(S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (APL) for use in the treatment of non-alcoholic steatohepatitis (NASH), hepatitis, hepatocellular ballooning, liver fibrosis and steatosis

Abstract

(S)-2-Amino-6-((3-aminopropyl)amino))hexanoic acid (APL) for use in the treatment of non-alcoholic steatohepatitis (NASH), hepatitis, hepatocellular ballooning, liver fibrosis, and steatosis. JPEG2023523424000094.jpg23145(I)

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 015,175, filed April 24, 2020, the entirety of which is incorporated by reference.

[0002] The present invention relates to methods and compositions for the treatment of non-alcoholic steatohepatitis (NASH). [Background technology]

[0003] Nonalcoholic steatohepatitis (NASH) is a non-benign disorder characterized by substantial health risks. Subjects diagnosed with NASH are at high risk of morbidity and mortality. More specifically, NASH is characterized by an increased risk of cardiovascular and liver-related death. NASH can lead to cirrhosis, which can result in fluid retention, muscle wasting, intestinal bleeding, and liver failure. Liver transplantation is the only treatment for advanced cirrhosis with liver failure, and NASH is currently the second leading reason for liver transplantation.

[0004] Therefore, there is a continuing need for therapeutic agents for the treatment of NASH. Summary of the Invention

[0005] Described herein are new methods for treating liver diseases and disorders, including nonalcoholic steatohepatitis (NASH).

[0006] In one aspect, the invention features a method of treating nonalcoholic steatohepatitis (NASH), the method comprising administering to a subject in need thereof an effective amount of a compound according to Formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, —CN, —NO2, —NH2, —NH(C1-C6)alkyl, —N[(C1-C6)alkyl)]2, —OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, thio(C1-C6)alkyl, -SONH2, -SO2NH2, selected from the group consisting of -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 2 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, -CN, -NO2, -NH2, -OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, Thio(C1-C6)alkyl, -SONH2, -SO2NH2, -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 13 , and R 14 is independently selected from the group consisting of hydrogen and -(C1-C6)alkyl; R 5 and R 6are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 5 and R 6 cannot both be -OH; R 11 and R 12 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 11 and R 12 cannot both be -OH; m is an integer from 1 to 4, n is an integer of 0 to 4. o is an integer from 0 to 4, p is an integer from 1 to 4, q is an integer from 0 to 4, r is an integer of 0 to 4.

[0007] In another aspect, the invention features a compound according to Formula I, or a pharmaceutically acceptable salt thereof, for use in treating NASH in a subject.

[0008] In another aspect, the invention features a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of NASH.

[0009] In embodiments, the compound of formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid, [ka] (1) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride, [ka] is.

[0010] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid, [ka] (2) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0011] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid, [ka] (3) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0012] In some embodiments, R 1 and R 2 is independently selected from the group consisting of hydrogen and —(C1-C8)alkyl.

[0013] In some embodiments, R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are each independently selected from hydrogen and —(C1-C8)alkyl.

[0014] In some embodiments, R 9 , R 10 , R 11 , R 12, R 13 , and R 14 are each independently selected from hydrogen and —(C1-C8)alkyl.

[0015] In some embodiments, R 3 , R 4 , R 5 , R 6 , R 7 and R 8 each is independently selected from hydrogen and —(C-C)alkyl; R 9 , R 10 , R 11 , R 12 , R 13 and R 14 each is independently selected from hydrogen and —(C 1 -C 8 )alkyl.

[0016] In embodiments, or a pharmaceutically acceptable salt thereof, the sum of m+n+o is in the range of 2-10, and the sum of p+q+r is in the range of 2-10.

[0017] In some embodiments, m is 3, p is 4, and n, o, q, and r are each zero. 3 , R 4 , R 9 , and R 10 is independently selected from hydrogen and —(C1-C8) alkyl. In some embodiments, R 1 and R 2 is independently selected from the group consisting of hydrogen and —(C1-C8)alkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is hydrogen.

[0018] In some embodiments, m is 4, n is 2, o is zero, p is 3, q ​​is 1, and r is zero. In some embodiments, R 3 , R 4 , R 5 , R 6 , R9 , R 10 , R 11 , and R 12 is independently selected from hydrogen and —(C1-C8) alkyl. In some embodiments, R 1 and R 2 is independently selected from the group consisting of hydrogen and —(C1-C8)alkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 , and R 12 is hydrogen.

[0019] In some embodiments, m is 4, n is 1, o is zero, p is 3, q ​​is 1, and r is zero. In some embodiments, R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 , and R 12 is independently selected from hydrogen and —(C1-C8) alkyl. In some embodiments, R 1 and R 2 is independently selected from the group consisting of hydrogen and —(C1-C8)alkyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 , and R 12 is hydrogen.

[0020] In embodiments, the subject is a human.

[0021] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject for at least about 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks.

[0022] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject for at least about 4 to 6, 4 to 8, 4 to 10, 4 to 12, 4 to 14, or 4 to 16 weeks.

[0023] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject at a dose of about 2-1000, 10-1000, or 10-100 mg / kg per day.

[0024] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject at a dose of about 10 mg / kg or more per day.

[0025] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject at a dose of about 2 mg / kg or more per day.

[0026] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject at a total daily dose of about 100-5000, 500-5000, or 600-3000 mg per day.

[0027] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is orally administered to a subject.

[0028] In embodiments, the subject has a NAFLD activity score (NAS) of 4 or greater.

[0029] In embodiments, the subject has a NAFLD activity score (NAS) of 5 or greater.

[0030] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, results in a NAFLD activity score (NAS) of less than 4.

[0031] In embodiments, the subject has non-cirrhotic NASH.

[0032] In embodiments, the subject has cirrhosis NASH.

[0033] In embodiments, the subject has hepatitis. In embodiments, the hepatitis is a lobular inflammation.

[0034] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a reduction in hepatitis.

[0035] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a hepatitis score of 0 or 1.

[0036] In embodiments, the subject's liver is characterized by hepatocyte ballooning.

[0037] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a reduction in hepatocyte ballooning.

[0038] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a ballooning score of 0.

[0039] In embodiments, the subject has elevated liver alanine aminotransferase (ALT) levels.

[0040] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a decrease in hepatic alanine aminotransferase (ALT) levels.

[0041] In embodiments, the subject has elevated hepatic aspartate aminotransferase (AST) levels.

[0042] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a decrease in hepatic aspartate aminotransferase (AST) levels.

[0043] In embodiments, the subject has liver fibrosis. In embodiments, the subject has stage 2, stage 3, or stage 4 liver fibrosis. In embodiments, the subject has cirrhosis.

[0044] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in stabilization of liver fibrosis in the subject.

[0045] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in the reversal of liver fibrosis in the subject.

[0046] In embodiments, the subject has a steatosis score of 1, 2, or 3.

[0047] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a reduction of liver hypertrophy in a subject.

[0048] In one aspect, the invention features a method of reducing hepatitis, the method comprising administering to a subject in need thereof an effective amount of a compound according to Formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, —CN, —NO2, —NH2, —NH(C1-C6)alkyl, —N[(C1-C6)alkyl)]2, —OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, thio(C1-C6)alkyl, -SONH2, -SO2NH2, selected from the group consisting of -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 2 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, -CN, -NO2, -NH2, -OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, Thio(C1-C6)alkyl, -SONH2, -SO2NH2, -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 13 , and R 14 is independently selected from the group consisting of hydrogen and -(C1-C6)alkyl; R 5 and R 6 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 5 and R 6 cannot both be -OH; R 11 and R 12 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 11 and R 12 cannot both be -OH; m is an integer from 1 to 4, n is an integer of 0 to 4. o is an integer from 0 to 4, p is an integer from 1 to 4, q is an integer from 0 to 4, r is an integer of 0 to 4.

[0049] In embodiments, the compound of formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid, [ka] (1) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride, [ka] is.

[0050] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid, [ka] (2) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0051] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid, [ka] (3) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0052] In embodiments, the hepatitis is lobular inflammation.

[0053] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)) or a pharmaceutically acceptable salt thereof results in a hepatitis score of 0 or 1.

[0054] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0055] In one aspect, the invention features a method for reducing hepatocyte ballooning, the method comprising administering to a subject in need thereof an effective amount of a compound according to Formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, —CN, —NO2, —NH2, —NH(C1-C6)alkyl, —N[(C1-C6)alkyl)]2, —OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, thio(C1-C6)alkyl, -SONH2, -SO2NH2, selected from the group consisting of -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 2 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, -CN, -NO2, -NH2, -OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, Thio(C1-C6)alkyl, -SONH2, -SO2NH2, -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 13 , and R 14 is independently selected from the group consisting of hydrogen and -(C1-C6)alkyl; R 5 and R 6 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 5 and R 6 cannot both be -OH; R 11 and R 12 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 11 and R 12 cannot both be -OH; m is an integer from 1 to 4, n is an integer of 0 to 4. o is an integer from 0 to 4, p is an integer from 1 to 4, q is an integer from 0 to 4, r is an integer of 0 to 4.

[0056] In embodiments, the compound of formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid, [ka] (1) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride, [ka] is.

[0057] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid, [ka] (2) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0058] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid, [ka] (3) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0059] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a ballooning score of 0.

[0060] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0061] In one aspect, the invention features a method of treating liver fibrosis, the method comprising administering to a subject in need thereof an effective amount of a compound according to Formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, —CN, —NO2, —NH2, —NH(C1-C6)alkyl, —N[(C1-C6)alkyl)]2, —OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, thio(C1-C6)alkyl, -SONH2, -SO2NH2, selected from the group consisting of -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 2 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, -CN, -NO2, -NH2, -OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, Thio(C1-C6)alkyl, -SONH2, -SO2NH2, -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 13 , and R 14 is independently selected from the group consisting of hydrogen and -(C1-C6)alkyl; R 5 and R 6 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 5 and R 6 cannot both be -OH; R 11 and R 12 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 11 and R 12 cannot both be -OH; m is an integer from 1 to 4, n is an integer of 0 to 4. o is an integer from 0 to 4, p is an integer from 1 to 4, q is an integer from 0 to 4, r is an integer of 0 to 4.

[0062] In embodiments, the compound of formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid, [ka] (1) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride, [ka] is.

[0063] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid, [ka] (2) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride, JPEG0007807079000026.jpg18107.

[0064] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid, [ka] (3) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0065] In embodiments, the subject has stage 2, stage 3, or stage 4 liver fibrosis.

[0066] In embodiments, the subject has cirrhosis.

[0067] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in stabilization of liver fibrosis in the subject.

[0068] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in the reversal of liver fibrosis in the subject.

[0069] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0070] In one aspect, the invention features a method of treating steatosis, the method comprising administering to a subject in need thereof an effective amount of a compound according to Formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, —CN, —NO2, —NH2, —NH(C1-C6)alkyl, —N[(C1-C6)alkyl)]2, —OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, thio(C1-C6)alkyl, -SONH2, -SO2NH2, selected from the group consisting of -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 2represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, -CN, -NO2, -NH2, -OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, Thio(C1-C6)alkyl, -SONH2, -SO2NH2, -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSO2NH2; R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 13 , and R 14 is independently selected from the group consisting of hydrogen and -(C1-C6)alkyl; R 5 and R 6 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 5 and R 6 cannot both be -OH; R 11 and R 12 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 11 and R 12 cannot both be -OH; m is an integer from 1 to 4, n is an integer of 0 to 4. o is an integer from 0 to 4, p is an integer from 1 to 4, q is an integer from 0 to 4, r is an integer of 0 to 4.

[0071] In embodiments, the compound of formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid, [ka] (1) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride, [ka] is.

[0072] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid, [ka] (2) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0073] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid, [ka] (3) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0074] In embodiments, the subject has a steatosis score of 1, 2, or 3.

[0075] In embodiments, administering the compound of Formula I or a pharmaceutically acceptable salt thereof results in a reduction of adiposity in the subject.

[0076] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0077] As contemplated in this disclosure with respect to the materials and methods of the disclosed compositions, in one aspect, embodiments of the disclosure comprise the components and / or steps disclosed herein. In another aspect, embodiments of the disclosure consist essentially of the components and / or steps disclosed herein. In yet another aspect, embodiments of the disclosure consist of the components and / or steps disclosed herein. [Brief explanation of the drawings]

[0078] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0079] The accompanying drawings, which are provided to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure.

[0080] [Figure 1]Figure 1 shows data on the effect of APL on glucose intolerance in a rat model of NASH. Figure 1 is a plot of the area under the curve (AUC) of the glucose tolerance test (GTT) in Zucker lean (Fa / fa) and obese (fa / fa) rats treated twice daily for 78 days with either vehicle (positive control) or APL (treatment). Two-way ANOVA and Bonferroni multiple comparison post-hoc test were performed. Significant statistical differences were observed between the positive control and treatment groups (**p<0.01).

[0081] [Figure 2] Figure 2, including Figures 2A and 2B, shows data on the effect of APL on glucose intolerance in a diet-induced mouse model of NASH. Figure 2A shows a plot of glucose levels in C57BL / 6J mice fed either a chow diet (CHOW) and vehicle (negative control), a high-fat / high-fructose diet (HFD) and vehicle (positive control), or an HFD and APL (treatment) for 131 days. After this, the animals were fasted, orally administered glucose (2 mg / g body weight), and blood glucose levels were measured. Two-way ANOVA with Bonferroni multiple comparison post-hoc test was performed. Significant statistical differences were observed between the positive control and treatment groups at 15 minutes (****p<0.0001), 60 minutes (*p<0.05), and 120 minutes (***p<0.001). Significant statistical differences were also observed between the negative control and positive control groups at 0 and 30 minutes (*p<0.05), 45 minutes (**p<0.01), and 15, 30, 60, and 120 minutes (****p<0.0001). There were no significant differences between the negative control and treatment groups at any time point. Figure 2B is a plot of the AUC of glucose levels in C57BL / 6J mice fed either CHOW or HFD. CHOW animals were treated twice daily with either vehicle (negative control) or APL for 120 days. HFD animals were treated with either vehicle (positive control), APL (treatment), or pioglitazone (Pio). Significant statistical differences were observed between the positive control and treatment groups (**p<0.01).

[0082] [Figure 3] Figure 3 shows liver biopsies at 20x magnification from C57BL / 6J mice fed either CHOW and vehicle (negative control), HFD and vehicle (positive control), or HFD and APL (TB-019, treatment) for 131 days. Sections were stained with hematoxylin and eosin (H&E). Sections from the positive control group showed severe hepatocellular fat accumulation, inflammatory infiltration, and pathological signs of steatohepatitis, especially in zone 3 of the liver, as well as ballooning. In contrast, liver sections from the treated groups showed minimal or no HFD-induced ballooning. Thus, APL (TB-019) was effective in reducing diet-induced microscopic liver injury.

[0083] [Figure 4] FIG. 4 shows liver biopsies at 100× magnification from C57BL / 6J mice fed either CHOW and vehicle (negative control), HFD and vehicle (positive control), or HFD and APL (treatment) for 131 days.

[0084] [Figure 5]Figure 5, including Figures 5A-5D, shows data on hepatic steatosis in a mouse model of NASH. C57BL / 6J mice were fed either CHOW or HFD. Mice were treated with vehicle or APL (po bid). The APL dose was 200 mg / kg / day, administered orally (po) twice daily (bid). Animals were fed the vehicle for APL treatment for 20 weeks. CHOW (vehicle): Mice fed chow and administered vehicle (negative control); HFD (vehicle): Mice fed HFD and administered vehicle (positive control); HFD (APL): Mice fed HFD and administered APL (treatment). Figure 5A contains representative images of hematoxylin and eosin (H&E)-stained liver tissue samples, and Figure 5B contains representative images of liver tissue samples immunostained with BODIPY and DAPI. Figures 5C and 5D show plots of the number of nuclei (Figure 5C) and fat deposits (Figure 5D). Measurements were performed on 20x magnification fields. More than 21 fields were analyzed from three different animals in each group: negative control (26 fields), positive control (25 fields), and treatment (23 fields). Fat deposits were calculated using indirect measurements using ImageJ (Schneider et al., 2012, Nature Methods 9(7):671-675). Fields containing large blood vessels were excluded from the count. Nuclei counts were assessed using Harmony® 4.6 High-Content Imaging and Analysis Software of Operetta CLS™ (PerkinElmer, Waltham, MA) and a PerkinElmer confocal microscope. One-way ANOVA and Tukey's multiple comparison post-hoc test were performed in both experiments. A significant statistical decrease in hepatocyte count was observed in the positive control (***p<0.001). There was no statistical difference between the negative control group and the treatment group. Furthermore, there was a significant statistical difference in fat deposition between the positive and negative controls (**p<0.01). No significant statistical difference in fat deposition was observed between the negative control group and the treatment group.

[0085] [Figure 6] Figure 6, including Figures 6A and 6B, shows data on nonalcoholic fatty liver disease activity scores (NAS) in diet-induced obesity (the DIO mouse model of NASH). C57BL / 6J mice were fed either a chow diet (CHOW) or a high-fat / high-fructose diet (HFD). Mice were treated with vehicle or APL (200 mg / kg body weight) pobid for 130 days. CHOW Vehicle: Mice fed chow and administered vehicle (negative control); HFD Vehicle: Mice fed HFD and administered vehicle (positive control); HFD APL: Mice fed HFD and administered APL (treated). Figure 6A includes representative images of liver tissue samples stained with hematoxylin and eosin (H&E). Arrow 1: Macrovesicular steatosis. Arrow 2: Microvesicular steatosis. Arrow 3: Hypertrophy. Arrow 4: Inflammatory foci. Figure 6B shows NAS component scores for steatosis (liver fat), inflammation, and hypertrophy in various treatment groups. Two-way ANOVA with Bonferroni multiple comparison post-hoc test was performed. Significant statistical differences were observed between the positive control group and the treatment groups (***p<0.001).

[0086] [Figure 7]Figure 7, including Figures 7A and 7B, shows data on NAS in the DIO mouse model of NASH. C57BL / 6J mice were fed either CHOW or HFD. Mice were treated with vehicle or APL (TB-019; 200 mg / kg body weight) pobid for 130 days. CHOW Vehicle: Mice fed chow and administered vehicle (negative control); HFD Vehicle: Mice fed HFD and administered vehicle (positive control); HFD APL: Mice fed HFD and administered APL (TB-019, treatment). Histopathological analysis of nonalcoholic steatohepatitis, NASH activity score (NAS), was performed. Figure 7A shows the mean total NAS score for the groups (negative control = 0.28 ± 0.46, positive control = 6 ± 1.76, treatment group = 1.2 ± 1.06). One-way ANOVA and Tukey's multiple comparison post-hoc test were performed. ANOVA analysis showed significant differences between groups (****p<0.0001). Post-hoc multiple comparison analysis (Tukey's test) also showed significant differences between all group pairs (negative control vs. positive control, ***q=23.38; negative control vs. treatment group, *q=3.55; and positive control vs. treatment group, ***q=18.5). Figure 7B shows data on pathological changes in the liver of the DIO mouse model of NASH, specifically (1) steatosis (st), (2) lobular inflammation (li), and (3) hepatic ballooning (hb). Two-way ANOVA RM and Bonferroni multiple comparison post-hoc test were performed. Significant statistical differences were observed between the negative control group and the positive control group in all comparisons (****p<0.0001). Significant statistical differences were observed between the negative control group and the treatment group for lobular inflammation (****p<0.0001). In all comparisons, significant statistical differences were observed between the treatment groups and the positive control (****p<0.0001).

[0087] [Figure 8]Figure 8, including Figures 8A, 8B, and 8C, shows data on liver collagen fibers in a rat NASH model. Figure 8A shows representative images (40x magnification) of hematoxylin and eosin-stained liver sections from Zucker (Fa / fa) lean rats, Zucker fa / fa obese rats administered vehicle, and Zucker (fa / fa) obese rats administered vehicle and APL. Figure 8B shows representative images (100x magnification) of Mason's trichrome-stained liver sections. Figure 8C shows an enlargement of the area within the red box in the image of Figure 8B. The arrows point to collagen fibers in the mesenchymal liver tissue of vehicle-treated Zucker obese rats.

[0088] [Figure 9] Figure 9, including Figures 9A, 9B, and 9C, shows data on hepatic 4-hydroxynonenal (4-HNE) in a rat NASH model. Figure 9A shows representative images of 4-HNE immunofluorescent liver sections from Zucker (Fa / fa) lean rats, Zucker (fa / fa) obese rats treated with vehicle (negative control), and Zucker (fa / fa) obese rats treated with vehicle (positive control) or APL (treatment). Confocal microscopy of liver tissue at 63x magnification. Figures 9B and 9C show plots of quantification of 4HNE / immunofluorescence intensity (4HNE intensity per μm2 of tissue area) and total tissue area, respectively. One-way ANOVA and Tukey's multiple comparison post-hoc test were performed in both experiments. Significant statistical differences were observed between the control and APL-treated groups (***p<0.001). No significant statistical differences were observed between the negative control group and the treatment groups (Ns = non-statistical difference).

[0089] [Figure 10]Figure 10, including Figures 10A and 10B, shows data on hepatic 4-hydroxynonenal (4-HNE) in the DIO mouse model of NASH. Figure 10A shows representative images of 4-HNE immunofluorescent liver sections from C57BL / 6 mice fed CHOW and treated with vehicle (negative control; left image), and C57BL / 6 mice fed an HFD and treated with either vehicle (positive control; middle image) or APL (TB-019; treatment; right image). Confocal microscopy of liver tissue at 20x and 63x magnification. Figure 10B shows a plot of quantification of 4HNE / immunofluorescence intensity (4HNE intensity per μm2 of tissue area). One-way ANOVA and Tukey's multiple comparison post-hoc test were performed. Significant statistical differences were observed between the positive control group and the treatment group (***p<0.001). No statistical differences were observed between the negative control group and the treatment group.

[0090] [Figure 11]Figure 11, including Figures 11A, 11B, 11C, 11D, 11E, and 11F, shows data on surrogate serum biomarkers in the AMLN diet mouse model of NASH. C57BL / 6J mice were fed either a CHOW or modified AMLN diet and switched to an HFD after 20 weeks. At week 50, mice were treated with vehicle or APL (po bid). APL doses of 25, 50, or 100 mg / kg were administered orally (po) twice daily (bid) (total dose of 50, 100, or 200 mg / kg / day). After 16 weeks of treatment with vehicle or APL, samples were collected for analysis. Dunnett's multiple comparison test was performed. Figure 11A shows serum alanine aminotransferase (ALT) levels. Figure 11B shows serum aspartate aminotransferase (AST) levels. Figure 11C shows serum alkaline phosphatase (ALP) levels. Figure 11D shows serum triglyceride levels. Figure 11E shows serum non-esterified fatty acid (NEFA) levels. Figure 11F shows serum cholesterol levels. All results shown were obtained from mice administered 50 mg / kg / day of APL, and for cholesterol only, from mice administered 200 mg / kg / day of APL. Significant statistical differences were observed between the positive control group and the treatment groups (*p<0.02; **p=0.002; ***p=0.0008, and ****p<0.0001).

[0091] [Figure 12]Figure 12, including Figures 12A and 12B, shows data on NAS and fibrosis in the AMLN diet mouse model of NASH. C57BL / 6J mice were fed either a CHOW or modified AMLN diet and switched to an HFD after 20 weeks. At week 50, mice were treated with vehicle or APL (po bid). APL doses of 25, 50, or 100 mg / kg were administered orally (po) twice daily (bid) (total dose of 50, 100, or 200 mg / kg / day). After 16 weeks of treatment with vehicle or APL, biopsies were collected, stained with hematoxylin and eosin (H&E), and evaluated for steatosis, inflammation, and hepatocyte ballooning. Figure 12A shows a reduction in NAS in the treatment group compared to the positive control. Significant statistical differences were observed between the positive control and treatment groups (***p<0.001). Figure 12B shows a reduction in liver fibrosis in the treatment group compared to the positive control. A significant statistical difference was observed between the positive control group and the treatment group (****p<0.0001).

[0092] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0093] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0094] The articles "a" and "an" are used in this disclosure to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, reference to a "cell," for example, includes multiple cells of the same type.

[0095] As used herein, the term "about," when referring to a measurable value, e.g., amount, duration, etc., is intended to encompass variations of ±20%, or ±10%, ±5%, ±1%, or ±0.1% from the particular value, where such variations are appropriate for the practice of the invention.

[0096] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbyl having the specified number of carbon atoms (i.e., C-C means 1 to 6 carbons). Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, and hexyl. In embodiments, alkyl is a (C-C) alkyl, such as a (C-C) alkyl (e.g., methyl and ethyl).

[0097] The term "alkenyl," used alone or in combination with other terms, means, unless otherwise stated, a straight or branched chain hydrocarbyl having the specified number of carbon atoms and containing one or more double bonds. Examples include ethenyl (vinyl), propenyl (allyl), crotyl, isopentenyl, butadienyl, 1,3-pentadienyl, and 1,4-pentadienyl. A functional group representing alkenyl is exemplified by -CH-CH=CH-.

[0098] The term "alkynyl," employed alone or in combination with other terms, means, unless otherwise stated, straight or branched chain hydrocarbyl having the stated number of carbon atoms and containing one or more triple bonds.

[0099] The term "alkoxy," employed alone or in combination with other terms, means, unless otherwise stated, an alkyl group, as defined above, attached to the remainder of the molecule via an oxygen atom, including, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy), and higher homologs and isomers thereof. The alkyl portion of the alkoxy group can have the specified number of carbon atoms, as defined above for the alkyl group. In embodiments, the alkoxy group is a (C-C)alkoxy, such as a (C-C)alkoxy (e.g., methoxy and ethoxy).

[0100] As used herein, the terms "APL" and "TB-019" refer to the compound of formula I, which is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (compound (1)), or a pharmaceutically acceptable salt thereof.

[0101] The term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings that have aromatic properties (i.e., have (4n+2) delocalized π (pi) electrons, where n is an integer).

[0102] The term "aryl" refers to an aromatic hydrocarbon ring system containing at least one aromatic ring. The aromatic ring may optionally be fused or otherwise attached to other aromatic or non-aromatic hydrocarbon rings. Examples of aryl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene, and biphenyl. Examples of aryl groups include phenyl and naphthyl.

[0103] The term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0104] As used herein, "effective amount" means an amount of a compound that, when administered to a subject in need thereof (e.g., a patient suffering from or at risk of developing a disease or condition such as NASH), provides a therapeutic benefit in alleviating one or more symptoms of the disease or condition (e.g., preventing, inhibiting, treating, or reducing the symptoms of a particular disorder or disease (e.g., NASH)). However, it is understood that the full therapeutic effect may not necessarily occur by administering a single dose, but may occur after administering a series of doses. Thus, an effective amount may be administered in one or more administrations. In the context of therapeutic (including prophylactic) applications, the amount of active agent administered to a subject will depend on the type and severity of the disease or condition, as well as the subject's characteristics, such as general health, age, sex, weight, and tolerance to drugs. It will also depend on the degree, severity, and type of the disease or condition. One of skill in the art will be able to determine appropriate dosages depending on these and other factors. The compounds of Formula I can also be administered in combination with one or more additional therapeutic compounds.

[0105] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. In embodiments, halogen includes fluorine, chlorine, or bromine. In embodiments, halogen is fluorine or chlorine.

[0106] The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group.

[0107] The terms "heterocycle" or "heterocyclyl" or "heterocyclic," by themselves or as part of another substituent, mean, unless otherwise stated, an unsubstituted or substituted mono- or polycyclic heterocyclic ring system consisting of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S. Heterocycles typically contain 5 to 10 ring atoms. A heterocyclic ring system may be attached to another atom at any heteroatom or carbon atom of the heterocyclic ring system, resulting in structural isomers, unless otherwise stated.

[0108] The terms "heteroaryl" or "heteroaromatic" refer to a heterocycle having aromatic character.

[0109] Unless otherwise stated, the term "hydrocarbyl," by itself or as part of another substituent, means a straight or branched chain hydrocarbon having the specified number of carbon atoms (i.e., C1-C6 means 1 to 6 carbons). Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, and hexyl. In embodiments, the hydrocarbyl is a (C1-C6) alkyl, such as a (C1-C3) alkyl, such as methyl and ethyl. The term "unsaturated hydrocarbyl" means a hydrocarbyl containing at least one double or triple bond.

[0110] The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom is replaced by a halogen atom. The term "perhaloalkyl" refers to a haloalkyl group in which all hydrogen atoms are replaced by halogen atoms. Perhaloalkyl includes perfluoroalkyl, such as -(C-C)perfluoroalkyl (e.g., -(C-C)perfluoroalkyl group, e.g., -CF).

[0111] The term "haloalkoxy" refers to an alkoxy group in which at least one hydrogen atom is replaced by a halogen atom. The term "perhaloalkoxy" refers to a haloalkoxy group in which all hydrogen atoms are replaced by halogen atoms. Perhaloalkoxy groups include perfluoroalkoxy groups such as -(C-C)perfluoroalkoxy (e.g., -(C-C)perfluoroalkoxy, e.g., -OCF).

[0112] As used herein, "individual" or "patient" or "subject" (as in the case of a subject of treatment) refers to both mammals and non-mammals. Mammals include, for example, humans; non-human primates, such as apes and monkeys; dogs; cats; cows; horses; sheep; and goats. Non-mammals include, for example, fish and birds. In one embodiment, the individual is a human.

[0113] As used herein, the term "pharmaceutically acceptable" refers to a formulation of a compound that does not significantly interfere with the biological activity, pharmacological activity, and / or other properties of the compound when the formulated compound is administered to a patient. In embodiments, a pharmaceutically acceptable formulation does not cause significant irritation to a patient.

[0114] The term "substituted" means that an atom or group of atoms replaces a hydrogen atom as a substituent attached to another group. For aryl and heteroaryl groups, the term "substituted" refers to any level of substitution, i.e., mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. Substituents are independently selected, and substitution can occur at any chemically accessible position. Substituents can include, for example, one of the moieties from the group: halo, oxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, aryl, and amino. Substituents containing carbon chains can contain 1 to 6, 1 to 3, or 1 to 2 carbon atoms.

[0115] As used herein, the terms "treat" and "treatment" in reference to a disease or condition (e.g., NASH) are used interchangeably and refer to taking measures to obtain a beneficial or desired clinical result in an individual's prevalence or risk of a disease or condition (e.g., NASH), including preventing, maintaining, inhibiting, treating, or reducing the symptoms of the disease or condition. Thus, treating a patient can include preventing or postponing (e.g., preventing or maintaining) further disease progression, preventing or reducing the severity of existing or predicted to develop symptoms, improving existing symptoms and preventing further symptoms, and / or prophylactic treatment of a subject at risk of developing a condition (e.g., NASH), resulting in a reduced likelihood that the subject will develop the condition.

[0116] Ranges: Throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. DETAILED DESCRIPTION OF THE INVENTION

[0117] Embodiments of the present disclosure are described below. However, it should be particularly noted that the present disclosure is not limited to these embodiments, but is intended to include modifications and equivalents thereof that are apparent to those skilled in the art.

[0118] Nonalcoholic fatty liver disease (NAFLD) generally describes a range of conditions in which excess fat accumulates in the liver and is not caused by excessive alcohol consumption. For example, simple fatty liver (nonalcoholic fatty liver or NAFL) is a condition in which the liver is fatty and there is little or no hepatitis or cellular damage. This condition usually does not progress to the point of causing liver damage or other complications. However, nonalcoholic steatohepatitis (NASH) is a separate condition in which subjects have liver inflammation (hepatitis) and / or cellular damage in addition to fatty liver, potentially leading to fibrosis (including cirrhosis) or liver cancer. Only a subset of subjects with NAFLD (approximately 20%) have NASH, whereas approximately 3-12% of US adults have NASH. However, NASH has been identified as the second leading cause of liver transplant waiting list enrollment and liver transplantation. Therefore, the severity of outcomes in patients with NASH indicates the urgent need for effective treatment.

[0119] Currently, treatment of NAFLD and NASH relies on dietary and other lifestyle modifications rather than drug therapy. In particular, the development of effective therapeutic agents for NASH and related pathologies or complications (e.g., fibrosis and cirrhosis) has presented considerable challenges.

[0120] Surprisingly, it has been found that compounds of formula I are effective in treating liver diseases and disorders, including non-alcoholic steatohepatitis (NASH), as well as conditions such as liver fibrosis (including cirrhosis), hepatic steatosis, hepatitis (e.g., lobular inflammation), and hepatocellular ballooning, which may be associated with NASH or may occur independently of NASH.

[0121] For example, the compound of formula I, or its pharmaceutically acceptable salt, has been found to be effective in treating non-alcoholic steatohepatitis (NASH), as shown in art-recognized rodent models of NASH.In particular, the compound of formula I has been found to improve diabetes-related impaired glucose tolerance (IGT), reduce hepatic fat deposition, attenuate hepatic steatosis, attenuate hepatic fibrosis, hepatic inflammation, hepatic hypertrophy, and treat NASH.Therefore, the compound of formula I, or any pharmaceutically acceptable salt thereof, can be useful for treating (for example, preventing, inhibiting, reducing, or maintaining) any of these conditions, including those described herein.

[0122] For example, individuals suffering from or at risk of non-alcoholic steatohepatitis (NASH) or other liver pathologies, including steatosis, hepatitis (e.g., lobular inflammation), hepatocyte ballooning, and / or fibrosis (e.g., cirrhosis), may benefit from treatments comprising administration of a compound according to Formula I or a pharmaceutically acceptable salt thereof.

[0123] Compounds of Formula I The compound for use in the method of treating nonalcoholic steatohepatitis (NASH) is a compound according to Formula I: [ka] (I) and pharmaceutically acceptable salts thereof, During the ceremony, R 1 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C 8) Alkynyl, unsubstituted or substituted -ar(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, -CN, -NO2, -NH2, selected from the group consisting of -NH(C1-C6)alkyl, -N[(C1-C6)alkyl)]2, -OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, thio(C1-C6)alkyl, -SONH2, -SON2NH2, -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSON2NH2; R 2 represents hydrogen, -(C1-C8) alkyl, -(C1-C8) alkenyl, -(C1-C8)alkynyl, unsubstituted or substituted -(C1-C6)alkyl, unsubstituted or substituted -heteroara(C1-C6)alkyl, wherein the substituents on the substituted ara(C1-C6)alkyl and substituted heteroara(C1-C6)alkyl are selected from the group consisting of halogen, -CN, -NO2, -NH2, -OH, halo(C1-C6)alkyl, -(C1-C6)alkoxy, halo(C1-C6)alkoxy, -SH, thio(C1-C6)alkyl, -SONH2, -SON2NH2, -SO-(C1-C6)alkyl, -SO2-(C1-C6)alkyl, -NHSO2(C1-C6)alkyl, and -NHSON2NH2; R 3 , R 4 , R 7 , R 8 , R 9 , R 10 , R 13 , and R 14 is independently selected from the group consisting of hydrogen and -(C1-C6)alkyl; R 5 and R 6 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 5 and R 6 cannot both be -OH; R 11 and R 12 are independently hydrogen, -(C1-C6) alkyl, and -OH, where R 11 and R12 cannot both be -OH; m is 1, 2, 3, or 4; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, 3, or 4; p is 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; r is 0, 1, 2, 3, or 4.

[0124] In some embodiments, R 1 and / or R 2 wherein halo(C1-C6)alkyl and / or halo(C1-C6)alkoxy are selected from perhalo(C1-C6)alkyl and perhalo(C1-C6).

[0125] In some embodiments, R 1 is selected from hydrogen and —(C1-C8) alkyl. In some embodiments, R 2 is selected from hydrogen or -(C1-C8) alkyl. In some embodiments, R 1 and R 2 is independently selected from hydrogen and -(C1-C8)alkyl. In the above embodiments, -(C1-C8)alkyl is -(C1-C6)alkyl, -(C1-C3)alkyl, or methyl or ethyl. In some embodiments, R 1 and R 2 is hydrogen.

[0126] In some embodiments, R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 is independently selected from hydrogen and -(C-C) alkyl. -(C-C) alkyl is -(C-C) alkyl, -(C-C) alkyl, or methyl or ethyl. In some embodiments, R 3 , R 4 , R 5 , R 6 , R 7 , and R8 is hydrogen.

[0127] In some embodiments, R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 is independently selected from hydrogen and -(C-C) alkyl. -(C-C) alkyl is -(C-C) alkyl, -(C-C) alkyl, or methyl or ethyl. In some embodiments, R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 is hydrogen.

[0128] In some embodiments, R 3 ~R 14 are each independently selected from hydrogen and —(C1-C8)alkyl according to the scheme above. In some embodiments, R 3 ~R 14 is hydrogen.

[0129] In embodiments of compounds of Formula I, the sum of m+n+o ranges from 2 to 10, 9, 8, 7, 6, 5, 4, or 3; from 3 to 10, 9, 8, 7, 6, 5, or 4; or from 4 to 10, 9, 8, 7, 6, or 5. In embodiments, the sum of m+n+o is 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0130] In embodiments of compounds of Formula I, the sum of p+q+r ranges from 1 to 10, 9, 8, 7, 6, 5, 4, 3, or 2; from 2 to 10, 9, 8, 7, 6, 5, 4, or 3; from 3 to 10, 9, 8, 7, 6, 5, or 4; or from 4 to 10, 9, 8, 7, 6, or 5. In embodiments, the sum of p+q+r is 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0131] In some of the foregoing embodiments that define the sum of m+n+o and / or that define the sum of p+q+r, R 3 ~R 14 Each of R is independently selected from hydrogen and —(C-C) alkyl. 3 ~R 14 is hydrogen.

[0132] In embodiments of the compound of Formula I, m is 3; p is 4; and each of n, o, q, and r is zero. In embodiments, R 3 , R 4 , R 9 , and R 10 is independently selected from hydrogen and —(C1-C8) alkyl. In some embodiments, R 3 , R 4 , R 9 , and R 10 are independently hydrogen. In some embodiments, R 1 and R 2 may be independently selected from hydrogen and —(C-C) alkyl. In some embodiments, R 1 and R 2 may independently be hydrogen. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is hydrogen. In embodiments, the compound of Formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (compound (1)), or a pharmaceutically acceptable salt thereof.

[0133] In some embodiments of the compound of Formula I, m is 4; n is 1 or 2; p is 3; and each of o, q, and r is zero. In some embodiments, R 3 , R 4 , R 9 , and R 10 is independently selected from hydrogen and —(C1-C8) alkyl. In some embodiments, R 3 , R4 , R 9 , and R 10 are independently hydrogen. In some embodiments, R 1 and R 2 may be independently selected from hydrogen and —(C-C) alkyl. In some embodiments, R 1 and R 2 may be independently selected from hydrogen. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is hydrogen. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid, or a pharmaceutically acceptable salt thereof, or (S)-2-amino-5-((6-aminopentyl)amino)pentanoic acid, or a pharmaceutically acceptable salt thereof.

[0134] In some embodiments, R 1 and R 2 Each is hydrogen. In some embodiments, R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 Each is hydrogen. In some embodiments, R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 Each is hydrogen. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , and R 14are each hydrogen. In embodiments, (m+n+o) is 3. In embodiments, (m+n+o) is 4. In embodiments, (m+n+o) is 5. In embodiments, (m+n+o) is 6. In embodiments, (p+q+r) is 3. In embodiments, (p+q+r) is 4. In embodiments, (p+q+r) is 5. In embodiments, (p+q+r) is 6.

[0135] In some embodiments, a pharmaceutically acceptable salt of a compound of Formula I is used in the methods described herein. Exemplary pharmaceutically acceptable salts are described herein. In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt of a compound of Formula I (e.g., a monohydrochloride, dihydrochloride, or trihydrochloride salt of a compound of Formula I).

[0136] In embodiments, the compound of formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid, [ka] (1) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride, [ka] is.

[0137] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid, [ka] (2) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0138] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid, [ka] (3) or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride, [ka] is.

[0139] Compound synthesis Compounds of Formula I may be prepared according to methods known in the art. Exemplary syntheses are described herein.

[0140] Compound (1) An exemplary method for preparing the dihydrochloride salt of the compound of Formula I, (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (compound (1), dihydrochloride salt), is as follows. A. Preparation of tert-butyl (S)-(2-oxoazepan-3-yl)carbamate: [ka]

[0141] Di-tert-butyl dicarbonate (733 μL, 3.189 mmol) was added to a suspension of L-(-)-α-amino-ε-caprolactam hydrochloride (500 mg, 3.037 mmol) and triethylamine (847 μL, 6.074 mmol) in anhydrous tetrahydrofuran (4 mL). The resulting suspension was stirred overnight at room temperature and concentrated. The residual white solid was partitioned between ethyl acetate and water. The aqueous layer was removed. The organic layer was washed twice with 1N aqueous hydrochloric acid, twice with saturated aqueous sodium bicarbonate, and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated. Pure title compound (tert-butyl (S)-(2-oxoazepan-3-yl)carbamate) was obtained as a white solid. 1 H NMR(400 MHz,CD3OD)δ 6.45(bd,J = 5.8 Hz,1H),4.18-4.30(m,1H),3.17-3.30(m,2H),1.70-2.03(m,4H),1.48-1.57(m,1H),1.45(s,9H),1.28-1.42(m,1H);MS(ESI):m / z 250.8(M+Na) + . B. Preparation of tert-butyl (S)-(1-(3-((tert-butoxycarbonyl)amino)propyl)-2-oxoazepan-3-yl)carbamate: [ka]

[0142] Bis(trimethylsilyl)amide (2.524 mmol; 2.5 mL of a 1.0 M solution in tetrahydrofuran) was added to a solution of tert-butyl (S)-(2-oxoazepan-3-yl)carbamate (288 mg, 1.262 mmol) in anhydrous tetrahydrofuran (12 mL).

[0143] The resulting suspension was stirred at room temperature for 30 minutes. 3-(Boc-amino)propyl bromide (2.524 mmol; 470 μL) was added all at once and the reaction was stirred at room temperature for 28 hours.

[0144] The reaction mixture was concentrated on a rotary evaporator, and the residue was partitioned between ethyl acetate and water. The aqueous layer was removed. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography on silica gel using a gradient solvent system of 0 to 100% ethyl acetate in hexane to give the title compound (tert-butyl (S)-(1-(3-((tert-butoxycarbonyl)amino)propyl)-2-oxoazepan-3-yl)carbamate) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ5.96(bd,J=5.0Hz,1H),5.32(bs,1H),4.36(m,1H),3.45-3.62(m,2H),3.33-3.41(m,1H),3.08-3.22(m,2H),2.97-3. 06(m,1H),2.02-2.09(m,1H),1.92-2.00(m,1H),1.76-1.87(m,2H),1.61-1.70(m,2H),1.40-1.50(m,19H),1.31-1.38(m,1H);MS(ESI):m / z 407.8(M+Na) + . C. Preparation of (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride (Compound (1), dihydrochloride salt): [ka]

[0145] (S)-tert-Butyl (1-(3-((tert-butoxycarbonyl)amino)propyl)-2-oxoazepan-3-yl)carbamate (100 mg, 0.2596 mmol) was dissolved in 12 N aqueous hydrochloric acid (4 mL). The resulting solution was stirred at room temperature until all effervescence subsided. The solution was transferred to a microwave reaction vial and heated at 160° C. for 90 minutes. Concentration afforded the pure title compound ((S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride) as a light tan solid. 1H NMR(400MHz,D2O)δ4.00(t,J=6.3Hz,1H),3.08-3.20(m,6H),1.90-2.15(m,4H),1.72-1.83(m,2H),1.43-1.62(m,2H);MS(ESI):m / z 203.9(M+H) + .

[0146] The solubility, liver microsomal stability, and solution stability of (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid ("APL") have been determined. See WO2018 / 049019 and U.S. Publication No. 2019 / 0192462.

[0147] Compound (2) An exemplary method for preparing the compound of Formula I, (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride (compound (2), trihydrochloride salt), is as follows. A. Preparation of tert-butyl (6-oxohexyl)carbamate: [ka]

[0148] Anhydrous dimethyl sulfoxide (83 μL) was added dropwise to a stirred solution of oxalyl chloride (50 μL) in anhydrous dichloromethane (2 mL) at −78° C. After stirring for 15 minutes, a solution of 6-(tert-butoxy-carbonylamino)-1-hexanol (115 mg, 0.53 mmol) in anhydrous dichloromethane (1 mL) was added dropwise. The resulting mixture was stirred at −78° C. for 45 minutes. Triethylamine (368 μL) was added, and the reaction was allowed to warm to room temperature. The solution was concentrated on a rotary evaporator to give the title compound (tert-butyl (6-oxohexyl)carbamate) as an off-white solid (86 mg, 75% yield), which was used without further purification. B. Preparation of (S)-2-(((benzyloxy)carbonyl)amino)-5-((6-((tert-butoxycarbonyl)amino)hexyl)amino)-pentanoic acid [ka]

[0149] To a stirred suspension of 94 mg (0.352 mmol) of N-alpha-benzyloxycarbonyl-L-ornithine in anhydrous methanol (2 mL) containing acetic acid (100 μL) was added a solution of tert-butyl (6-oxohexyl)carbamate (114 mg, 0.528 mmol) in anhydrous methanol (1.9 mL). The resulting mixture was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (66 mg, 1.057 mmol) was then added, and the reaction was stirred at room temperature overnight. After concentration on a rotary evaporator, the residue was partitioned between ethyl acetate and 1 M aqueous potassium hydrogen sulfate. The aqueous layer was removed. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The resulting residue was purified by reverse-phase chromatography (C18 column) using a gradient of 10 to 100% acetonitrile in water containing 0.1% formic acid modifier. The title compound ((S)-2-(((benzyloxy)carbonyl)amino)-5-((6-((tert-butoxycarbonyl)amino)hexyl)amino)-pentanoic acid) (87 mg, 53% yield) was obtained as a pale yellow oil. 1 H NMR(400MHz,D2O)δ3.94(t,J=5.92Hz,0.5H),3.63(m,0.5H),2.99-3.13(m,6H),1.65-2.04(m,8H),1.43(m,4H);MS(ESI):m / z 466.2[(M+H) + ]. C. Preparation of (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride [ka]

[0150] A solution of (S)-2-(((benzyloxy)carbonyl)amino-5-((6-((tert-butoxycarbonyl)amino)hexyl)amino)pentanoic acid (18 mg, 0.039 mmol) in 6 N aqueous hydrochloric acid (4 mL) was refluxed for 2 hours. The solution was concentrated on a rotary evaporator to give the title compound ((S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride) (12 mg, 90% yield) as a pale yellow oil. 1 H NMR(400MHz,D2O)δ4.27(m,0.5H),3.95(m,0.5H),3.33-3.48(m,6H),2.00-2.37(m,8H),1.77(m,4H);MS(ESI):m / z 232.2[(M+H) + ].

[0151] Compound (3) An exemplary method for preparing the compound of Formula I, (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride (compound (3), trihydrochloride salt), is as follows. A. Preparation of tert-butyl (5-oxopentyl)carbamate [ka]

[0152] Anhydrous dimethyl sulfoxide (58 μL) was added dropwise to a stirred solution of oxalyl chloride (35 μL) in anhydrous dichloromethane (1.5 mL) at −78° C. After stirring for 15 minutes, a solution of 6-(tert-butoxy-carbonylamino)-1-pentanol (75 mg, 0.37 mmol) in anhydrous dichloromethane (0.75 mL) was added dropwise. The resulting mixture was stirred at −78° C. for 45 minutes. Triethylamine (257 μL) was added, and the reaction was allowed to warm to room temperature. The solution was concentrated on a rotary evaporator to afford the title compound (tert-butyl (5-oxopentyl)carbamate) as an off-white solid (52 mg, 70% yield), which was used without further purification. B. Preparation of (S)-2-(((benzyloxy)carbonyl)amino)-5-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-pentanoic acid [ka]

[0153] To a stirred suspension of 35 mg (0.13 mmol) of N-alpha-benzyloxycarbonyl-L-ornithine in anhydrous methanol (1 mL) containing acetic acid (38 μL) was added a solution of tert-butyl (5-oxopentyl)carbamate (40 mg, 0.20 mmol) in anhydrous methanol (1.0 mL). The resulting mixture was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (25 mg, 0.40 mmol) was then added, and the reaction was stirred at room temperature overnight. After concentration on a rotary evaporator, the residue was partitioned between ethyl acetate and 1 M aqueous potassium hydrogen sulfate. The aqueous layer was removed. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The resulting residue was purified by reverse-phase chromatography (C18 column) using a gradient of 10 to 100% acetonitrile in water containing 0.1% formic acid modifier. The title compound ((S)-2-(((benzyloxy)carbonyl)amino)-5-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-pentanoic acid) (34 mg, yield 58%) was obtained as a colorless oil. 1 H NMR(400MHz,CD3OD)δ7.26-7.38(m,5H),5.08(s,2H),4.03(m,1H),2.90-3. 07(m,6H),1.87(m,1H),1.65-1.79(m,5H),1.34-1.54(m,13H);MS(ESI):m / z 452.30[(M+H) + ]. C. Preparation of (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride [ka]

[0154] A solution of (S)-2-(((benzyloxy)carbonyl)amino-5-((5-((tert-butoxycarbonyl)amino)pentyl)amino)pentanoic acid (20 mg, 0.044 mmol) in 6 N aqueous hydrochloric acid (4 mL) was refluxed for 2 h. The solution was concentrated on a rotary evaporator to give the title compound ((S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride) (12 mg, 88% yield) as a pale yellow oil. 1 H NMR(400MHz,CD3OD)δ4.06(t,J=5.36Hz,1H),3.06(m,4H),2.96(t,J=7.52Hz ,2H),1.88-2.10(m,4H),1.68-1.83(m,4H),1.47-1.55(m,2H);MS(ESI):m / z 218.2[(M+H) + ].

[0155] Those skilled in the art will understand that the processes illustrated in Schemes 1-16 of WO 2018 / 049019 and U.S. Publication No. 2019 / 0192462 are not the only means by which compounds of Formula I may be synthesized, and that there is a repertoire of synthetic organic reactions available that may be employed in synthesizing the compounds of the present disclosure. Those skilled in the art will know how to select and implement appropriate synthetic routes. Suitable synthetic methods may be found in, for example, Comprehensive Organic Synthesis, Ed. B.M. Trost and I. Fleming (Pergamon Press, 1991), Comprehensive Organic Functional Group Transformations, Ed. A.R. Katrittzky, O. Meth-Cohn, and C.W. Rees (Pergamon Press, 1996), Comprehensive Organic Functional Group Transformations II, Ed. A.R. Katrittzky and R.J.K. Taylor (Editors) (Elsevier, 2002). ndHeterocyclic compounds may be identified by reference to literature, including reference sources such as A.R. Katrittzky and C.W. Rees (Pergamon Press, 1984), Comprehensive Heterocyclic Chemistry, Eds. A.R. Katrittzky and C.W. Rees (Pergamon Press, 1984), and Comprehensive Heterocyclic Chemistry II, Eds. A.R. Katrittzky, C.W. Rees, and E.F.V. Scriven (Pergamon Press, 1996).

[0156] Compounds of formula I and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid-phase extraction, distillation, recrystallization, or chromatography.

[0157] It will be understood that when the compounds of Formula I contain one or more chiral centers, the compounds may exist or be isolated as pure enantiomeric or diastereomeric forms, or as racemic mixtures. Accordingly, the present disclosure includes any possible enantiomers, diastereomers, racemates, or mixtures thereof of the compounds of the present disclosure that are biologically active in the treatment of NASH.

[0158] A chiral center occurs at the α-carbon of the α-amino acid functional group of the compound of formula I. The compound of formula I is characterized by the (S) absolute configuration with respect to the α-carbon of the α-amino acid functional group involved, according to the Cahn-Ingold-Prelog rules. [ka] As exemplified by the compound of formula I, the compound (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (compound (1)). [ka] (1) (S)-2-Amino-6-((3-aminopropyl)amino)hexanoic acid

[0159] According to embodiments, the compound of Formula I is an isolated (S) enantiomer, relative to the configuration around the α-carbon of the α-amino acid functional group involved. "Isolated enantiomer" refers to a compound that has been substantially purified from the corresponding enantiomer of the same formula. In embodiments, the isolated isomer is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least about 99% pure by weight, with the remainder being the corresponding (R) enantiomer. In embodiments, the isolated (S) enantiomer is free of the corresponding (R) enantiomer, except for minor amounts of the (R) enantiomer.

[0160] Pharmaceutically acceptable salts The compounds of Formula I may take the form of salts when appropriately substituted with groups or atoms capable of forming salts. Such groups or atoms are known to those skilled in the art of organic chemistry. The term "salt" encompasses addition salts of free acids or free bases that are compounds of the present disclosure. The term "pharmaceutically acceptable salt" refers to salts that possess a toxicity profile within a range that makes them useful in pharmaceutical applications. Pharmaceutically unacceptable salts may possess properties such as high crystallinity, but still have utility in the practice of the present disclosure, for example, usefulness in the process of synthesizing, purifying, or formulating the compounds of the present invention.

[0161] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids may be selected from aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carbocyclic, and sulfonic organic acids, examples of which include formic acid, acetic acid, pivalic acid, propionic acid, furoic acid, mucic acid, isethionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthracite, and the like. Examples of pharmaceutically unacceptable acid addition salts include benzoic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, camphorsulfonic acid, and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.

[0162] In some embodiments, a suitable pharmaceutically acceptable salt is a hydrochloride salt of a compound described herein (e.g., the hydrochloride salt of any one of Compound (1), Compound (2), and Compound (3)). In some embodiments, the hydrochloride salt is a monochloride salt. In some embodiments, the hydrochloride salt is a dihydrochloride salt. In some embodiments, the hydrochloride salt is a trihydrochloride salt.

[0163] Suitable pharmaceutically acceptable base addition salts of the compounds of the present disclosure include, for example, metallic salts, including alkali metal, alkaline earth metal, and transition metal salts, such as calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts formed from basic amines, such as N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, tromethamine, meglumine (N-methylglucamine), and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts.

[0164] All of these salts can be prepared by conventional means from the corresponding compound of formula I, for example, by reacting the compound of formula I with an appropriate acid or base. The salts can be in crystalline form and can be prepared by crystallizing the salt from an appropriate solvent. A person skilled in the art will know how to prepare and select suitable salt forms, for example, as described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use by P.H. Stahl and C.G. Wermuth (Wiley-VCH 2002).

[0165] Treatment method Nonalcoholic steatohepatitis (NASH) In one aspect, the invention features a method of treating nonalcoholic steatohepatitis (NASH), the method comprising administering to a subject in need thereof an effective amount of a compound according to Formula I, or a pharmaceutically acceptable salt thereof. In another aspect, the invention features a compound according to Formula I, or a pharmaceutically acceptable salt thereof, for use in treating NASH in a subject. In another aspect, the invention features a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of NASH.

[0166] Nonalcoholic fatty liver disease (NAFLD) has become one of the most prominent forms of chronic liver disease worldwide, reflecting the obesity epidemic. Hepatic steatosis, or "fatty liver," is the accumulation of fat in the liver. Apart from NAFLD, nonalcoholic steatohepatitis (NASH) is a disorder characterized by substantial health risks. In addition to the presence of excess fat in the liver, NASH can be characterized by histological evidence of liver inflammation and hepatocellular injury (ballooning), with or without fibrosis. Subjects diagnosed with NASH are at significantly increased risk of morbidity and mortality and continually require therapeutic agents for the treatment of NASH.

[0167] More specifically, NASH can be characterized by an increased risk of cardiovascular and liver-related mortality. NASH can lead to cirrhosis, in which the liver becomes permanently damaged and scarred. Cirrhosis results in fluid retention, muscle wasting, intestinal bleeding, and liver failure. Liver transplantation is the only treatment for advanced cirrhosis with liver failure. Transplants are increasingly being performed in patients with NASH. NASH is currently the second leading reason for liver transplantation and is very likely to become the leading cause in 10 years as new antiviral drugs suppress hepatitis C, currently the leading cause of liver failure.

[0168] In some embodiments, NASH can be diagnosed by liver biopsy (e.g., histological evidence of, e.g., steatosis, inflammation, and hepatocellular ballooning, in the absence of other causes of liver disease or substantial alcohol consumption). For example, the NAFLD Activity Score (NAS) can be useful in identifying patients with NASH. See Kleiner et al., "Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease," Hepatology, 41(6):1313-1321 (2005).

[0169] The NAS is the sum of separate scores for steatosis (0-3), hepatocellular ballooning (0-2), and lobular inflammation (0-3), with a maximum score of 8. The NAS score can be generated at the time of biopsy according to the criteria described above in Kleiner et al. See also Table 1. [Table 1]

[0170] In embodiments, subjects are selected based on a liver biopsy (eg, a liver biopsy used to determine a NAS score).

[0171] In some embodiments, subjects are selected based on NAS score. In some embodiments, the presence of NASH is established by an NAS score of 4 or more (i.e., an NAS score of 4 or more). In some embodiments, the presence of NASH can be established by a liver biopsy revealing an NAS score of 5 or more (i.e., an NAS score of 5 or more).

[0172] In embodiments, subjects are selected based on a NAS score determined prior to treatment.

[0173] In embodiments, the presence of NASH is established by a pre-treatment NAS score of 4 or greater (i.e., an NAS score of 4 or greater). In embodiments, the presence of NASH can be established by a liver biopsy revealing a pre-treatment NAS score of 5 or greater (i.e., an NAS score of 5 or greater).

[0174] In some embodiments, NASH may be characterized by a NAFLD activity score (NAS) of 5 or greater, where the NAS is the sum of the individual scores for steatosis (range: 0-3), hepatocyte ballooning (range: 0-2), and lobular inflammation (range: 0-3). See Kleiner et al., supra. Steatosis is the abnormal retention of lipids in the liver. The steatosis score represents the percentage of hepatocytes containing lipid droplets (steatosis) as 0 (<5%), 1 (5-33%), 2 (33-66%), and 3 (>66%). Patients treated for NASH according to the present disclosure may have a NAS steatosis score of 1, 2, or 3. Hepatocyte ballooning is a type of cell death visually characterized by enlargement and localization of the cell nucleus at or near the center of the cell. In some embodiments, hepatocyte ballooning is scored as 0 (none), 1 (few), or 2 (many cells with significant ballooning). In some embodiments, lobular inflammation is scored according to the number of inflammatory foci: 0 (no foci), 1 (less than 2 foci / 200x field), and 2 (2-4 foci / 200x field). In some embodiments, subjects have a lobular inflammation score of 0, 1, 2, or 3 and a ballooning score of 0, 1, or 2, provided that the sum of the lobular inflammation score and ballooning score is at least 2.

[0175] In embodiments, the methods and uses described herein comprise an improvement in NAS score, hi embodiments, the improvement occurs without worsening fibrosis.

[0176] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, results in a NAFLD activity score (NAS) of less than 4.

[0177] In embodiments, the methods and uses described herein result in a decrease of at least 1 (≧1) in the subject's NAS score.

[0178] In embodiments, the methods and uses described herein result in a decrease of at least two (≧2) in the subject's NAS score.

[0179] In embodiments, the methods and uses described herein result in a reduction of at least three (≧3) in the subject's NAS score.

[0180] In embodiments, the methods and uses described herein result in a one (1) to three (3) reduction in a subject's hepatic steatosis score.

[0181] In embodiments, the methods and uses described herein result in a decrease of one (1) or two (2) in the hepatocellular ballooning score of a subject.

[0182] In embodiments, the methods and uses described herein result in a one (1) to three (3) reduction in the lobular inflammation score of a subject.

[0183] In embodiments, the subject has a steatosis score of 1, 2, or 3.

[0184] In embodiments, steatosis comprises macrovesicular steatosis. In embodiments, steatosis comprises microvesicular steatosis. In embodiments, steatosis comprises macrovesicular and microvesicular steatosis.

[0185] In some embodiments, the methods and uses described herein result in the resolution of steatohepatitis without worsening of fibrosis in a patient in need thereof, hi some embodiments, resolution includes the absence of hepatocellular ballooning (e.g., a ballooning score of 0), no inflammation or mild inflammation (e.g., a ballooning score of 0-1), and / or the presence or absence of steatosis (e.g., a steatosis score of 0-3).

[0186] In some embodiments, NASH is steatohepatitis characterized by at least one of lobular inflammation and hepatocellular ballooning. In some embodiments, NASH occurs in the absence of other causes of liver disease and / or substantial alcohol consumption.

[0187] In embodiments, the subject has hepatitis. In embodiments, the hepatitis is a lobular inflammation.

[0188] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a reduction in hepatitis.

[0189] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)) or a pharmaceutically acceptable salt thereof results in a hepatitis score of 0 or 1 (e.g., as described herein).

[0190] In embodiments, the subject's liver is characterized by hepatocyte ballooning.

[0191] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a reduction in hepatocyte ballooning.

[0192] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a ballooning score of 0.

[0193] In some embodiments, treatment is initiated without regard to determining the NAS score.

[0194] In embodiments, subjects are selected based on surrogate markers that parallel histological assessment of NAS scores (eg, biomarkers).

[0195] In embodiments, the methods and uses described herein result in at least a two-point improvement in NAS without worsening fibrosis in a patient in need thereof.

[0196] In embodiments, the subject has liver fibrosis.

[0197] In some embodiments, fibrosis is scored / staged on a scale of 0 to 4 (Table 2). [Table 2]

[0198] In embodiments, the subject has non-cirrhotic NASH.

[0199] In embodiments, the subject has cirrhosis NASH.

[0200] In some embodiments, the subject has a fibrosis stage score of 0 to 3. In some embodiments, the subject has a fibrosis stage score of 0. In some embodiments, the subject has a fibrosis stage score of 1. In some embodiments, the subject has a fibrosis stage score of 2. In some embodiments, the subject has a fibrosis stage score of 3. In some embodiments, the subject has a fibrosis stage score of 4 (cirrhosis). In some embodiments, the patient after treatment may have a fibrosis stage score that is at least no worse than the baseline score before treatment, or may have a reduction in the fibrosis stage score of at least one level, or at least two or three levels.

[0201] In embodiments, the methods and uses described herein do not result in an increase in the subject's liver fibrosis score (stabilization of the subject's liver fibrosis).

[0202] In embodiments, the methods and uses described herein result in a decrease of at least one (≧1) in the subject's liver fibrosis score (reversal of liver fibrosis in the subject).

[0203] In some embodiments, liver fibrosis is characterized by using Enhanced Liver Fibrosis Test (ELF) score. In some embodiments, liver fibrosis is characterized by an ELF score of less than 7.7. In some embodiments, liver fibrosis is characterized by an ELF score of 7.7 or more and less than 9.8. In some embodiments, liver fibrosis is characterized by an ELF score of more than 9.8.

[0204] In embodiments, the methods and uses described herein result in the maintenance (no worsening) of the fibrosis stage in a patient in need thereof.

[0205] In embodiments, the methods and uses described herein result in regression of fibrosis in a patient in need thereof.

[0206] In embodiments, the methods and uses described herein result in a reduction in liver enlargement in a subject.

[0207] In embodiments, the methods and uses described herein result in a reduction of liver collagen levels in a subject.

[0208] In embodiments, the methods and uses described herein result in a decrease in liver tissue alpha-smooth muscle actin (α-SMA) levels in a subject.

[0209] In embodiments, the methods and uses described herein result in a reduction of hepatocyte apoptosis in a subject.

[0210] In embodiments, the methods and uses described herein result in a reduction of hyaluronic acid levels in a subject.

[0211] In embodiments, the methods and uses described herein result in a decrease in tissue inhibitor of metalloproteinases (TIMP-1) levels in a subject.

[0212] In embodiments, the methods and uses described herein result in a reduction in procollagen III terminal peptide (PIIINP) levels in a subject.

[0213] In embodiments, the methods and uses described herein result in a decrease in soluble Fas ligand levels in a subject.

[0214] In embodiments, the methods and uses described herein result in a decrease in leptin levels in a subject.

[0215] In embodiments, the methods and uses described herein result in a decrease in the aspartate aminotransferase (AST) to platelet index (APRI) in a subject.

[0216] In embodiments, the methods and uses described herein result in a reduction in the subject's Fibrosis 4 (FIB-4) score.

[0217] In embodiments, the methods and uses described herein result in a reduction in liver stiffness in a subject.

[0218] In embodiments, the methods and uses described herein result in an increase in adiponectin levels in a subject.

[0219] Serum markers and biomarkers In embodiments, any of the methods and uses described herein result in a change in a serum marker (e.g., a serum marker of a liver pathology such as liver fibrosis or NASH) in a patient in need thereof. Thus, in embodiments, the patient is selected based on a particular expression level of a serum marker (including any described herein). For example, the methods described herein may be particularly beneficial for patients with a particular (e.g., threshold level) of a serum marker (e.g., any described herein). Furthermore, the methods described herein can result in a favorable change in the serum marker (e.g., modulating the level of any serum marker described herein). In embodiments, the methods described herein can result in a reduction of an elevated serum marker (e.g., any described herein).

[0220] For example, noninvasive measurement of fibrosis to monitor treatment effectiveness can be useful to avoid the need for repeat liver biopsies (e.g., to identify patients who could benefit from the methods described herein, including patients with any of the liver pathologies described herein, such as NASH). In some embodiments, the serum marker is FIB-4. In some embodiments, the serum marker is APRI. APRI and FIB-4 scores are calculated by the following published formula (Kim et al., Hepatology 2013, 57:1357), where "PLT count" is platelet count, "AST" is aspartate transaminase, with an upper limit of normal of 40 IU / mL, and "ALT" is alanine aminotransferase. APRI=([AST / normal upper limit] / PLT number[109 / L]) FIB-4 = (age [years] × AST [IU / L]) / (PLT [109 / L] × (ALT [IU / L]) 1 / 2).

[0221] Thus, exemplary serum markers include enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), or gamma-glutamyltransferase (GGT), or any combination thereof. In some embodiments, the patient has at least one elevated liver enzyme.

[0222] Still other exemplary serum markers include total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, bilirubin, albumin, C-peptide, apolipoprotein A1, apolipoprotein B, leptin, adiponectin, free fatty acids, ghrelin, and tumor necrosis factor alpha (TNF-α).

[0223] In some embodiments, the subject has elevated liver alanine aminotransferase (ALT) levels. In some embodiments, the methods and uses described herein result in a decrease in liver alanine aminotransferase (ALT) levels. In some embodiments, the subject has ALT levels that fall within normal levels (e.g., about 10-40 IU / L). In some embodiments, the subject has ALT levels that are greater than about 40 IU / L. In some embodiments, the ALT does not exceed about 30 IU / L (e.g., for male patients). In some embodiments, the ALT exceeds about 30 IU / L (e.g., for male patients). In some embodiments, the ALT does not exceed about 19 IU / L (e.g., for female patients). In some embodiments, the ALT exceeds about 19 IU / L (e.g., for female patients).

[0224] In some embodiments, the subject has elevated hepatic aspartate aminotransferase (AST) levels. In some embodiments, the subject has AST levels that fall within normal levels (e.g., about 10-35 IU / L). In some embodiments, the subject has AST levels greater than about 30 IU / L. In some embodiments, the subject has AST levels greater than about 35 IU / L. In some embodiments, the methods and uses described herein result in a decrease in hepatic aspartate aminotransferase (AST) levels.

[0225] In some embodiments, the ratio of aspartate aminotransferase (AST) to alanine aminotransferase (AST) is determined. In some embodiments, the patient has an AST / ALT ratio greater than 1. In some embodiments, the patient has an AST / ALT ratio less than 1.

[0226] In embodiments, the methods and uses described herein result in a decrease in the ratio of aspartate aminotransferase (AST) to alanine aminotransferase (ALT) in a subject.

[0227] In embodiments, the methods and uses described herein result in a change in the ratio of aspartate aminotransferase (AST) to alanine aminotransferase (ALT) in a subject so that it is closer to one.

[0228] In some embodiments, the subject has elevated alkaline phosphatase (ALP) levels. In some embodiments, the subject has an ALP level that falls within the normal range (e.g., about 20-140 or about 37-116 IU / L). In some embodiments, the subject has an ALP level greater than about 120 IU / L or about 140 IU / L. In some embodiments, the subject has an ALP level greater than about 150 IU / L. In some embodiments, the methods and uses described herein result in a decrease in alkaline phosphatase (ALP) levels.

[0229] In some embodiments, the subject has elevated gamma-glutamyltransferase (GGT) levels. In some embodiments, the subject has GGT levels that fall within the normal range (e.g., about 5-30 IU / L or about 9-48 IU / L). In some embodiments, the subject has GGT. In some embodiments, the subject has elevated GGT levels that are at least about 50 IU / L. The GGT levels are up to about 90 IU / L or about 100 IU / L. In some embodiments, the methods and uses described herein result in a decrease in GGT levels.

[0230] In embodiments, the subject has elevated triglyceride levels, hi embodiments, the methods and uses described herein result in a decrease in triglyceride levels.

[0231] In embodiments, the subject has elevated non-esterified fatty acid (NEFA) levels. In embodiments, the methods and uses described herein result in a decrease in non-esterified fatty acid (NEFA) levels.

[0232] In embodiments, the subject has elevated cholesterol levels, hi embodiments, the methods and uses described herein result in a reduction of cholesterol levels.

[0233] In embodiments, the subject has reduced HDL-cholesterol levels.

[0234] hepatitis In one aspect, the invention features a method for treating (e.g., alleviating) hepatitis, the method including administering to a subject in need thereof an effective amount of a compound according to formula I, or a pharmaceutically acceptable salt thereof.

[0235] In embodiments, the hepatitis is lobular inflammation.

[0236] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)) or a pharmaceutically acceptable salt thereof results in a hepatitis score of 0 or 1.

[0237] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0238] In embodiments, the compound of Formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (Compound (1)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride (Compound (1), dihydrochloride).

[0239] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid (Compound (2)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride (Compound (2), trihydrochloride).

[0240] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid (Compound (3)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride (Compound (3), trihydrochloride).

[0241] Hepatocyte ballooning In one aspect, the invention features a method for treating (e.g., reducing) hepatocellular ballooning, the method including administering to a subject in need thereof an effective amount of a compound according to Formula I, or a pharmaceutically acceptable salt thereof.

[0242] Hepatocyte ballooning is a type of cell death that is visually characterized by enlargement and localization of the cell nucleus at or near the center of the cell.

[0243] In some embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in a ballooning score of 0.

[0244] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0245] In embodiments, the compound of Formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (Compound (1)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride (Compound (1), dihydrochloride).

[0246] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid (Compound (2)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride (Compound (2), trihydrochloride).

[0247] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid (Compound (3)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride (Compound (3), trihydrochloride).

[0248] Liver fibrosis (including cirrhosis) In one aspect, the invention features a method of treating liver fibrosis, the method including administering to a subject in need thereof an effective amount of a compound according to Formula I, or a pharmaceutically acceptable salt thereof.

[0249] In embodiments, the subject has stage 2, stage 3, or stage 4 liver fibrosis.

[0250] In embodiments, the subject has cirrhosis of the liver (stage 4 liver fibrosis).

[0251] In some embodiments, the subject has a fibrosis stage score of 1 or greater.

[0252] In some embodiments, the subject has a fibrosis stage score of 0-3.

[0253] In some embodiments, the subject has a Fibrosis Stage Score of 0. In some embodiments, the subject has a Fibrosis Stage Score of 1. In some embodiments, the subject has a Fibrosis Stage Score of 2. In some embodiments, the subject has a Fibrosis Stage Score of 3. In some embodiments, the subject has a Fibrosis Stage Score of 4 (cirrhosis).

[0254] In some embodiments, the patient after treatment may have a fibrosis stage score that is at least no worse than the pre-treatment baseline score, or may have a reduction in the fibrosis stage score of at least one level, or at least two or three levels.

[0255] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in stabilization of liver fibrosis in the subject.

[0256] In embodiments, administering a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or a pharmaceutically acceptable salt thereof, results in the reversal of liver fibrosis in the subject.

[0257] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0258] In embodiments, the compound of Formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (Compound (1)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride (Compound (1), dihydrochloride).

[0259] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid (Compound (2)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride (Compound (2), trihydrochloride).

[0260] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid (Compound (3)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride (Compound (3), trihydrochloride).

[0261] steatosis In one aspect, the invention features a method of treating steatosis, the method including administering to a subject in need thereof an effective amount of a compound according to Formula I, or a pharmaceutically acceptable salt thereof.

[0262] In embodiments, administering the compound of Formula I or a pharmaceutically acceptable salt thereof results in a reduction of adiposity in the subject.

[0263] In embodiments, steatosis comprises macrovesicular steatosis. In embodiments, steatosis comprises microvesicular steatosis. In embodiments, steatosis comprises macrovesicular and microvesicular steatosis.

[0264] In embodiments, the subject has non-alcoholic steatohepatitis (NASH).

[0265] In embodiments, the compound of Formula I is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (Compound (1)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula (I) is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride (Compound (1), dihydrochloride).

[0266] In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid (Compound (2)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((6-aminohexyl)amino)pentanoic acid trihydrochloride (Compound (2), trihydrochloride).

[0267] In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid (Compound (3)), or a pharmaceutically acceptable salt thereof. In embodiments, the compound of Formula I is (S)-2-amino-5-((5-aminopentyl)amino)pentanoic acid trihydrochloride (Compound (3), trihydrochloride).

[0268] Comorbidities The methods described herein may be useful for treating subjects with co-morbidities.

[0269] In embodiments, the subject is pre-diabetic.

[0270] In some embodiments, the subject has diabetes. In some embodiments, the subject has type 2 diabetes.

[0271] In embodiments, the subject does not have diabetes. In embodiments, the subject does not have type 2 diabetes.

[0272] In embodiments, the subject is obese. In embodiments, the subject is not obese. In embodiments, the subject has a body mass index (BMI) greater than 30. In embodiments, the subject has a BMI less than 30. In embodiments, the subject has a BMI less than 25.

[0273] In embodiments, the subject has dyslipidemia. In embodiments, the subject does not have dyslipidemia.

[0274] In embodiments, the subject has hypertension. In embodiments, the subject does not have hypertension.

[0275] In embodiments, the subject is insulin resistant. In embodiments, the subject is not insulin resistant.

[0276] In embodiments, the subject has cardiovascular disease. In embodiments, the subject is at risk for cardiovascular disease. In embodiments, the subject does not have cardiovascular disease.

[0277] Pharmaceutical Composition In another aspect, the invention features a pharmaceutical composition including a compound according to Formula I (e.g., any of compounds (1), (2), and (3)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant any carrier, diluent, or excipient that is compatible with the other ingredients of the formulation and not deleterious to the recipient.

[0278] In some embodiments of the methods and uses described herein, the compound may be combined with a pharmaceutically acceptable carrier and administered in the form of a pharmaceutical composition.

[0279] The active agent may be formulated into dosage forms according to standard practice in the pharmaceutical arts. See Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Edition (1990), Mack Publishing Co., Easton, PA. Suitable dosage forms may include, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.

[0280] The compound of formula I may be administered in a convenient manner. Suitable local routes include oral, rectal, inhalation (including nasal), topical (including buccal and sublingual), transdermal, and vaginal, for example, through the epidermis. The compound of formula I may also be used for parenteral administration (including subcutaneous, intravenous, intramuscular, intradermal, intraarterial, intrathecal, and epidural). It will be apparent that the selected route may vary, for example, depending on the condition of the recipient.

[0281] For parenteral administration, the active agent may be mixed with a suitable carrier or diluent, such as water, oil (especially vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or glycols, such as propylene glycol or polyethylene glycol. Solutions for parenteral administration may contain a water-soluble salt of the active agent. Stabilizers, antioxidants, and preservatives may also be added. Suitable antioxidants include sulfites, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. Compositions for parenteral administration may take the form of aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions.

[0282] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is orally administered to a subject.

[0283] For oral administration, the active agent may be combined with one or more solid inactive ingredients to prepare tablets, capsules, pills, powders, granules, or other suitable oral dosage forms. For example, the active agent may be combined with at least one excipient such as a filler, binder, humectant, disintegrant, solution retarder, absorption accelerator, wetting agent, absorbent, or lubricant. According to one tablet embodiment, the active agent may be combined with carboxymethylcellulose (CMC) calcium, magnesium stearate, mannitol, and starch, and then formed into tablets by conventional tablet-forming methods.

[0284] The pharmaceutical compositions of the present disclosure may also be formulated to provide delayed or controlled release of the active ingredient contained therein using various proportions of hydropropyl methylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes and / or microspheres to provide the desired release profile.

[0285] Generally, controlled-release preparations are pharmaceutical compositions that release active ingredients at a required rate and maintain constant pharmacological activity for a desired period of time. Such dosage forms provide a supply of drug to the body for a predetermined period of time, thus maintaining drug levels in the therapeutic range for a longer period of time than conventional non-controlled formulations.

[0286] U.S. Patent No. 5,674,533 discloses a liquid controlled-release pharmaceutical composition for the administration of moguisteine, a potent peripheral antitussive. U.S. Patent No. 5,059,595 describes the controlled release of an active agent through the use of a gastroresistant tablet for the treatment of organic psychiatric disorders. U.S. Patent No. 5,591,767 describes a liquid reservoir transdermal patch for controlled release. U.S. Patent No. 5,120,548 discloses a controlled-release drug delivery device composed of a swellable polymer. U.S. Patent No. 5,073,543 describes a controlled-release formulation containing nutrients encapsulated in a ganglioside-liposome vehicle. U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. Biodegradable microparticles are known for use in controlled-release formulations. US Patent No. 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions.

[0287] The controlled release of an active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. Various mechanisms of drug release exist. For example, in one embodiment, the controlled-release component may swell after administration to a patient, forming porous openings large enough to release the active ingredient. In the context of this disclosure, the term "controlled-release component" is defined herein as a compound, such as a polymer, polymer matrix, gel, permeable membrane, liposome, and / or microsphere, that facilitates the controlled release of an active ingredient in a pharmaceutical composition. In another embodiment, the controlled-release component is biodegradable and is triggered by exposure to an aqueous environment, pH, temperature, or enzymes in the body. In another embodiment, a sol-gel may be used, in which the active ingredient is incorporated into a sol-gel matrix that is solid at room temperature. This matrix is ​​implanted into a patient, e.g., a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient.

[0288] Components used in formulating pharmaceutical compositions are of high purity and substantially free of potentially harmful contaminants (e.g., at least national food grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Particularly for human consumption, compositions may be manufactured or formulated under Good Manufacturing Practice standards as defined by applicable U.S. Food and Drug Administration regulations. For example, suitable formulations may be sterile and / or substantially isotonic and / or in full compliance with all U.S. Food and Drug Administration Good Manufacturing Practices.

[0289] Dosage and Administration Regimen The physician will determine the most appropriate dosage of the active agent. Dosages will vary with the form of administration and the particular compound selected. Furthermore, dosages will vary depending on various factors, including, but not limited to, the patient being treated, the patient's age, the severity of the condition being treated, the route of administration, etc. Physicians will generally wish to initiate treatment with a low dosage, much less than the optimal dose of the compound, and increase the dosage by small, incremental increases until the optimal effect under the circumstances is achieved. In many cases, when the composition is administered orally, it will be found that a larger amount of the active agent is required to achieve the same effect as when a smaller amount is administered parenterally. The compounds are useful in the same manner as comparable therapeutic agents, and dosage levels are of the same order of magnitude as those commonly employed with other therapeutic agents.

[0290] For example, the daily dose is about 2 mg / kg / day to about 1000 mg / kg / day, about 10 mg / kg / day to about 1000 mg / kg / day, or about 10 mg / kg / day to about 100 mg / kg / day. In some embodiments, the daily dose is about 2 mg / kg / day to about 1000 mg / kg / day. In some embodiments, the daily dose is about 10 mg / kg / day to about 1000 mg / kg / day. In some embodiments, the daily dose is about 10 mg / kg / day to about 100 mg / kg / day.

[0291] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject (e.g., a human) at a dose of about 2-1000, 10-1000, or 10-100 mg / kg per day. In some embodiments, the dose is about 10-1000 mg / kg per day. In some embodiments, the dose is about 10-1000 mg / kg per day.

[0292] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject (e.g., a human) at a total daily dose of about 10 mg / kg or more per day. In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject (e.g., a human) at a total daily dose of about 2 mg / kg or more per day.

[0293] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject (e.g., a human) at a total daily dose of about 100-5000, 500-5000, or 600-3000 mg per day. In some embodiments, the total daily dose is about 500-5000 mg / day. In some embodiments, the total daily dose is about 600-3000 mg / day.

[0294] Treatment may be carried out over an extended period of time, either in a single continuous session or in separate sessions, as needed. In some embodiments, the subject undergoes treatment.

[0295] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject (e.g., a human) for at least about 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 16 weeks.

[0296] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject (e.g., a human) for at least 14 weeks.

[0297] In some embodiments, a compound of Formula I (e.g., any one of Compound (1), Compound (2), and Compound (3)), or any pharmaceutically acceptable salt thereof, is administered to a subject (e.g., a human) for at least about 4 to 6, 4 to 8, 4 to 10, 4 to 12, 4 to 14, or 4 to 16 weeks.

[0298] The practice of the present disclosure is illustrated by the following non-limiting examples. [Example]

[0299] In the following examples, all protocols were approved by the Institutional Animal Care and Use Committee (IACUC). All measures were taken to ensure the welfare, safety, health, and comfort of the animals and to minimize stress and pain. Procedures such as necropsies were performed using an IACUC-approved euthanasia protocol.

[0300] Example 1 - APL and glucose tolerance in a rodent model of NASH This experiment was designed to determine the effect of (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid (APL) on impaired glucose tolerance (IGT) in an art-recognized model of NASH. The following rodent models were used: Zucker fa / fa obese rats and Zucker Fa / fa lean rats, and C57BL / 6J mice. C57BL / 6J mice are a model of diet-induced obesity (DIO), and these mice are highly susceptible to obesogenic diets and prone to developing diet-induced hepatic necroinflammation and fibrosis. See, e.g., Liang et al., 2014, PLoS One, 9(12):e115922; and London & George, 2007, Clinics in Liver Disease, 11(1):55-74.

[0301] Zucker fa / fa obese rats and Zucker Fa / fa lean rats (7-week-old male animals obtained from Charles Rivers Laboratories, Wilmington, MA, USA) were housed in individual cages (one rat per cage to promote sedentary activity) and had free access to standard rodent chow (catalog number 5053, obtained from Labdiet, St. Louis, MO, USA) and water (filtered and filled using a Hydrapak system) for 7 days prior to the start of the experiment. Zucker fa / fa obese rats were randomly assigned to two groups: 1) vehicle po bid (administered orally twice daily) for 78 days (n = 7), and 2) 100 milligrams per kilogram (mg / kg) of body weight APL po bid for 78 days (n = 8). Zucker Fa / fa lean rats (n = 4) were administered vehicle po bid for 78 days. Phosphate-buffered saline (PBS), the solution used to dissolve APL, was used as the vehicle solution. APL powder was dissolved in PBS at a concentration of 100 mg / mL under sterile conditions, and the volume was adjusted according to the body weight of each animal. Animals had free access to food and water except during the glucose tolerance test (GTT) procedure.

[0302] C57BL / 6J mice (5-week-old male mice obtained from Jackson Laboratories, Bar Harbor, Maine, USA) were housed in cages (5 mice per cage) with free access to standard rodent chow (catalog number 5053, obtained from Labdiet, St. Louis, MO) and water (filtered and filled using a Hydrapak system) for 5 days prior to the start of the experiment. Animals were then randomly assigned to five groups (n=5 per group) and received different diets and treatments as shown in Table A for 130 days. Each treatment was administered orally twice daily. The APL dose administered to each animal was 200 milligrams per kilogram of body weight administered by oral gavage twice daily, and the pioglitazone dose administered to each animal was 20 milligrams per kilogram of body weight administered by oral gavage twice daily. "HFD" is a high-fat / high-fructose diet containing 58% fat, 25% carbohydrate, and 17% protein. HFD was obtained from Research Diets, Inc. "Chow" refers to standard rodent chow (catalog number 5053, obtained from Labdiet, St. Louis, MO). Animals were fed ad libitum except during the GTT experiment. [Table 3]

[0303] At the end of the experimental period (78 days for rats and 130 days for mice), animals underwent a glucose tolerance test (GTT) as follows: Animals were fasted for 5 hours, weighed, and their tails were clipped to measure fasting blood glucose levels. Immediately afterward, glucose (2 mg / g body weight) was administered orally. Glucose levels were measured 0, 15, 30, 45, 60, and 120 minutes after glucose administration. Glucose was measured using an Abbott AlphaTRAK 2 blood glucose meter.

[0304] The area under the curve (AUC) of the glucose tolerance test data was calculated. These data are shown in Figures 1 and 2. Figure 1 shows rat data. The AUC of the GTT was significantly higher in all vehicle-treated obese rats compared to control rats. The AUC of the GTT in obese rats treated with APL was significantly lower than in vehicle-treated obese rats (**p<0.01). In mice, the GTT was performed at the beginning of week 18 (130 days). Figure 2 shows mouse data. Figure 2A shows the GTT data over time. Significant statistical differences were observed between the positive control group and the treatment groups at 15 minutes (****p<0.0001), 60 minutes (*p<0.05), and 120 minutes (***p<0.001). Significant statistical differences were also observed between the negative control group and the positive control group at 0 and 30 minutes (*p<0.05), 45 minutes (**p<0.01), and 15, 30, 60, and 120 minutes (****p<0.0001). There were no significant differences between the negative control group and the treatment groups at any time point. Figure 2B shows the integrated AUC data. Mice fed an HFD diet and treated with vehicle solution had significantly higher AUCs of the GTT compared to both control mice (chow-fed and vehicle-treated) and HFD-fed and APL-treated mice (**p<0.01).

[0305] Impaired glucose tolerance (IGT) and diabetes are significant comorbidities of NASH. These data demonstrate that APL significantly improves IGT in both rat and mouse NASH models.

[0306] Example 2 - APL and Hepatic Steatosis in a High Fat / High Fructose Rodent Model of NASH As in Example 1, C57BL / 6 mice (same mouse samples as in Example 1) were housed in cages (5 mice per cage) with free access to standard rodent chow (catalog number 5053, obtained from Labdiet, St. Louis, MO) and water (filtered and filled using a Hydrapak system) for 5 days. C57BL / 6J mice were fed either CHOW or a high-fat / high-fructose diet (HFD) and treated with either vehicle or APL twice daily (bid) orally (po) for 18 weeks. The APL dose was 200 milligrams per kilogram (mg / kg) of body weight per day, with two doses (po bid) of APL. Animals were allowed to eat ad libitum. Animals were humanely euthanized, and livers were removed for histological analysis. Liver samples were fixed in formalin for 48 hours and processed into FFPE blocks after fixation. Slides were stained with hematoxylin and eosin (H&E) and immunostained with BODIPY and DAPI, and fat deposits and nuclei number were quantified using confocal microscopy. See, e.g., Rico et al., 2007, J Cell Physiol, 211(2):504-12, and Daemen et al., 2016, Mol Metab, 5(3)153-63. Data are shown in Figures 3, 4, and 5.

[0307] Figures 3 and 4 show liver samples stained with hematoxylin and eosin (H&E). Figure 3 shows liver samples at 20x magnification. The leftmost column shows liver samples from mice fed CHOW (negative control), the middle column shows liver samples from mice fed an HFD with vehicle (positive control), and the rightmost column shows liver samples from mice fed an HFD and treated with APL (treated). Figure 4 shows similar liver sections at 100x magnification. The leftmost column shows livers from the negative control group, the middle column shows livers from the positive control group, and the rightmost column shows livers from the treatment groups.

[0308] Liver sections from animals treated with APL exhibited less severe hepatocellular fat accumulation, inflammatory infiltrates, and hepatocellular ballooning. These data demonstrate that APL ameliorates the characteristic liver injury associated with NASH.

[0309] Figure 5A shows multiple images of hematoxylin and eosin (H&E) staining of liver sections from C57BL / 6J mice fed either a CHOW or high-fat / high-fructose diet (HFD) and treated twice daily with either vehicle or APL. The left image in Figure 5A shows the normal anatomy of the liver observed in mice fed a CHOW diet. The normal anatomy is completely altered by fat accumulation (large white spots), a characteristic change observed in nonalcoholic steatohepatitis (NASH) in mice fed a HFD and treated with vehicle solution (Figure 5A, center image). In contrast, as shown in the right image in Figure 5A, mice fed a HFD and treated with APL retained the normal anatomy of the liver.

[0310] Figure 5B is a diagram of multiple images of immunofluorescent staining with BODIPY and DAPI of liver sections from the same mouse, and Figure 5C is quantification of nuclei from immunofluorescent slides stained with DAPI. Quantification of nucleic acid counts was performed using Harmony® 4.6 High-Content Imaging and Analysis Software from Operetta CLS™ (PerkinElmer, Waltham, MA). HFD-fed HFD mice had statistically significantly fewer nuclei than either CHOW-fed mice or HFD-fed mice treated with APL (***p<0.001). These data indicate that APL ameliorates the hepatocyte death (i.e., ballooning) pathology of NASH.

[0311] Figure 5D shows a plot of fat deposition assessed using confocal microscopy. Measurements were performed at 20x magnification. More than 21 fields were analyzed from three different animals in each group: Chow-vehicle (field count = 26), HFD-vehicle (field count = 25), and HFD-APL (field count = 23). Fat deposition was calculated using indirect measurements using ImageJ (Schneider et al., 2012, Nature Methods 9(7):671-675). Fat deposition was increased 3-fold in HFD / vehicle-treated mice compared with CHOW control and HFD / APL-treated mice (***p<0.001). These data demonstrate that APL reduces hepatic fat deposition and attenuates hepatic steatosis, both hallmarks of NASH.

[0312] Hepatocyte ballooning and steatosis are pathological features of NASH and are indicative of liver damage associated with this condition. These data indicate that APL significantly reduces these features and is effective in treating NASH.

[0313] Example 3 - APL and Non-Alcoholic Fatty Liver Disease Activity Score (NAS) in a High Fat / High Fructose Rodent NASH Model C57BL / 6J mice were housed in cages (5 mice per cage) with free access to standard rodent chow (catalog number 5053, obtained from Labdiet, St. Louis, MO) and water (filtered and filled using a Hydrapak system) for 5 days prior to the start of the experiment. Animals were then randomly assigned to three groups (n=5 in each group) and received different diets and treatments as shown in Table B for 130 days. Each treatment was administered orally twice daily. The dose of APL was 200 mg / kg body weight. "HFD" refers to the high-fat / high-fructose diet described in Example 1. "Chow" refers to the standard rodent chow as described in Example 1. Animals were allowed to eat ad libitum. [Table 4]

[0314] At the end of the study period, animals were humanely euthanized, and livers were removed for histological analysis. Liver samples were fixed in formalin for 48 hours and processed into FFPE blocks after fixation. Slides were stained with hematoxylin and eosin (H&E). Figure 6A shows multiple images of H&E stained liver sections. These data demonstrate that APL attenuates the morphological changes associated with NASH liver pathology. The positive control group showed increased both macrovesicular and microvesicular steatosis (arrows 1 and 2, respectively). Furthermore, the positive control group showed advanced hypertrophy (i.e., ballooning) and inflammatory foci (arrows 3 and 4, respectively).

[0315] Liver samples were evaluated for hepatic fat (steatosis), inflammation, and hypertrophy according to the NAS scoring system. The NAS scoring is shown in Figure 6B. Compared with CHOW / vehicle-treated mice, the NAS scores of mice in HFD / vehicle-treated mice were increased for all pathological parameters, including macrovesicular steatosis, microvesicular steatosis, hepatic hypertrophy, and inflammatory lesions. Notably, APL treatment showed statistically significant improvements in all parameters (***p<0.001). Thus, APL attenuates hepatic steatosis, liver inflammation, and hypertrophy, which are the pathologies of liver injury and NASH, respectively.

[0316] Figure 7 shows additional NASH activity score (NAS) data in the DIO mouse model of NASH. Figure 7A shows total NAS for mice fed CHOW and vehicle, mice fed HFD and vehicle, and mice fed HFD and treated with APL (200 mg / kg / day, divided into two doses). These data demonstrate that treatment with APL significantly reduces NAS (****p<0.0001). Figure 7B shows NAS component scores for steatosis, lobular inflammation, and liver ballooning. Again, these data demonstrate that treatment with APL significantly reduces each component of NAS, including steatosis, lobular inflammation, and liver ballooning (****p<0.0001). Thus, APL attenuates each feature of NASH (hepatic steatosis, liver inflammation, and hypertrophy), reduces total NAS, and is effective in treating NASH.

[0317] Example 4 - APL and Liver Fibrosis in a Rodent Model of NASH Zucker (fa / fa) obese rats and Zucker (Fa / fa) lean rats (7-week-old male animals obtained from Charles Rivers Laboratories, Wilmington, MA, USA) were housed in individual cages (one rat per cage to promote sedentary activity) and had free access to standard rodent chow (catalog number 5053, obtained from Labdiet, St. Louis, MO, USA) and water (filtered and filled using a Hydrapak system) for 7 days prior to the start of the experiment. Zucker (fa / fa) obese rats were randomly assigned to two groups: 1) vehicle po bid (administered orally twice daily) for 78 days, and 2) 100 milligrams per kilogram (mg / kg) of body weight APL po bid for 78 days. Zucker (Fa / fa) lean rats served as controls. The APL dose was 100 mg / kg / day. Animals were allowed to eat ad libitum. Animals were humanely euthanized, and livers were removed for histological analysis. Liver samples were fixed in formalin for 48 hours and processed into FFPE blocks after fixation. Slides were stained with hematoxylin and eosin (H&E) and Mason's trichrome stain.

[0318] The data are shown in Figure 8. Figure 8A is one of several photographs of hematoxylin and eosin-stained liver sections from Zucker rats. The left image is of a liver from a control Zucker (Fa / fa) lean rat, showing normal liver morphology. The middle image is of a liver from a vehicle-treated Zucker (fa / fa) obese rat, and the right image is of a liver from an APL-treated Zucker (fa / fa) obese rat. The images clearly show significant changes in liver tissue due to fat accumulation in the vehicle-treated Zucker (Fa / fa) obese rat (middle image). In contrast, the APL-treated Zucker (Fa / fa) obese rats maintained normal liver morphology (right image).

[0319] Figure 8B shows a Mason's trichrome-stained liver section from the same Zucker rat in Figure 8A. Figure 8C is an enlargement of one region of Figure 8B. In the enlargement of a vehicle-treated Zucker (Fa / fa) obese rat (center image), multiple blue-stained collagen fibers are evident, whereas in an APL-treated Zucker (Fa / fa) obese rat (right image), blue staining is minimal. Thus, APL attenuates the increase in liver collagen fibers.

[0320] These data indicate that APL attenuates liver fibrosis, a hallmark of liver injury associated with advanced and / or severe NASH, and therefore APL attenuates the development of liver fibrosis and is effective in treating NASH.

[0321] Example 5 - APL and liver protein carbonyls in a rodent (rat) NASH model Numerous oxidative stress and antioxidant biomarkers, including 4-hydroxynonenal (4-HNE), have been investigated for their potential use in assessing the pathological state and progression of NASH. See, for example, Ore et al., 2019, Medicina (Kaunas) 55(2):26. Oxidative stress can directly or indirectly induce irreversible damage to proteins by forming reactive carbonyl groups, primarily aldehydes and ketones. The most common reactive aldehyde in the indirect carbonylation process is 4-HNE. APL has a high affinity for 4-HNE and can prevent the harmful effects of 4-HNE on proteins, making it effective in reducing insulin resistance. See WO 2018 / 049019 and U.S. Patent Application Publication No. 2019 / 0192462.

[0322] Liver samples from Zucker rats in Example 4 were stained with anti-4HNE antibody from Abcam (Ab46545) for immunofluorescence analysis of the presence of 4-HNE. The data are shown in Figure 9. Figure 9A shows representative images of 4-HNE immunofluorescent liver sections from Zucker (Fa / fa) lean rats, Zucker (fa / fa) obese rats administered vehicle, and Zucker (fa / fa) obese rats administered vehicle and APL. Figure 9B shows a plot of 4HNE immunofluorescence intensity per tissue area, and Figure 9C shows a plot of total tissue area. These data show that APL reduces protein carbonyls to a statistically significant extent in the livers of Zucker (Fa / fa) obese rats and preserves total liver tissue area compared with Zucker (Fa / fa) obese rats administered vehicle (***p<0.001). Cytotoxic lipids, such as 4-HNE, are known to be elevated in human NASH livers compared with healthy livers. See, for example, Serviddio et al., Uncoupling protein-2 (UCP2) induces mitochondrial proton leak and increases susceptibility of non-alcoholic steatohepatitis (NASH) liver to ischemia-reperfusion injury. Gut. 2008 Jul;57(7):957-65. doi:10.1136 / gut.2007.147496. Epub 2008 Feb 28. This data suggests that 4-HNE plays an important role in the pathogenesis of NASH. Furthermore, these data demonstrate that treatment with APL significantly reduces the presence of 4-HNE (***p<0.001). As disclosed herein, this reduction in 4-HNE and the amelioration of NASH hallmarks such as steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis indicate that reduction of 4-HNE is an important therapeutic approach for attenuating NASH.

[0323] Example 6 - APL and liver protein carbonyls in a rodent (mouse) NASH model Liver samples from chow-fed, vehicle-treated C57BL / 6J mice, HFD-fed, vehicle-treated C57BL / 6J mice, and HFD-fed, APL-treated C57BL / 6J mice were prepared and stained with Abcam's anti-4HNE antibody (catalog no. ab46545) for immunofluorescence analysis of the presence of 4-HNE, as described in Example 1. The dose, dosing frequency, and treatment duration of APL were as described in Example 1 (the dose of APL administered to each animal was 200 milligrams per kilogram of body weight by oral gavage, administered twice daily; treatment period was 130 days; animals were fed ad libitum). The data are shown in Figure 10. Figure 10A shows representative images of 4-HNE immunofluorescent liver sections at 20x or 63x magnification from C57BL / 6J mice fed a chow diet and treated with vehicle, and from C57BL / 6J mice fed an HFD and treated with either vehicle or APL(TB-019). Figure 10B shows plots of 4-HNE immunofluorescence intensity per tissue area. These data show that APL(TB-019) statistically significantly reduced protein carbonyls in the livers of HFD-fed C57BL / 6J mice compared with HFD-fed C57BL / 6J mice administered vehicle (***p<0.001). As disclosed herein, this reduction in protein carbonylation and the amelioration of NASH hallmarks, such as steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis, indicate that reducing 4-HNE is an important therapeutic approach for attenuating NASH.

[0324] The pathophysiological mechanism of NASH involves an increase in free fatty acids in hepatocytes, which subsequently leads to an increase in lipotoxic lipids, including 4-hydroxynonenal (4-HNE). Without being bound by theory, the mechanism of APL's effectiveness in treating NASH is thought to be based on APL's high affinity for 4-HNE, which reduces or prevents the deleterious effects of 4-NHE on hepatocyte proteins, including carbonylation.

[0325] Example 7 - APL and liver function serum biomarkers in a diet-induced mouse model C57BL / 6 mice were housed in cages and given free access to standard rodent chow (catalog number 5053, obtained from Labdiet, St. Louis, MO) and water. C57BL / 6J mice were fed either a CHOW (negative control) or an amylin liver NASH (AMLN) diet (58% high fat, 25% carbohydrate, 2% cholesterol) for 20 weeks. Beginning at week 20, animals fed the AMLN diet were switched to an HFD. At week 50, animals were treated orally (po) twice daily (bid) with either vehicle (positive control) or APL (treatment). APL doses were 50, 100, or 200 milligrams per kilogram (mg / kg) per day and administered in two doses (i.e., 25 mg / kg bid, 50 mg / kg bid, or 100 mg / kg bid). After 66 weeks, serum samples were collected and assayed for ALT, AST, APL, triglycerides, non-esterified fatty acids, and cholesterol. All treatment groups were administered 50 mg / kg / day, except for the cholesterol test group, which received 200 mg / kg / day. These results are summarized in Figure 11. Animals treated with APL showed significant decreases in ALT, AST, ALP, triglycerides, non-esterified fatty acids, and cholesterol compared to positive control animals (*p<0.02; **p=0.002; ***p=0.0008, and ****p<0.0001). Results regarding the reduction of NAS and liver fibrosis are summarized in Figure 12. Animals treated with APL showed significant decreases in NAS (***p<0.001) and liver fibrosis (****p<0.0001). This data indicates that APL attenuates NASH, serum liver function biomarkers, and fibrosis, and is therefore effective in treating NASH.

[0326] The disclosures of each patent, patent application, GenBank record and publication cited herein are hereby incorporated by reference in their entirety.

[0327] Although the present embodiments have been described in detail with reference to the examples above, it will be understood that various modifications can be made without departing from the spirit of these embodiments, as would be readily apparent to one skilled in the art, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. 1. A composition for treating non-alcoholic steatohepatitis (NASH) in a subject in need thereof, said composition comprising a compound according to formula I: 【Chemistry 1】 (I), or a pharmaceutically acceptable salt thereof.

2. (i) the subject has a NAFLD activity score (NAS) of ≧4, and / or (ii) administration of the compound of formula I or a pharmaceutically acceptable salt thereof results in a NAFLD activity score (NAS) of <4; The therapeutic composition of claim 1.

3. The therapeutic composition of claim 1 or 2, wherein the subject has non-cirrhotic NASH or cirrhotic NASH.

4. (i) the subject has hepatitis, wherein (a) the hepatitis may be lobular inflammation, and / or (b) administration of the compound of Formula I or a pharmaceutically acceptable salt thereof may result in a reduction in hepatitis, and / or (c) administration of the compound of Formula I or a pharmaceutically acceptable salt thereof may result in a hepatitis score of 0 or 1, and / or (ii) the subject's liver is characterized by hepatocellular ballooning, and wherein administration of the compound of Formula I or a pharmaceutically acceptable salt thereof may result in a reduction in hepatocellular ballooning, and wherein administration of the compound of Formula I or a pharmaceutically acceptable salt thereof may result in a ballooning score of 0; and / or (iii) the subject has elevated hepatic alanine aminotransferase (ALT) levels, and wherein administration of the compound of Formula I or a pharmaceutically acceptable salt thereof may result in a decrease in hepatic alanine aminotransferase (ALT) levels; and / or (iv) the subject has elevated hepatic aspartate aminotransferase (AST) levels, and wherein administration of the compound of Formula I or a pharmaceutically acceptable salt thereof may result in a decrease in hepatic aspartate aminotransferase (AST) levels; and / or (v) the subject has liver fibrosis, wherein (a) the subject may have stage 2, stage 3, or stage 4 liver fibrosis, and / or (b) administration of the compound of Formula I or a pharmaceutically acceptable salt thereof may result in stabilization of liver fibrosis in the subject or reversal of liver fibrosis in the subject, and / or (vi) the subject has a hepatic steatosis score of 1, 2, or 3, and wherein administration of the compound of formula I or a pharmaceutically acceptable salt thereof may result in a reduction of hepatic steatosis in the subject; and / or (vii) The therapeutic composition of claim 1 or 2, wherein administration of the compound of formula I or a pharmaceutically acceptable salt thereof results in a reduction of liver hypertrophy in the subject.

5. 1. A composition for treating hepatitis in a subject in need thereof, said composition comprising a compound according to formula I: 【Chemistry 2】 (I), or a pharmaceutically acceptable salt thereof.

6. (i) the hepatitis is lobular inflammation, and / or (ii) The therapeutic composition of claim 5, wherein administration of said compound of formula I or a pharmaceutically acceptable salt thereof results in a Hepatitis Score of 0 or 1.

7. 1. A composition for treating hepatocellular ballooning in a subject in need thereof, said composition comprising a compound according to formula I: 【Transformation 3】 (I), or a pharmaceutically acceptable salt thereof.

8. 8. The therapeutic composition of claim 7, wherein administration of the compound of formula I or a pharmaceutically acceptable salt thereof results in a ballooning score of 0.

9. 1. A composition for treating liver fibrosis in a subject in need thereof, said therapeutic composition comprising a compound according to formula I: 【Chemistry 4】 (I), or a pharmaceutically acceptable salt thereof.

10. (i) the subject has (a) stage 2, stage 3, or stage 4 liver fibrosis or (b) cirrhosis; and / or (ii) administration of the compound of formula I or a pharmaceutically acceptable salt thereof results in stabilization of liver fibrosis or reversal of liver fibrosis in the subject; The therapeutic composition of claim 9.

11. A composition for treating hepatic steatosis in a subject in need thereof, said composition comprising a compound according to formula I: 【Transformation 5】 (I), or a pharmaceutically acceptable salt thereof.

12. (i) the subject has a hepatic steatosis score of 1, 2, or 3, and / or (ii) The therapeutic composition of claim 11, wherein administration of said compound of formula I or a pharmaceutically acceptable salt thereof results in a reduction of hepatic steatosis in said subject.

13. The therapeutic composition of any one of claims 5 to 12, wherein the subject has non-alcoholic steatohepatitis (NASH).

14. (i) the subject is a human, and / or (ii) the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered to a subject (a) for a period of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks; (b) for a period of at least 4 to 6, 4 to 8, 4 to 10, 4 to 12, 4 to 14, or 4 to 16 weeks; (c) at a dose of 2 to 1000, 10 to 1000, or 10 to 100 mg / kg per day; (d) at a dose of 10 mg / kg or more per day; or (e) at a total daily dose of 100 to 5000, 500 to 5000, or 600 to 3000 mg per day; and / or (iii) A therapeutic composition according to any one of claims 1 to 13, wherein said compound of formula I is administered orally to a subject.

15. The compound is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid 【Transformation 6】 The therapeutic composition according to any one of claims 1 to 14, wherein 16. The pharmaceutically acceptable salt is (S)-2-amino-6-((3-aminopropyl)amino)hexanoic acid dihydrochloride. 【Transformation 7】 The therapeutic composition according to any one of claims 1 to 14, wherein

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