide synthesis can be achieved using an automated synthesizer using standard reagents.

[0128] Alternatively, the GLP-1 / glucagon agonist peptides provided herein may be produced recombinantly using a convenient vector / host cell combination known to those skilled in the art. A variety of methods are available for recombinantly producing GLP-1 / glucagon agonist peptides. In general, a polynucleotide sequence encoding the GLP-1 / glucagon agonist peptide is inserted into a suitable expression vehicle (e.g., a vector containing the necessary elements for transcription and translation of the inserted coding sequence). A nucleic acid encoding the GLP-1 / glucagon agonist peptide is inserted into the vector in the proper reading frame. The expression vector is then transfected into a suitable host cell that expresses the GLP-1 / glucagon agonist peptide. Suitable host cells include, but are not limited to, bacteria, yeast, or mammalian cells. A variety of commercially available host expression vector systems may be utilized to express the GLP-1 / glucagon agonist peptides described herein.

[0129] ii. Modifications, conjugates, fusions, and derivatizations. In some embodiments, the peptides described herein include modifications to amino acids in the amino acid sequence. In certain embodiments, the GLP-1 / glucagon agonist peptides provided herein are stabilized by amino acid modifications. In certain embodiments, the carboxyl group of the C-terminal amino acid is amidated. In certain embodiments, the C-terminal amino acid is an amidated glycine, such as G730, G797, G849, G865, G796, G812, and G380. In certain embodiments, for example, G933 (i.e., the C-terminal glycine) is an unmodified acid. In certain embodiments, GLP-1 / glucagon agonist peptides are provided in which one or more amino acid residues are acylated (i.e., the addition of an acyl moiety). For example, in certain embodiments, the GLP-1 / glucagon agonist peptides provided herein contain one or more lysine residues, in which a palmitoyl moiety is attached to the N(ε) group. In certain embodiments, a linker is incorporated between the lysine and the palmitoyl group. This linker can be a gamma glutamic acid group or another linker, such as, but not limited to, beta alanine and aminohexanoic acid. Different acylation methods, such as the addition of cholesterol or myristoyl groups, may be used. In certain embodiments, the palmitoyl moiety is added at position 13 (e.g., G730). In certain embodiments, the palmitoyl moiety is added at position 10 (e.g., G797, G849, G933, G865, G796, and G812). In certain embodiments, the palmitoyl moiety is added at position 17 (e.g., G380).

[0130] The GLP-1 / glucagon agonist peptides provided herein (e.g., G730, G797, G849, and G933) may be palmitoylated to increase half-life through association with serum albumin, thereby reducing the propensity for renal clearance, as described in Example 1 of U.S. Pat. No. 9,765,130, incorporated herein by reference.

[0131] Alternatively, or in addition, the GLP-1 / glucagon agonist peptides disclosed herein may be associated with a heterologous moiety, e.g., to increase half-life, which may be a protein, peptide, protein domain, linker, organic polymer, inorganic polymer, polyethylene glycol (PEG), biotin, albumin, human serum albumin (HSA), HSA FcRn binding moiety, antibody, antibody domain, antibody fragment, single chain antibody, domain antibody, albumin binding domain, enzyme, ligand, receptor, binding peptide, non-FnIII scaffold, epitope tag, recombinant polypeptide polymer, cytokine, and combinations of two or more of such moieties.

[0132] For example, GLP-1 / glucagon agonist peptides can be fused with heterologous polypeptides. The peptides can be fused with proteins either by recombinant gene fusion and expression or chemical conjugation. Suitable proteins for fusion partners include, but are not limited to, human serum albumin, antibodies, and antibody fragments, including fusions with the Fc portion of antibodies. GLP-1 has been fused with these proteins while retaining potency (L.Baggio et al,Diabetes 53 2492-2500(2004);P.Barrington et al Diabetes,Obesity and Metabolism 13 426-433(2011);P.Paulik et al American Diabetes Association 2012,Poster 1946). Extended recombinant peptide sequences have also been described to confer high molecular weight to peptides (V. Schellenberger et al Nature Biotechnol 27 1186-1190 (2009); PASylation (EP 2173890)). In certain embodiments, the GLP-1 / glucagon agonist peptide is incorporated as the N-terminal part of a fusion protein with a fusion partner (e.g., albumin or Fc part) at the C-terminus. The GLP-1 / glucagon agonist peptides described herein can also be fused to peptide or protein domains such as "Albudabs" that have affinity for human serum albumin (MSDennis et al J Biol Chem 277 35035-35043 (2002); A. Walker et al Protein Eng Design Selection 23 271-278 (2010)). Methods for fusing the GLP-1 / glucagon agonist peptides disclosed herein to heterologous polypeptides (eg, albumin or Fc regions) are known to those of skill in the art.

[0133] Other heterologous moieties may be conjugated to the GLP-1 / glucagon agonist peptide to further stabilize or further extend half-life. In the case of chemical fusion, certain embodiments feature the maintenance of a free N-terminus, but alternative points for derivatization may be created. Yet another method is to derivatize the peptide with a large chemical moiety, such as high molecular weight polyethylene glycol (PEG). A "pegylated GLP-1 / glucagon agonist peptide" has a PEG chain covalently attached thereto. Derivatization (e.g., pegylation) of the GLP-1 / glucagon agonist peptide may be performed at palmitoylated lysine or at residues such as cysteine ​​that have been substituted or incorporated by extension to allow derivatization. The aforementioned GLP-1 / glucagon agonist peptide formats may be characterized in vitro and / or in vivo for relative potency and balance between GLP-1 and glucagon receptor activation.

[0134] The general term "polyethylene glycol chain" or "PEG chain" refers to a mixture of condensation polymers of ethylene oxide and water, which have the general formula H(OCH2CH2) n PEG chains are branched or linear, represented by the formula: OH (n is an integer of 3, 4, 5, 6, 7, 8, 9 or more). The PEG chain comprises a polymer of ethylene glycol having an average total molecular weight selected from the range of about 500 to about 40,000 daltons. The average molecular weight of the PEG chain is indicated by a numerical value, for example, PEG-5,000 refers to a polyethylene glycol chain having a total average molecular weight of about 5,000.

[0135] Pegylation can be carried out by any of the pegylation reactions known in the art. See, for example, Focus on Growth Factors, 3:4-10, 1992, and European Patent Application Publication Nos. 0 154 316 and 0 401 384. Pegylation can be carried out using an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).

[0136] Methods for preparing PEGylated GLP-1 / Glucagon agonist peptides generally include (a) reacting a GLP-1 / Glucagon agonist peptide with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions such that the molecule is attached to one or more PEG groups, and (b) obtaining the reaction product.

[0137] Administration The present disclosure provides a method for treating liver disease in a subject by administering to the subject an inhibitor of PNPLA3 expression and an agonist of glucagon receptor and / or GLP-1 receptor. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered simultaneously. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 1 hour of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 24 hours of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 72 hours of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 1 week of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 2 weeks of each other. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered within 1 month of each other, within 2 months of each other, or within 3 months or more of each other.

[0138] Various modes of administration are known to those skilled in the art and can be used to administer the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor. For example, modes of administration can include oral, parenteral, inhalation, or topical. "Parenteral administration" can mean administration via injection or infusion. Parenteral administration can include subcutaneous, intravenous, intramuscular, intraarterial, intraperitoneal, or intracranial administration, such as intrathecal or intraventricular, vaginal, or rectal administration. Another example form for administration is an injection solution, particularly a solution for intravenous or intraarterial injection or instillation. The inhibitor of PNPLA3 expression and / or GLP-1 / glucagon agonist peptide provided herein can be administered as a single dose or multiple doses. In certain embodiments, the inhibitor of PNPLA3 expression and / or GLP-1 / glucagon agonist peptide is administered by subcutaneous injection.

[0139] In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered in the same mode of administration. In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are administered in different modes of administration. In some embodiments, the inhibitor of PNPLA3 expression is administered parenterally. In some embodiments, the agonist of glucagon receptor and / or GLP-1 receptor is administered parenterally.

[0140] Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed by a maintenance dose. These compositions can be administered at specific fixed or variable intervals, for example, once a day, or can be administered "as needed." Dosage regimens can also be adjusted to achieve the optimal response desired (e.g., therapeutic or prophylactic response).

[0141] The administration frequency of the inhibitor of PNPLA3 expression and / or the agonist of glucagon receptor and / or GLP-1 receptor can be determined by those skilled in the art without undue experimentation. In some embodiments, the administration frequency of the inhibitor of PNPLA3 expression is the same as the administration frequency of the agonist of glucagon receptor and / or GLP-1 receptor. In some embodiments, the administration frequency of the inhibitor of PNPLA3 expression is different from the administration frequency of the agonist of glucagon receptor and / or GLP-1 receptor, e.g., more frequently or less frequently. In some embodiments, the inhibitor of PNPLA3 expression is administered once a day, twice a day, or three times a day. In some embodiments, the inhibitor of PNPLA3 expression is administered once a week, twice a week, or three times a week. In some embodiments, the inhibitor of PNPLA3 expression is administered once a month, twice a month, or three times a month. In some embodiments, the inhibitor of PNPLA3 expression is administered no more than once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, or once every seven weeks. In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered once a day, twice a day, or three times a day. In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered once a week, twice a week, or three times a week. In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered once a month, twice a month, or three times a month. In some embodiments, the glucagon receptor and / or GLP-1 receptor agonist is administered no more than once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, or once every seven weeks.

[0142] Indications The methods, compounds, peptides, and compositions described herein can be used to treat liver disease in subjects. The term "subject" refers to any subject (especially a mammalian subject) that requires treatment with the combination of an inhibitor of PNPLA3 expression and a GLP-1 / glucagon agonist peptide provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, cows, apes, monkeys, orangutans, and chimpanzees. In one embodiment, the subject is a human subject. In some embodiments, the subject is a human subject. In one embodiment, the subject is a male subject.

[0143] As used herein, "subject in need thereof" refers to an individual for whom treatment is desirable, for example, a subject with liver disease. In some embodiments, the present disclosure provides a method for treating liver disease (e.g., NASH and / or NAFLD). Nonalcoholic fatty liver disease (NAFLD) can include a variety of liver diseases, ranging from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD can be defined as the accumulation of more than 5% fat by weight in the liver in the absence of significant alcohol intake, lipogenic drug therapy, or genetic disorders (see, for example, Kotronen et al, Arterioscler Thromb.Vasc.Biol.2008,28:27-38).

[0144] Nonalcoholic steatohepatitis (NASH) can be NAFLD with signs of inflammation and liver damage. In some embodiments, NASH can be histologically defined by macrovesicular steatosis, hepatocyte ballooning, and lobular inflammatory infiltration (Sanyal, Hepatol. Res. 2011. 41:670-4). Some studies estimate that NASH affects 2-3% of the general population. In the presence of other conditions such as obesity or diabetes, some studies report that the estimated prevalence increases to 7% and 62%, respectively (see, for example, Hashimoto et al, J. Gastroenterol. 2011. 46(1):63-69).

[0145] In some embodiments, the methods provided herein are suitable for treating liver disease, NAFLD, fatty liver, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis.Certain embodiments provided herein are directed to compounds and compositions that reduce liver damage, steatosis, liver fibrosis, hepatitis, liver scarring or cirrhosis, liver failure, hepatomegaly, elevated transaminases, or liver fat accumulation in animals.

[0146] In some embodiments, the subject may have a secondary indication (e.g., an obese or obese-prone subject in which it is desirable to promote weight or body fat loss, weight or body fat maintenance, or to prevent or minimize weight gain over a certain period of time). In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the liver disease is non-alcoholic steatohepatitis. In some embodiments, the liver disease is liver fibrosis.

[0147] In some embodiments, the present disclosure provides a method for reducing fatty liver in the liver of a subject with liver disease, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of glucagon receptor and / or glucagon-like peptide-1 (GLP-1) receptor. In some embodiments, the total fatty liver of the subject is reduced compared to the total fatty liver of the subject when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone. In some embodiments, the total fatty liver of the subject is reduced by at least 30% compared to the total fatty liver of the subject when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone. In some embodiments, the subject's total hepatic steatosis is reduced by at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% compared to the total hepatic steatosis observed when an inhibitor of PNPLA3 expression or an agonist of the glucagon receptor GLP-1 receptor is administered alone.

[0148] In some embodiments, the total fatty liver of the subject is reduced by at least 30% compared to the total fatty liver of the subject when an inhibitor of PNPLA3 expression or an agonist of the glucagon receptor GLP-1 receptor is administered alone. In some embodiments, the total fatty liver of the subject is reduced by at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% compared to the total fatty liver of the subject when an inhibitor of PNPLA3 expression or an agonist of the glucagon receptor GLP-1 receptor is administered alone.

[0149] In some embodiments, the present disclosure provides a method for reducing inflammation in the liver of a subject with non-alcoholic fatty liver disease, comprising administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of glucagon receptor and / or glucagon-like peptide-1 (GLP-1) receptor. In some embodiments, inflammation in the liver of the subject is reduced by at least 50% compared to inflammation in the liver when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone. In some embodiments, inflammation in the liver of the subject is reduced by at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% compared to inflammation in the liver when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone.

[0150] In some embodiments, a method for reducing liver collagen in a subject with liver disease comprises administering to the subject i) an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and ii) an agonist of glucagon receptor and / or glucagon-like peptide-1 (GLP-1) receptor. In some embodiments, the liver collagen of the subject is reduced by at least 25% compared to the liver collagen when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone. In some embodiments, the liver collagen of the subject is reduced by at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to the liver collagen when the inhibitor of PNPLA3 expression or the agonist of glucagon receptor GLP-1 receptor is administered alone.

[0151] Pharmaceutical preparations In some embodiments, the present disclosure provides a pharma- ceutically acceptable composition comprising an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression and an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor, and at least one pharma- ceutically acceptable excipient.

[0152] The term "composition" or "pharmaceutical composition" refers to a composition comprising an inhibitor of PNPLA3 expression provided herein and an agonist of the glucagon receptor and / or the GLP-1 receptor, together with a pharma- ceutical acceptable carrier, excipient, or diluent, for example, to be administered to a subject in need of treatment (e.g., a human subject having liver disease).

[0153] The term "pharmaceutical acceptable" refers to a composition that is suitable for contact with human and animal tissues without excessive toxicity or other complications commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. A "pharmaceutical composition" or "pharmaceutical formulation" may include a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition may include one or more compounds or salts thereof and a sterile aqueous solution. In some embodiments, a pharmaceutical formulation includes a pharmaceutical acceptable carrier or diluent. A "pharmaceutical acceptable carrier or diluent" refers to any substance suitable for use in administration to an individual. For example, a pharmaceutical acceptable carrier may be a sterile aqueous solution, such as PBS or water for injection.

[0154] In some embodiments, the pharmaceutical formulation comprises a pharma- ceutically acceptable salt. "Pharmaceutically acceptable salt" refers to a physiologically and pharma- ceutically acceptable salt of a compound, such as an oligomeric compound or oligonucleotide, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects.

[0155] Further provided is a composition (e.g., pharmaceutical composition) comprising an effective amount of an inhibitor of PNPLA3 expression and an agonist of glucagon receptor and / or GLP-1 receptor provided herein, which is formulated for the treatment of metabolic disease (e.g., liver disease). An "effective amount" refers to the amount of an inhibitor of PNPLA3 expression and an agonist of glucagon receptor and / or GLP-1 receptor provided herein that is effective for treatment (e.g., treatment of liver disease) when administered to a subject, either as a single dose or as part of a series. This amount may be a fixed dose for all subjects to be treated, or may vary depending on the weight, health, and physical condition of the subjects to be treated, the degree of weight loss or weight maintenance desired, the formulation of the peptide, a professional evaluation of the medical situation, and other relevant factors.

[0156] The composition of the present disclosure can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), which is incorporated herein by reference in its entirety. The composition can be in various forms, including, but not limited to, aqueous solutions, emulsions, gels, suspensions, lyophilized forms, or any other forms known in the art. In addition, the composition can include pharma- ceutically acceptable additives, including, for example, diluents, binders, stabilizers, and preservatives. Once formulated, the composition of the present disclosure can be administered directly to a subject.

[0157] Carriers that can be used in the compositions of the present disclosure are known in the art, and include, but are not limited to, thyroglobulin, albumin, such as human serum albumin, tetanus toxoid, and polyamino acids, such as poly-L-lysine, poly-L-glutamic acid, influenza, and Hepatitis B virus core proteins. A variety of aqueous carriers can be used, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. The compositions can be sterilized by conventional known sterilization techniques or can be sterile filtered. The resulting compositions can be packaged for use as is, or lyophilized, and the lyophilized preparations are combined with a sterile solution before administration. The compositions can contain pharma-ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, isotonicity agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. In some embodiments, the composition is formulated for parenteral administration.

[0158] In some embodiments, the present disclosure provides a kit comprising i) an inhibitor of PNPLA3 expression and ii) an agonist of glucagon receptor and / or GLP-1 receptor.In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are in the same dosage form in the kit.In some embodiments, the inhibitor of PNPLA3 expression and the agonist of glucagon receptor and / or GLP-1 receptor are in different dosage forms in the kit.

[0159] All references cited herein (e.g., patents, patent applications, articles, textbooks, etc.), and the references cited therein, are incorporated by reference in their entirety, unless they have already been cited. EXAMPLES

[0160] Example 1 - PNPLA3 silencing inhibition in combination with incretin-based therapy, dual GLP-1 and glucagon receptor agonist, has superior efficacy for improving non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver fibrosis Introduction Nonalcoholic fatty liver disease (NAFLD) and its more advanced form, nonalcoholic steatohepatitis (NASH), are unmet medical needs that affect a large and growing population (Younossi et al Nat Rev Gastroenterol Hepatol, 2018, DOI: 10.1038 / nrgastro.2017.109). NAFLD is defined as excess hepatic fat accumulation (fatty liver) caused by causes other than alcohol consumption and includes NAFL, NASH, fibrosis, and cirrhosis. Fatty liver can progress to steatohepatitis, NASH, with or without fibrosis, depending on the individual, and ultimately, in susceptible individuals, cirrhosis, liver failure, and hepatocellular carcinoma (Friedman et al Nat Med, 2018, DOI: 10.1038 / s41591-018-0104-9).

[0161] NAFLD and NASH have a strong genetic component. The most common mutation associated with these conditions is the rs738409 variant (148M) in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene (Carlsson et al Aliment Pharmacol Ther, 2020, DOI: 10.1111 / apt.15738). Pnpla3 silencing in the liver ameliorates NAFLD, NASH, and associated liver fibrosis in mouse models genetically engineered to carry the human risk allele variant (148M) of the mouse Pnpla3 gene (Linden et al Mol Metab, 2019, DOI: 10.1016 / j.molmet.2019.01.013).

[0162] Obesity and type 2 diabetes mellitus (T2DM) are major risk factors for the development of NAFLD and NASH, and treatment with incretin hormones, which reduce body weight and improve glucose homeostasis, has been shown to ameliorate NAFLD and NASH. Such incretin hormones include peptide analogs that bind to the glucagon-like peptide-1 (GLP-1) receptor, both the GLP-1 and glucagon receptors, or the GLP-1 and gastric inhibitory polypeptide (GIP) receptors (Newsome et al NEJM, 2020, DOI: 10.1056 / NEJMoa2028395; Ambry et al Lancet, 2018, DOI: 10.1016 / S0140-6736(18)30726-8; Boland et al Nat Metab, 2020, DOI: 10.1038 / s42255-020-0209-6+Hartman et al Diabetes Care, 2020, DOI: 10.2337 / dc19-1892; Kannt et al Diabetes Obes Metab,2020,DOI:10.1111 / dom.14035).

[0163] It is hypothesized that the combined treatment of PNPLA3 silencing inhibition and incretin hormone receptor activation can obtain additive beneficial effects on the treatment of NAFLD, NASH, and liver fibrosis.To investigate this, a genetically engineered mouse model of human PNPLA3 I148M NASH risk allele is used while feeding on NASH-inducing diet.Then, the mouse is treated with: 1) control antisense oligonucleotide (ASO); 2) Pnpla3 ASO; 3) balanced GLP-1 receptor and glucagon receptor dual agonist peptide cotadutide; or 4) the combination of both Pnpla3 ASO and cotadutide.

[0164] This study demonstrates for the first time that the combination of PNPLA3 ASO silencing inhibition treatment and incretin mimetic treatment (exemplified by cotaduthide) can provide surprising and beneficial treatment effects against NAFLD, NASH, and liver fibrosis. The results obtained by this combination treatment show synergistic effects compared with each treatment alone.

[0165] Materials and Methods Mouse Pnpla3 cEt 5'-GalNAc3-conjugated cEt ASO and cotadutide An optimal and potent mouse S-restricted ethyl (cEt) modified 16mer ASO targeting the mouse Pnpla3 gene (5'-TATTTTTGGTGTATCC-3') (SEQ ID NO: 37) was used (Linden et al Mol Metab, 2019, DOI: 10.1016 / j.molmet.2019.01.013). This mouse Pnpla3 ASO was modified by 5'-conjugation with tri-branched N-acetylgalactosamine (GalNAc3) to further enhance hepatocyte targeting in vivo after subcutaneous administration. A chemically matched scrambled control GalNAc3-conjugated ASO (5'-GGCCAATACGCCGTCA-3') (SEQ ID NO: 38) was used to demonstrate the specificity of the target knockdown. Cotadutide (MEDI0382) is engineered to balance GLP-1 and glucagon receptor agonism (with an approximately 5:1 bias towards GLP-1 receptor affinity) (Henderson et al Diabetes Obes Metab, 2016, DOI:10.1111 / dom.12735).

[0166] animal All animal experiments were performed under humane protection and approved by the Gothenburg Ethics Committee for Experimental Animals, Sweden. The facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The human PNPLA3 I148M mutation was introduced into the mouse Pnpla3 gene by replacing the isoleucine codon at amino acid position 148 of the mouse Pnpla3 gene with a methionine codon using homologous recombination as previously described (Linden et al Mol Metab, 2019, DOI: 10.1016 / j.molmet.2019.01.013). Heterozygous Pnpla3 148I / M Mice were crossed to generate experimental homozygous Pnpla3 148M / M Knock-in mice were generated. All experimental animals were verified to have the correct genotype using PCR before the start of the study and again using PCR after completion as previously described (Linden et al Mol Metab, 2019, DOI: 10.1016 / j.molmet.2019.01.013). All animals were housed in clear makrolon cages with aspen wood chip bedding and nesting material, and the holding facility was temperature (21 ± 1 °C) and humidity (50 ± 10%) controlled. Mice were provided with tap water and food ad libitum with a 12-h day / night cycle.

[0167] Male Pnpla3 148M / MMice (6-8 weeks old) were fed a diet high in fat (40 kcal % fat (non-trans-fat Primex Shortening), fructose (20 kcal %), and cholesterol (2%) (NASH diet; D16022301, Research Diets, New Brunswick, NJ) for 22 weeks. The mice were then assigned to study groups based on body weight, fed the same NASH diet, and administered either 1) control ASO + saline, 2) Pnpla3 ASO + saline, 3) control ASO + cotadutide, or 4) Pnpla3 ASO + cotadutide for 14 weeks. ASO was administered at 5 mg / kg / week by subcutaneous injection twice per week using saline as vehicle. Cotadutide was administered at 1 nmol / kg by daily subcutaneous injection using saline as vehicle. Mice not treated with cotadutide were injected daily with vehicle (saline) and all animals received the same number of subcutaneous injections. Body weights were recorded throughout the study. At the time of sacrifice, non-fasted mice were euthanized with isoflurane (Forene, Abbot Scandinavia AB, Sweden), blood was collected to isolate plasma, livers were harvested and sections (same location of the left lateral lobe for all mice) were fixed in 4% formaldehyde in PBS for histological examination or snap frozen in liquid N2 and stored at -80°C.

[0168] Liver histology Formaldehyde-fixed paraffin-embedded mouse liver sections (one liver section per mouse) 4 μm thick were routinely stained with hematoxylin and eosin (HE). Adjacent sections were immunohistochemically stained for collagen 1A1 (Col1A1, LS-C343921, BioSite, USA). Slides were scanned with a Panoramic Scan II (3Dhistech, Hungary) and digital images were analyzed by detecting the stained area per total section area for galectin-3 or collagen 1A1 using the image analysis program Visiopharm (version 2020.03.0.7300, Visiopharm, Horsholm, Denmark).

[0169] Fatty liver was determined by evaluation of HE stained liver sections. Total fatty liver was determined by measuring the total amount of lipid droplets as a percentage of the area of ​​the section. Macrodroplet steatosis was determined by measuring the amount of large lipid droplets as a percentage of the area of ​​the section. Microdroplet steatosis was determined by measuring the amount of small lipid droplets as a percentage of the area of ​​the section.

[0170] Hepatic macrophages were determined by staining various liver sections with galectin-3 (Mac2) and determining the percentage of sections stained.

[0171] NAFLD activity score (NAS) was determined according to the method reported by Kleiner et al. (Kleiner et al Hepatology, 2005, DOI:10.1002 / hep.20701). NAS was based on a combination of fatty liver score (steatosis less than 5% = score 0, 5%-33% = score 1, >33%-66% = score 2, >66% = score 3) and inflammation score (no lesions = score 0, <2 lesions per 200× field = score 1, 2-4 lesions per 200× field = score 2, >4 lesions per 200× field = score 3). Hepatocellular swelling degeneration was not found in any of the mouse livers, which was not unexpected since it is rarely observed in preclinical rodent NASH models in contrast to human NASH pathology. All histological evaluations were performed by a board-certified veterinary pathologist blinded to the treatments.

[0172] RNA preparation and qPCR RNA was isolated from snap-frozen liver tissue using the RNeasy Mini Kit (Qiagen, Germany). cDNA templates were generated by reverse transcription with a cDNA kit (ThermoFisher Scientific, Stockholm, Sweden) and used for real-time quantitative PCR with a QuantStudio 7 Flex instrument (Applied Biosystems, Stockholm, Sweden). Mouse Pnpla3 mRNA expression was analyzed using a commercially available complete assay (Mm00504420_m1, TaqMan, Life Technologies Europe, Stockholm, Sweden). Results were normalized to mouse ribosomal protein large P0 (Rplp0, 36B4) using the forward primer 5'-GAGGAATCAGATGAGGATATGGGA-3' (SEQ ID NO: 39), reverse primer 5'-AAGCAGGCTGACTTGGTTGC-3' (SEQ ID NO: 40), and FAM-TAM-labeled probe 5'-TCGGTCTCTTCGACTAATCCCGCCAA-3' (SEQ ID NO: 41) (Sigma-Aldrich) as the reference gene.

[0173] statistical analysis Differences between treatment groups were examined using one-way ANOVA followed by Tukey's post-hoc test (GraphPad Prism v.8.0.1., GraphPad Software, CA). Differences between treatment groups for NAFLD activity score (NAS) derived from liver tissue were analyzed by ordinal regression analysis followed by correction of family-wise error rate using Sidak method. p-values ​​less than 0.05 were considered significant. Data are presented as individual values ​​and mean ± standard error of the mean (SEM).

[0174] Results and Conclusions Combined treatment with Pnpla3 ASO and cotadutide improved Pnpla3 mice fed a NASH-inducing diet 148M / MTo test the combined treatment of Pnpla3 ASO and cotadutide, which has additive effects on improving NAFLD, NASH, and liver fibrosis in mice, homozygous Pnpla3 mice were used. 148M / M Knock-in mice were fed a NASH-inducing diet for 36 weeks. During the final 14 weeks, mice were also treated with Pnpla3 ASO or cotadutide, alone or in combination, and compared to control ASO-treated animals (all mice received the same number of subcutaneous injections). Cotadutide treatment, but not Pnpla3 ASO treatment, reduced weight gain during the treatment period compared to control ASO-treated animals (Figure 1A). Pnpla3 ASO treatment, but not cotadutide treatment, significantly reduced hepatic Pnpla3 mRNA levels by more than 97% (Figure 1B).

[0175] Total hepatic steatosis (Figure 2A), macrodroplet steatosis (Figure 2B), and microdroplet steatosis (Figure 2C) were determined as described. Stained sections are shown in Figure 2D, showing the percentage of total lipid droplets per area for each section. As seen in Figures 2A-C, combined treatment with Pnpla3 ASO and cotadutide significantly reduced all types of steatosis compared to either treatment alone. The percentage of hepatic macrophages (Figure 3A) and inflammation scores (Figure 3B) were determined for each treatment as described. As shown in Figures 3A and 3B, hepatic macrophages and inflammation scores were reduced in all treatment groups compared to controls.

[0176] NAFLD activity scores (NAS) were calculated as described above. Pnpla3 ASO treatment reduced NAS compared to control ASO-treated animals (p<0.005) (Figure 4). Cotadutide treatment also reduced NAS compared to control ASO-treated animals (p<0.001) (Figure 4). Importantly, combined treatment of Pnpla3 ASO and cotadutide reduced NAS compared to control ASO-treated animals (p<0.001), compared to Pnpla3 ASO-treated animals (p<0.001), and compared to cotadutide-treated animals (p<0.001) (Figure 4). These results indicate that combined treatment of Pnpla3 knockdown inhibitors with dual GLP-1 and glucagon receptor agonists improves the beneficial treatment effect on improving NAFLD and NASH.

[0177] Pnpla3 ASO treatment tended to reduce liver fibrosis measured as hepatic collagen 1A1 content, and cotadutide treatment also tended to reduce hepatic collagen 1A1 content (Figure 5). Importantly, combined treatment with Pnpla3 ASO and cotadutide significantly reduced hepatic collagen 1A1 content compared to control ASO-treated animals (p<0.005) (Figure 5). Thus, these results indicate that combined treatment with Pnpla3 knockdown inhibition and dual GLP-1 and glucagon receptor agonist has a significant beneficial treatment effect on improving liver fibrosis.

Claims

Claim 1 A pharmaceutical composition comprising an inhibitor of patatin-like phospholipase domain-containing 3 (PNPLA3) expression for treating a target liver disease, further comprising an agonist of a glucagon receptor and / or a glucagon-like peptide-1 (GLP-1) receptor, or administered in parallel or sequentially with an agonist of a glucagon receptor and / or a glucagon-like peptide-1 (GLP-1) receptor. Claim 2 The pharmaceutical composition according to claim 1, wherein the inhibitor of PNPLA3 expression is an antisense oligonucleotide complementary to a region of the nucleic acid encoding PNPLA3. Claim 3 The pharmaceutical composition according to claim 2, wherein the antisense oligonucleotide is complementary to a site within nucleotides 5567-5731, 5644-5731, 5567-5642, or 5567-5620 of the nucleic acid encoding PNPLA3. Claim 4 The pharmaceutical composition according to claim 2, wherein the nucleic acid encoding PNPLA3 is mRNA. Claim 5 The pharmaceutical composition according to claim 2, wherein the antisense oligonucleotide is 12-30 or 16-30 nucleosides in length. Claim 6 The pharmaceutical composition according to claim 2, wherein the antisense oligonucleotide comprises one or more modified sugar moieties, one or more modified bases, and / or one or more non-natural internucleoside linkages. Claim 7 (a) The one or more modified sugar moieties are 2'-deoxy, 2'-O-methyl, 2'-O-methoxymethyl, 2'-O-methoxyethyl, 2'-fluoro, 4'-CH(CH3)-O-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', or a combination thereof; and / or (b) The one or more modified bases are 5-methylcytosine; and / or (c) The one or more internucleoside linkages are phosphorothioate linkages, The pharmaceutical composition according to claim 6. Claim 8 (a) All cytosines in the antisense oligonucleotide are 5-methylcytosine; and / or (b) All internucleoside linkages are phosphorothioate linkages, The pharmaceutical composition according to claim 7. Claim 9 The pharmaceutical composition according to claim 2, wherein the antisense oligonucleotide comprises a sequence having at least 8 consecutive bases of any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

10. The pharmaceutical composition according to claim 2, wherein the antisense oligonucleotide comprises one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

11. The antisense oligonucleotide is a) a gap segment consisting of 10 linked deoxynucleosides; b) a 5' wing segment consisting of 3 linked nucleosides; and c) a 3' wing segment consisting of 3 linked nucleosides comprising, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment contains a constrained ethyl sugar, each internucleoside linkage is a phosphorothioate linkage, and each cytosine is 5-methylcytosine, The pharmaceutical composition according to claim 10.

12. The pharmaceutical composition according to claim 2, wherein the inhibitor of PNPLA3 expression further comprises a conjugate group.

13. (a) The conjugate group is present at the 5' end of the antisense oligonucleotide; and / or (b) The conjugate group is 【Chemical 1】 The pharmaceutical composition according to claim 12.

14. The inhibitor of PNPLA3 expression is the following formula (SEQ ID NO: 2): 【Chemical 2】 The pharmaceutical composition according to claim 1, which is a compound or a pharmaceutically acceptable salt thereof.

15. The pharmaceutical composition according to claim 1, wherein the agonist of the glucagon receptor and / or GLP-1 receptor is a peptide.

16. The peptide has the amino acid sequence: HX2QGTSDX10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30 (SEQ ID NO: 25) wherein, (1) X2 is S, X10 is Y, X12 is K, X13 is K, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is V, X28 is A, X30 is G (SEQ ID NO: 14); (2) Is X2 S, X10 K, X12 E, X13 Y, X15 D, X16 S, X17 E, X18 R, X20 R, X21 D, X23 V, X24 A, X27 E, X28 A, and X30 G (SEQ ID NO: 15); (3) Is X2 S, X10 K, X12 K, X13 Y, X15 E, X16 G, X17 Q, X18 A, X20 K, X21 E, X23 I, X24 A, X27 E, X28 K, and X30 R (SEQ ID NO: 20); (4) Is X2 S, X10 K, X12 S, X13 Y, X15 D, X16 S, X17 R, X18 S, X20 R, X21 D, X23 V, X24 A, X27 E, X28 A, and X30 G (SEQ ID NO: 18); (5) Is X2 S, X10 K, X12 E, X13 Y, X15 D, X16 S, X17 E, X18 R, X20 R, X21 D, X23 V, X24 A, X27 E, X28 A, and X30 G (SEQ ID NO: 33); or (6) X2 is S, X10 is K, X12 is S, X13 is Y, X15 is D, X16 is S, X17 is R, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 19), The pharmaceutical composition according to claim 15.

17. The pharmaceutical composition according to claim 15, wherein the peptide comprises the amino acid sequence HSQGTFTSDKSEYLDSEARADFVAWLEAGG (SEQ ID NO: 33).

18. The pharmaceutical composition according to claim 15, wherein the peptide further comprises a modification to the amino acids in the amino acid sequence.

19. The pharmaceutical composition according to claim 18, wherein the modification is the addition of an acyl moiety.

20. The pharmaceutical composition according to claim 19, wherein the modification is a palmitoyl moiety on the N(ε) group of a lysine residue.

21. The pharmaceutical composition according to claim 20, wherein the palmitoyl group is bonded to the lysine via a linker.

22. The pharmaceutical composition according to claim 21, wherein the linker is γ-glutamic acid.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the subject is obese and / or has type 2 diabetes.

24. The pharmaceutical composition according to any one of claims 1 to 22, wherein the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis or liver fibrosis.

25. A pharmaceutical composition comprising an inhibitor of PNPLA3 expression for (i) reducing fatty liver in the liver of a subject having a liver disease or (ii) reducing inflammation in the liver of a subject having non-alcoholic fatty liver disease or (iii) reducing liver collagen in a subject having a liver disease, further comprising an agonist of a glucagon receptor and / or a glucagon-like peptide-1 (GLP-1) receptor and administering.

26. The pharmaceutical composition according to claim 25, wherein the inhibitor of PNPLA3 expression is an antisense oligonucleotide complementary to a region of the nucleic acid encoding PNPLA3.

27. The pharmaceutical composition according to claim 26, wherein the antisense oligonucleotide is 16 to 30 nucleosides in length.

28. The pharmaceutical composition according to claim 26, wherein the antisense oligonucleotide comprises one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

29. The antisense oligonucleotide is a) a gap segment consisting of 10 linked deoxynucleosides; b) a 5' wing segment consisting of 3 linked nucleosides; and c) a 3' wing segment consisting of 3 linked nucleosides and the gap segment is located between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment contains a constrained ethyl sugar, each internucleoside bond is a phosphorothioate bond, and each cytosine is 5-methylcytosine. The pharmaceutical composition according to claim 28.

30. The pharmaceutical composition according to claim 25, wherein the agonist of the glucagon receptor and / or the GLP-1 receptor is a peptide.

31. The peptide has an amino acid sequence: HX2QGTTFTSD X10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30 (SEQ ID NO: 25) comprising, wherein (1) X2 is S, X10 is Y, X12 is K, X13 is K, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is V, X28 is A, X30 is G (SEQ ID NO: 14); (2) X2 is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, X30 is G (SEQ ID NO: 15); (3) X2 is S, X10 is K, X12 is K, X13 is Y, X15 is E, X16 is G, X17 is Q, X18 is A, X20 is K, X21 is E, X23 is I, X24 is A, X27 is E, X28 is K, X30 is R (SEQ ID NO: 20); (4) X2 is S, X10 is K, X12 is S, X13 is Y, X15 is D, X16 is S, X17 is R, X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, X30 is G (SEQ ID NO: 18); (5) X2 is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, X30 is G (SEQ ID NO: 33); or (6) X2 is S, X10 is K, X12 is S, X13 is Y, X15 is D, X16 is S, X17 is R, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, X30 is G (SEQ ID NO: 19), The pharmaceutical composition according to claim 30.

32. The pharmaceutical composition according to any one of claims 25 to 31, wherein the subject is obese and / or has type 2 diabetes.

33. The pharmaceutical composition according to any one of claims 25 to 31, wherein the liver disease is non-alcoholic steatohepatitis or liver fibrosis.

34. i) an inhibitor of the expression of patatin-like phospholipase domain-containing 3 (PNPLA3); ii) an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor; iii) at least one pharmaceutically acceptable excipient The pharmaceutical composition according to claim 1 or 25, comprising.

35. i) an inhibitor of the expression of patatin-like phospholipase domain-containing 3 (PNPLA3); ii) an agonist of the glucagon receptor and / or the glucagon-like peptide-1 (GLP-1) receptor A kit comprising.

36. The composition according to claim 34 or the kit according to claim 35, wherein the inhibitor of the expression of PNPLA3 is an antisense oligonucleotide complementary to a region of the nucleic acid encoding PNPLA3.

37. The composition according to claim 34 or the kit according to claim 35, wherein the agonist of the glucagon receptor and / or the GLP-1 receptor is a peptide.

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