Use of chloroquine and clemizole compounds to treat inflammatory and cancerous conditions

Abstract

To provide methods for treating and / or preventing inflammatory and / or cancerous conditions.SOLUTION: Disclosed herein are methods for use of R-chloroquine or clemizole or combinations of R-chloroquine and clemizole for the treatment of a subject in need thereof. Uses include methods of treating inflammatory conditions, treating liver cancer or reducing the risk of developing liver cancer in a subject. Uses also include methods of treating non-alcoholic steatohepatitis in a subject.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 187,061, filed June 30, 2015, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under R42 AI088793 / NIAID NIH HHS / USA awarded by the National Institutes of Health and the National Institute of Allergy and Infectious Diseases. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present invention is directed to the field of medicine, and more particularly to the use of certain compounds for the treatment and prevention of inflammatory and cancerous conditions, especially of the liver. [Background technology]

[0004] background The treatment and prevention of inflammatory conditions and cancerous conditions are a major unmet medical need.In particular, improved treatment methods are needed for inflammatory conditions of the liver, such as non-alcoholic steatohepatitis (NASH).Left untreated, NASH and other inflammatory liver disorders, such as those caused by viral infections, can lead to hepatocellular carcinoma.The present invention addresses these needs by providing a method for treating and / or preventing inflammatory conditions and / or cancerous conditions, as described below. Summary of the Invention

[0005] overview Disclosed herein are methods that include administering an effective amount of clemizole and / or chloroquine, or an analog thereof, to a subject in need thereof. In some aspects of the invention, the subject is administered either R-chloroquine, clemizole, or R-chloroquine in combination with clemizole.

[0006] In some aspects of the invention, the subject has an inflammatory condition.

[0007] In another aspect of the present invention, the subject has an inflammatory condition of the liver.In some aspects of the present invention, the subject suffers from non-alcoholic steatohepatitis, and the method further comprises treating the non-alcoholic steatohepatitis.In some aspects, the method reduces inflammation of the liver lobule.In other aspects, the method reduces the risk of liver cancer.In yet another aspect of the present invention, the subject suffers from liver cancer, and the method further comprises treating the liver cancer.

[0008] In some aspects of the present invention, clemizole and / or R-chloroquine are administered to a subject suffering from non-alcoholic steatohepatitis. In other aspects, administering clemizole or R-chloroquine to a subject reduces the level of plasma alanine aminotransferase compared to vehicle control. In other aspects, administering clemizole and / or R-chloroquine to a subject reduces the non-alcoholic fatty liver disease activity score determined by histological analysis of liver tissue. In some aspects, administering clemizole and / or R-chloroquine reduces steatosis. In other aspects, administering clemizole and / or R-chloroquine reduces hepatocyte ballooning. In still other aspects, administering clemizole and / or R-chloroquine reduces lobular inflammation.

[0009] In some aspects of the present invention, clemizole and / or R-chloroquine are administered to a subject to reduce the risk of developing liver cancer.In another aspect of the present invention, the subject is diagnosed with non-alcoholic steatohepatitis.In another aspect of the present invention, clemizole and / or R-chloroquine are administered to a subject suffering from liver cancer.In yet another aspect, administering clemizole and / or R-chloroquine reduces the tumor burden of the subject or increases the survival time of the subject.

[0010] In some aspects of the present invention, 0.5 to 50 mg / kg of clemizole is administered to a subject. In some aspects, clemizole is administered once daily, twice daily, or three times daily. In other aspects, clemizole is administered daily for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more.

[0011] In some aspects of the invention, 0.5 to 50 mg / kg of R-chloroquine is administered to a subject. In other aspects, R-chloroquine is administered once daily, once every other day, or once weekly. In some aspects, R-chloroquine is administered for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more. In yet another aspect, R-chloroquine is administered daily as a double loading dose for the first 2 days of treatment, followed by a single dose that is half the double loading dose for the remainder of treatment. [The present invention 1001] administering to a subject an effective amount of clemizole; A method for treating liver inflammation in a subject suffering from non-alcoholic steatohepatitis in need of such treatment. [The present invention 1002] 1001. The method of claim 10, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [The present invention 1003] 1001. The method of claim 1001, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, twice daily, or three times daily. [The present invention 1004] 1001. The method of claim 1001, wherein said administering significantly reduces the probability of developing liver cancer in the subject. [The present invention 1005] 1001. The method of claim 1001, wherein said administration is oral. [The present invention 1006] 1001. The method of claim 1001, wherein said administration is once daily, twice daily, or three times daily. [The present invention 1007] 1001. The method of claim 1001, wherein said inflammation is characterized by elevated plasma levels of alanine aminotransferase in the subject's plasma compared to levels associated with a normal healthy control population. [The present invention 1008] 1001. The method of claim 1001, wherein the inflammation in said subject is lobular inflammation characterized by multiple concentrations of infiltrating immune cells in a histological sample. [The present invention 1009] 1002. The method of claim 1001, wherein inflammation in said subject is diagnosed from examination of a sample obtained from a liver biopsy. [The present invention 1010] Inflammation in the subject is assessed by ultrasound, computed tomography, magnetic resonance imaging, blood tests, or hyperpolarized 13 1001. The method of claim 1001, wherein the diagnosis is by NMR spectroscopy, including C-NMR spectroscopy. [The present invention 1011] 1001. The method of claim 1001, wherein said treatment significantly reduces the subject's alanine aminotransferase plasma levels. [The present invention 1012] The method of claim 1001, wherein said subject is also treated with an effective amount of an anti-NASH agent. [The present invention 1013] The method of claim 1033, wherein the anti-NASH agent comprises an FXR agonist, a LOXL2 inhibitor, a caspase protease inhibitor, cysteamine bitartrate, a galectin-3 inhibitor, a CCR2 and CCR5 pathway inhibitor, a cysteine-depleting agent, an SGLT-2 inhibitor, GLP-1, a bile acid, a synthetic fatty acid and a conjugate of a bile acid, a sirtuin stimulator, an immunomodulator, or a PPAR agonist. [The present invention 1014] 1002. The method of claim 1001, further comprising the step of treating insulin resistance in said subject by administering to said subject an effective amount of an agent that increases insulin sensitivity in said subject. [The present invention 1015] 1015. The method of claim 1014, wherein said agent comprises metformin, a thiazolidinedione, pioglitazone, or rosiglitazone. [The present invention 1016] 1001. The method of claim 1001, further comprising the step of treating high cholesterol and / or high triglycerides in said subject by administering to said subject an effective amount of an agent that lowers cholesterol and / or high triglycerides in said subject. [The present invention 1017] 1016. The method of claim 1016, wherein the agent comprises a statin, a bile acid sequestrant, a cholesterol absorption inhibitor, a fibric acid derivative, or nicotinic acid. [The present invention 1018] 1001. The method of claim 1001, wherein said subject is also administered an effective amount of a CYP3A4 inhibitor. [The present invention 1019] The method of claim 1018, wherein the CYP3A4 inhibitor comprises ritonavir or cobicistat. [The present invention 1020] The method of claim 1001, wherein said subject is also treated with an effective amount of R-chloroquine. [The present invention 1021] 1001. The method of claim 1001, wherein said subject is also treated with an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [The present invention 1022] administering to a subject an effective amount of a deuterated analog of clemizole or a clemizole metabolite; A method for treating liver inflammation in a subject suffering from non-alcoholic steatohepatitis in need of such treatment. [The present invention 1023] The method of claim 1022, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [The present invention 1024] 1022. The method of claim 1022, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, twice daily, or three times daily. [The present invention 1025] The method of claim 1022, wherein said administration significantly reduces the probability of developing liver cancer in the subject. [The present invention 1026] 1023. The method of claim 1022, wherein said administration is oral. [The present invention 1027] 1023. The method of claim 1022, wherein said administration is once daily, twice daily, or three times daily. [The present invention 1028] 1023. The method of claim 1022, wherein said inflammation is characterized by elevated plasma levels of alanine aminotransferase in the subject's plasma compared to levels associated with a normal healthy control population. [The present invention 1029] The method of claim 1022, wherein the inflammation in said subject is lobular inflammation characterized by multiple concentrations of infiltrating immune cells in a histological sample. [The present invention 1030] The method of claim 1022, wherein inflammation in said subject is diagnosed from examination of a sample obtained from a liver biopsy. [The present invention 1031] Inflammation in the subject is assessed by ultrasound, computed tomography, magnetic resonance imaging, blood tests, or hyperpolarized 13 The method of claim 1022, wherein the diagnosis is by NMR spectroscopy, including C-NMR spectroscopy. [The present invention 1032] The method of claim 1022, wherein said treatment significantly reduces the subject's plasma levels of alanine aminotransferase. [The present invention 1033] The method of claim 1022, wherein said subject is also treated with an effective amount of an anti-NASH agent. [The present invention 1034] The method of claim 1033, wherein the anti-NASH agent comprises an FXR agonist, a LOXL2 inhibitor, a caspase protease inhibitor, cysteamine bitartrate, a galectin-3 inhibitor, a CCR2 and CCR5 pathway inhibitor, a cysteine-depleting agent, an SGLT-2 inhibitor, GLP-1, a bile acid, a synthetic fatty acid and a conjugate of a bile acid, a sirtuin stimulator, an immunomodulator, or a PPAR agonist. [This invention 1035] The method of claim 1022, further comprising treating insulin resistance in the subject by administering to the subject an effective amount of an agent that increases insulin sensitivity in the subject. [The present invention 1036] 1035. The method of claim 1035, wherein said agent comprises metformin, a thiazolidinedione, pioglitazone, or rosiglitazone. [This invention 1037] The method of claim 1022, further comprising treating high cholesterol and / or high triglycerides in the subject by administering to the subject an effective amount of an agent that lowers cholesterol and / or high triglycerides in the subject. [The present invention 1038] 1037. The method of claim 1037, wherein the agent comprises a statin, a bile acid sequestrant, a cholesterol absorption inhibitor, a fibric acid derivative, or nicotinic acid. [This invention 1039] The method of claim 1022, wherein the subject is also administered an effective amount of a CYP3A4 inhibitor. [The present invention 1040] The method of claim 1039, wherein the CYP3A4 inhibitor comprises ritonavir or cobicistat. [The present invention 1041] The method of claim 1022, wherein said subject is also treated with an effective amount of R-chloroquine. [The present invention 1042] The method of claim 1022, wherein the subject is also treated with an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [This invention 1043] 1. A method for treating liver inflammation in a subject suffering from nonalcoholic steatohepatitis in need of treatment, comprising administering to the subject an effective amount of R-chloroquine. [This invention 1044] The method of claim 1043, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1045] 1043. The method of claim 10, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, every other day, or weekly. [The present invention 1046] The method of claim 1043, which also includes a double loading dose daily for the first two days of treatment. [This invention 1047] The method of claim 1043, wherein said administering significantly reduces the probability of developing liver cancer in the subject. [This invention 1048] 1044. The method of claim 1043, wherein said administration is oral. [This invention 1049] 1044. The method of claim 1043, wherein said administration is daily, every other day, or weekly. [The present invention 1050] 1043. The method of claim 1043, wherein said inflammation is characterized by elevated plasma levels of alanine aminotransferase in the subject's plasma compared to levels associated with a normal healthy control population. [This invention 1051] The method of claim 1043, wherein the inflammation in said subject is lobular inflammation characterized by multiple concentrations of infiltrating immune cells in a histological sample. [This invention 1052] The method of claim 1043, wherein inflammation in said subject is diagnosed from examination of a sample obtained from a liver biopsy. [This invention 1053] Inflammation in the subject is assessed by ultrasound, computed tomography, magnetic resonance imaging, blood tests, or hyperpolarized 13 The method of claim 1043, wherein the method is diagnosed by NMR spectroscopy, including C-NMR spectroscopy. [This invention 1054] The method of claim 1043, wherein said treatment significantly reduces the subject's plasma level of alanine aminotransferase. [This invention 1055] The method of claim 1043, wherein said subject is also treated with an effective amount of an anti-NASH agent. [This invention 1056] The method of the present invention 1055, wherein the anti-NASH agent comprises an FXR agonist, a LOXL2 inhibitor, a caspase protease inhibitor, cysteamine bitartrate, a galectin-3 inhibitor, a CCR2 and CCR5 pathway inhibitor, a cysteine ​​depletor, an SGLT-2 inhibitor, GLP-1, a bile acid, a synthetic fatty acid and bile acid conjugate, a sirtuin stimulator, an immunomodulator, or a PPAR agonist. [This invention 1057] The method of claim 1043, wherein insulin resistance in the subject is also treated by administering to the subject an effective amount of an agent that increases insulin sensitivity in the subject. [This invention 1058] 1057. The method of claim 1057, wherein said agent comprises metformin, a thiazolidinedione, pioglitazone, or rosiglitazone. [This invention 1059] The method of claim 1043, wherein the subject is also treated for high cholesterol and / or high triglycerides, comprising administering to the subject an effective amount of an agent that lowers cholesterol and / or high triglycerides in the subject. [The present invention 1060] 1059. The method of claim 1059, wherein the agent comprises a statin, a bile acid sequestrant, a cholesterol absorption inhibitor, a fibric acid derivative, or nicotinic acid. [The present invention 1061] The method of claim 1043, wherein said subject is also treated with an effective amount of clemizole. [The present invention 1062] The method of claim 1043, wherein said subject is also treated with an effective amount of a deuterated analog of clemizole or a clemizole metabolite. [The present invention 1063] A method for treating liver inflammation in a subject suffering from non-alcoholic steatohepatitis in need of treatment, comprising administering to the subject an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [The present invention 1064] The method of claim 1063, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1065] 1063. The method of claim 1063, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, every other day, or weekly. [The present invention 1066] The method of claim 1063, which also includes a double loading dose daily for the first two days of treatment. [This invention 1067] The method of claim 1063, wherein said administration significantly reduces the probability of developing liver cancer in the subject. [The present invention 1068] 1064. The method of claim 1063, wherein said administration is oral. [The present invention 1069] 1064. The method of claim 1063, wherein said administration is daily, every other day, or weekly. [The present invention 1070] 1063. The method of claim 1063, wherein said inflammation is characterized by elevated plasma levels of alanine aminotransferase in the subject's plasma compared to levels associated with a normal healthy control population. [This invention 1071] The method of claim 1063, wherein the inflammation in the subject is lobular inflammation characterized by multiple concentrations of infiltrating immune cells in a histological sample. [This invention 1072] The method of claim 1063, wherein inflammation in said subject is diagnosed from examination of a sample obtained from a liver biopsy. [This invention 1073] Inflammation in the subject is assessed by ultrasound, computed tomography, magnetic resonance imaging, blood tests, or hyperpolarized 13 The method of claim 1063, wherein the diagnosis is by NMR spectroscopy, including C-NMR spectroscopy. [This invention 1074] The method of claim 1063, wherein said treatment significantly reduces the subject's plasma level of alanine aminotransferase. [This invention 1075] The method of claim 1063, wherein said subject is also treated with an effective amount of an anti-NASH agent. [This invention 1076] The method of the present invention 1075, wherein the anti-NASH agent comprises an FXR agonist, a LOXL2 inhibitor, a caspase protease inhibitor, cysteamine bitartrate, a galectin-3 inhibitor, a CCR2 and CCR5 pathway inhibitor, a cysteine-depleting agent, an SGLT-2 inhibitor, GLP-1, a bile acid, a synthetic fatty acid and a conjugate of a bile acid, a sirtuin stimulator, an immunomodulator, or a PPAR agonist. [This invention 1077] The method of claim 1063, wherein insulin resistance in the subject is also treated by administering to the subject an effective amount of an agent that increases insulin sensitivity in the subject. [This invention 1078] 1077. The method of claim 1077, wherein said agent comprises metformin, a thiazolidinedione, pioglitazone, or rosiglitazone. [This invention 1079] The method of claim 1063, wherein the subject is also treated for high cholesterol and / or high triglycerides, comprising administering to the subject an effective amount of an agent that lowers cholesterol and / or high triglycerides in the subject. [The present invention 1080] 1079. The method of claim 1079, wherein the agent comprises a statin, a bile acid sequestrant, a cholesterol absorption inhibitor, a fibric acid derivative, or nicotinic acid. [This invention 1081] The method of claim 1063, wherein said subject is also treated with an effective amount of clemizole. [This invention 1082] The method of claim 1063, wherein said subject is also treated with an effective amount of a deuterated analog of clemizole or a clemizole metabolite. [This invention 1083] A method for treating a subject suffering from liver cancer by administering to the subject an effective amount of clemizole. [This invention 1084] The method of claim 1083, wherein the liver cancer is hepatocellular carcinoma. [This invention 1085] The method of claim 1083, wherein the subject has been diagnosed with nonalcoholic steatohepatitis. [The present invention 1086] The method of claim 1083, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1087] 1083. The method of claim 1083, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered once daily, twice daily, or three times daily. [This invention 1088] 1083. The method of claim 1083, wherein said administration is once daily, twice daily, or three times daily. [This invention 1089] 1083. The method of claim 1083, wherein said administration is oral. [The present invention 1090] The method of claim 1083, wherein the result of said treatment is shrinking the tumor, inhibiting tumor growth, increasing the time to tumor progression, extending the disease-free survival of the subject, reducing metastasis, increasing the progression-free survival of the subject, or increasing the overall survival of the subject. [This invention 1091] The method of claim 1083, further comprising the step of introducing a chemotherapeutic agent into said subject. [This invention 1092] The method of claim 1083, wherein the subject is a human. [This invention 1093] A method for treating a subject suffering from liver cancer by administering to the subject an effective amount of a deuterated analog of clemizole or a clemizole metabolite. [This invention 1094] The method of claim 1093, wherein the liver cancer is hepatocellular carcinoma. [This invention 1095] The method of claim 1093, wherein the subject has been diagnosed with non-alcoholic steatohepatitis. [This invention 1096] The method of claim 1093, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1097] 1093. The method of claim 1093, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered once daily, twice daily, or three times daily. [This invention 1098] 1093. The method of claim 1093, wherein said administration is once daily, twice daily, or three times daily. [This invention 1099] 1093. The method of claim 1093, wherein said administration is oral. [The present invention 1100] The method of claim 1093, wherein the result of said treatment is shrinking the tumor, inhibiting tumor growth, increasing the time to tumor progression, extending the disease-free survival of the subject, reducing metastasis, increasing the progression-free survival of the subject, or increasing the overall survival of the subject. [The present invention 1101] The method of claim 1093, further comprising the step of introducing a chemotherapeutic agent into said subject. [The present invention 1102] The method of claim 1093, wherein the subject is a human. [The present invention 1103] administering to a subject an effective amount of a pharmaceutical composition comprising clemizole; A method for reducing the number of tumor cells in a subject suffering from liver cancer. [The present invention 1104] The method of claim 1103, wherein said administration shrinks said tumor, inhibits growth of said tumor, increases the time to progression of said tumor, extends the disease-free survival of said subject, reduces metastasis, increases the progression-free survival of said subject, or increases the overall survival of said subject. [This invention 1105] The method of claim 1103, further comprising the step of introducing a chemotherapeutic agent into said subject. [The present invention 1106] 1104. The method of claim 1103, wherein said subject is a human. [This invention 1107] The method of claim 1103, wherein the clemizole is modified so that it is activated after administration to a subject. [This invention 1108] administering to a subject an effective amount of a pharmaceutical composition comprising a deuterated analog of clemizole or a clemizole metabolite; A method for reducing the number of tumor cells in a subject suffering from liver cancer. [This invention 1109] The method of claim 1108, wherein said administration shrinks said tumor, inhibits growth of said tumor, increases the time to progression of said tumor, extends the disease-free survival of said subject, reduces metastasis, increases the progression-free survival of said subject, or increases the overall survival of said subject. [The present invention 1110] The method of claim 1108, further comprising the step of introducing a chemotherapeutic agent into said subject. [The present invention 1111] 1108. The method of claim 1108, wherein the subject is a human. [The present invention 1112] The method of claim 1108, wherein the clemizole is modified so that it is activated after administration to a subject. [The present invention 1113] administering to the subject a pharmaceutical composition further comprising an effective amount of clemizole. A method for significantly reducing the probability of developing liver cancer in a subject at risk of developing liver cancer. [This invention 1114] The method of claim 1113, wherein said subject has been diagnosed with non-alcoholic steatohepatitis. [This invention 1115] The method of claim 1113, wherein said subject has been diagnosed with hepatitis C. [The present invention 1116] The method of claim 1113, wherein said subject has been diagnosed with hepatitis B. [This invention 1117] 1114. The method of claim 1113, wherein said subject is suffering from cirrhosis. [This invention 1118] The method of claim 1113, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1119] 1113. The method of claim 1113, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, twice daily, or three times daily. [The present invention 1120] 1114. The method of claim 1113, wherein said administration is oral. [This invention 1121] 1114. The method of claim 1113, wherein said administration is once daily, twice daily, or three times daily. [This invention 1122] The method of claim 1113, wherein said treatment significantly reduces the subject's plasma level of alanine aminotransferase. [This invention 1123] The method of claim 1113, wherein said subject is also treated with an effective amount of an anti-NASH agent. [This invention 1124] The method of claim 1123, wherein the anti-NASH agent comprises an FXR agonist, a LOXL2 inhibitor, a caspase protease inhibitor, cysteamine bitartrate, a galectin-3 inhibitor, a CCR2 and CCR5 pathway inhibitor, a cysteine-depleting agent, an SGLT-2 inhibitor, GLP-1, a bile acid, a synthetic fatty acid and a conjugate of a bile acid, a sirtuin stimulator, an immunomodulator, or a PPAR agonist. [Invention 1125] The method of claim 1113, further comprising treating insulin resistance in said subject by administering to said subject an effective amount of an agent that increases insulin sensitivity in said subject. [The present invention 1126] 1125. The method of claim 1125, wherein said agent comprises metformin, a thiazolidinedione, pioglitazone, or rosiglitazone. [This invention 1127] 1113. The method of claim 1113, further comprising treating high cholesterol and / or high triglycerides in the subject by administering to the subject an effective amount of an agent that lowers cholesterol and / or high triglycerides in the subject. [This invention 1128] 1127. The method of claim 1127, wherein said agent comprises a statin, a bile acid sequestrant, a cholesterol absorption inhibitor, a fibric acid derivative, or nicotinic acid. [This invention 1129] The method of claim 1113, further comprising the step of treating the viral infection in the subject by administering to the subject an effective amount of an antiviral drug. [The present invention 1130] 1129. The method of claim 1129, wherein the antiviral agent comprises an interferon, a nucleoside analog, a direct acting antiviral, or other antiviral agent, including interferon alfa-2b, pegylated interferon alfa-2a, entecavir, lamivudine, adefovir, telbivudine, tenofovir, sofosbuvir, ledipasvir, ombitasvir, paritaprevir, ritonavir, dasabuvir, grazoprevir, elbasvir, asunaprevir, declatasvir, or beclabuvir. [This invention 1131] The method of claim 1113, further comprising the step of treating fibrosis in the subject by administering to the subject an effective amount of an anti-fibrotic agent. [This invention 1132] The method of claim 1131, wherein the antifibrotic agent reduces inflammation, decreases collagen 1 synthesis, increases collagen degradation, increases extracellular matrix degradation, inhibits angiotensin converting enzyme, inhibits AT1 receptor, inhibits ET-1 type A receptor, inhibits PPARγ, or inhibits TGF-β. [This invention 1133] The method of claim 1131, wherein the antifibrotic agent comprises corticosteroids, colchicine, pirfenidone, oltipraz, and silymarin. [This invention 1134] The method of claim 1113, wherein said subject is also treated with an effective amount of R-chloroquine. [This invention 1135] The method of claim 1113, wherein said subject is also treated with an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [This invention 1136] administering to the subject a pharmaceutical composition further comprising an effective amount of a deuterated analog of clemizole or a clemizole metabolite; A method for significantly reducing the probability of developing liver cancer in a subject at risk of developing liver cancer. [This invention 1137] The method of claim 1136, wherein said subject has been diagnosed with nonalcoholic steatohepatitis. [This invention 1138] The method of claim 1136, wherein the subject has been diagnosed with hepatitis C. [This invention 1139] The method of claim 1136, wherein said subject has been diagnosed with hepatitis B. [This invention 1140] The method of claim 1136, wherein the subject is suffering from cirrhosis. [This invention 1141] The method of claim 1136, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1142] 1136. The method of claim 1136, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, twice daily, or three times daily. [This invention 1143] 1136. The method of claim 1136, wherein said administration is oral. [This invention 1144] 1136. The method of claim 1136, wherein said administration is once daily, twice daily, or three times daily. [Invention 1145] 1136. The method of claim 1136, wherein said treatment significantly reduces the subject's plasma level of alanine aminotransferase. [Invention 1146] The method of claim 1136, wherein said subject is also treated with an effective amount of an anti-NASH agent. [This invention 1147] 1146. The method of claim 1146, wherein the anti-NASH agent comprises an FXR agonist, a LOXL2 inhibitor, a caspase protease inhibitor, cysteamine bitartrate, a galectin-3 inhibitor, a CCR2 and CCR5 pathway inhibitor, a cysteine-depleting agent, an SGLT-2 inhibitor, GLP-1, a bile acid, a synthetic fatty acid and a conjugate of a bile acid, a sirtuin stimulator, an immunomodulator, or a PPAR agonist. [Invention 1148] The method of claim 1136, further comprising treating insulin resistance in said subject by administering to said subject an effective amount of an agent that increases insulin sensitivity in said subject. [This invention 1149] 1148. The method of claim 1148, wherein said agent comprises metformin, a thiazolidinedione, pioglitazone, or rosiglitazone. [This invention 1150] 1136. The method of claim 1136, further comprising treating high cholesterol and / or high triglycerides in the subject by administering to the subject an effective amount of an agent that lowers cholesterol and / or high triglycerides in the subject. [This invention 1151] 1150. The method of claim 1150, wherein the agent comprises a statin, a bile acid sequestrant, a cholesterol absorption inhibitor, a fibric acid derivative, or nicotinic acid. [This invention 1152] The method of claim 1136, further comprising the step of treating a viral infection in the subject by administering to the subject an effective amount of an antiviral drug. [This invention 1153] 1152. The method of claim 1152, wherein the antiviral agent comprises an interferon, a nucleoside analog, a direct acting antiviral, or other antiviral agent, including interferon alfa-2b, pegylated interferon alfa-2a, entecavir, lamivudine, adefovir, telbivudine, tenofovir, sofosbuvir, ledipasvir, ombitasvir, paritaprevir, ritonavir, dasabuvir, grazoprevir, elbasvir, asunaprevir, declatasvir, or beclabuvir. [This invention 1154] The method of claim 1136, further comprising the step of treating fibrosis in the subject by administering to the subject an effective amount of an anti-fibrotic agent. [This invention 1155] The method of claim 1154, wherein the anti-fibrotic agent reduces inflammation, decreases collagen 1 synthesis, increases collagen degradation, increases extracellular matrix degradation, inhibits angiotensin converting enzyme, inhibits AT1 receptors, inhibits ET-1 type A receptors, inhibits PPARγ, or inhibits TGF-β. [Invention 1156] The method of claim 1154, wherein the antifibrotic agent comprises a corticosteroid, colchicine, pirfenidone, oltipraz, and silymarin. [This invention 1157] The method of claim 1136, wherein said subject is also treated with an effective amount of R-chloroquine. [This invention 1158] The method of claim 1136, wherein said subject is also treated with an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [This invention 1159] administering to a subject an effective amount of clemizole; A method for treating an inflammatory condition in a subject in need thereof. [The present invention 1160] 1159. The method of claim 1159, wherein said inflammatory condition comprises pancreatitis, inflammatory bowel disease, primary sclerosing cholangitis, primary biliary cirrhosis, arthritis, lupus, asthma, psoriasis, allergies, anemia, and fibromyalgia. [This invention 1161] 1159. The method of claim 1159, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1162] 1159. The method of claim 1159, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, twice daily, or three times daily. [This invention 1163] 1159. The method of claim 1159, wherein said administering significantly reduces the probability of developing cancer in the subject. [This invention 1164] 1159. The method of claim 1159, wherein said administration is oral. [Invention 1165] 1159. The method of claim 1159, wherein said administration is once daily, twice daily, or three times daily. [Invention 1166] 1159. The method of claim 1159, wherein said subject is also administered an effective amount of a CYP3A4 inhibitor. [This invention 1167] The method of claim 1166, wherein the CYP3A4 inhibitor comprises ritonavir or cobicistat. [Invention 1168] 1159. The method of claim 1159, wherein said subject is also treated with an effective amount of R-chloroquine. [This invention 1169] 1159. The method of claim 1159, wherein said subject is also treated with an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [This invention 1170] 1159. The method of claim 1159, wherein the subject is a human. [This invention 1171] administering to a subject an effective amount of a deuterated analog of clemizole or a clemizole metabolite; A method for treating an inflammatory condition in a subject in need thereof. [This invention 1172] The method of claim 1171, wherein the inflammatory condition comprises pancreatitis, inflammatory bowel disease, primary sclerosing cholangitis, primary biliary cirrhosis, arthritis, lupus, asthma, psoriasis, allergies, anemia, and fibromyalgia. [This invention 1173] 1171. The method of claim 1171, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1174] 1171. The method of claim 1171, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, twice daily, or three times daily. [This invention 1175] 1171. The method of claim 1171, wherein said administering significantly reduces the probability of developing cancer in the subject. [Invention 1176] 1171. The method of claim 1171, wherein said administration is oral. [This invention 1177] 1171. The method of claim 1171, wherein said administration is once daily, twice daily, or three times daily. [This invention 1178] The method of claim 1171, wherein said subject is also administered an effective amount of a CYP3A4 inhibitor. [This invention 1179] The method of claim 1178, wherein the CYP3A4 inhibitor comprises ritonavir or cobicistat. [This invention 1180] The method of claim 1171, wherein said subject is also treated with an effective amount of R-chloroquine. [This invention 1181] The method of claim 1171, wherein said subject is also treated with an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [This invention 1182] 1171. The method of claim 1171, wherein the subject is a human. [This invention 1183] 1. A method for treating an inflammatory condition in a subject in need thereof, comprising administering to the subject an effective amount of R-chloroquine. [This invention 1184] The method of claim 1183, wherein the inflammatory condition comprises pancreatitis, inflammatory bowel disease, primary sclerosing cholangitis, primary biliary cirrhosis, arthritis, lupus, asthma, psoriasis, allergies, anemia, and fibromyalgia. [This invention 1185] The method of claim 1183, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [Invention 1186] 1183. The method of claim 1183, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, every other day, or weekly. [This invention 1187] The method of claim 1183, wherein said administering significantly reduces the probability of developing cancer in the subject. [This invention 1188] 1183. The method of claim 1183, wherein said administration is oral. [This invention 1189] 1183. The method of claim 1183, wherein said administration is once daily, once every other day, or once weekly. [This invention 1190] 1183. The method of claim 1183, wherein the subject is a human. [This invention 1191] A method for treating an inflammatory condition in a subject in need of treatment, comprising administering to the subject an effective amount of a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite. [This invention 1192] The method of claim 1191, wherein the inflammatory condition comprises pancreatitis, inflammatory bowel disease, primary sclerosing cholangitis, primary biliary cirrhosis, arthritis, lupus, asthma, psoriasis, allergies, anemia, and fibromyalgia. [This invention 1193] The method of claim 1191, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg. [This invention 1194] 1191. The method of claim 1191, wherein said effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, every other day, or weekly. [This invention 1195] 1191. The method of claim 1191, wherein said administering significantly reduces the probability of developing cancer in the subject. [Invention 1196] 1191. The method of claim 1191, wherein said administration is oral. [This invention 1197] 1191. The method of claim 1191, wherein said administration is once daily, once every other day, or once weekly. [This invention 1198] 1191. The method of claim 1191, wherein the subject is a human. [This invention 1199] 1083. The method of claim 1083, wherein said effective amount comprises a dose of 200 mg administered orally three times daily. [The present invention 1200] 1083. The method of claim 1083, wherein said effective amount comprises a dose of 300 mg administered orally three times daily. [The present invention 1201] 1083. The method of claim 1083, wherein said effective amount comprises a dose of 400 mg administered orally three times daily. [This invention 1202] 1083. The method of claim 1083, wherein said effective amount comprises a dose of 500 mg administered orally three times daily. [Brief explanation of the drawings]

[0012] These and other features, aspects, and advantages of the present invention will be better understood with consideration of the following description and accompanying drawings. [Figure 1] A scheme of the synthesis process of R-chloroquine is illustrated. [Figure 2] The chemical structures of clemizole and clemizole metabolites are shown. [Figure 3] 1 illustrates a synthetic scheme for the synthesis of clemizole in accordance with Good Manufacturing Practice (GMP). [Figure 4] The chemical structures of chloroquine metabolites are shown. [Figure 5] The chemical structures of hydroxychloroquine (rac-1) and R-hydroxychloroquine (R)-1 are shown. [Figure 6] 1 illustrates a scheme of the synthesis process for the enantiomers of hydroxychloroquine. [Figure 7] The chemical structures of hydroxychloroquine metabolites are shown. [Figure 8] Representative images of mouse livers are shown demonstrating the suppression of tumor formation in mice treated with clemizole (Compound D). DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description Benefits and Usefulness In short, as described in more detail below, this paper describes compositions and methods for treating non-alcoholic steatohepatitis or reducing the risk or severity of liver cancer using clemizole and / or R-chloroquine.The advantages of this approach include but are not limited to, when patients are treated with clemizole and / or R-chloroquine, compared with patients who are not treated with clemizole and / or R-chloroquine, inflammation in subjects suffering from inflammatory conditions is reduced, inflammation in liver lobule is reduced, liver function is improved, and the risk of developing or progressing liver cancer is reduced.

[0014] definition Terms used in the claims and specification, unless otherwise specified, are defined as set forth below.

[0015] The term "treatment" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a disease state caused by an inflammatory condition of the liver, including prevention, reduction in severity or slowing of progression, remission, or cure.

[0016] The term "in situ" refers to processes that occur in living cells that are grown apart from an organism, for example, in tissue culture.

[0017] The term "in vivo" refers to processes that take place within an organism.

[0018] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0019] The term "effective amount" means an amount sufficient to produce a desired effect, eg, an amount sufficient to prevent or reduce the amount of liver cancer in a subject.

[0020] The term "subject" refers to cells, human and non-human animals.

[0021] The term "therapeutically effective amount" is an amount effective for alleviating the symptoms of a disease. Since prevention can be considered a treatment, a therapeutically effective amount can be a "prophylactically effective amount."

[0022] The term "administration" refers to the introduction of the agent of the present disclosure into a host. One preferred route of administration of the agent is oral administration. Another preferred route is intravenous administration. However, any route of administration can be used, such as topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into cerebrospinal fluid, or instillation into a body compartment.

[0023] The term "vehicle control" refers broadly to any inert medium in which an active ingredient is administered, including, but not limited to, a solvent, carrier, or binder for the active ingredient.

[0024] The term "deuterated analog" refers to a modified form or analog of a compound of the invention in which the compound contains at least one deuterium isotope.

[0025] The term "clemizole metabolites" refers to clemizole metabolite compounds M1, M12, and M14 as illustrated in Figure 1 of the present application.

[0026] The term "R-chloroquine metabolite" refers to a metabolite compound of chloroquine as illustrated in Figure 6 of the present application.

[0027] The term "R-hydroxychloroquine metabolite" refers to a metabolite compound of hydroxychloroquine as illustrated in Figure 7 of the present application.

[0028] The term "anti-NASH agent" refers to a drug or compound used to treat non-alcoholic steatohepatitis or a condition related to non-alcoholic steatohepatitis, and includes FXR agonists (e.g., obeticholic acid and PX-104), LOXL2 inhibitors (e.g., simtuzumab), caspase protease inhibitors (e.g., emricasan and icosapentaenoic acid ethyl ester), cysteamine bitartrate (e.g., Proscysbi), and steroid hormone receptor agonists (e.g., steroid hormone receptor agonists). or RP103), galectin-3 inhibitors (e.g., GR-MD-02 and LJPC-1010), CCR2 and CCR5 pathway inhibitors (e.g., Cenicriviroc), PPAR agonists (e.g., GFT505, DUR-928, Saroglitazar, and Pioglitazone), cysteine ​​depleting agents (e.g., RP103), SGLT-2 inhibitors (e.g., Remogliflozin etabonate), etabonate), GLP-1 (e.g., liraglutide), bile acids (ursodeoxycholic acid), conjugates of synthetic fatty acids and bile acids (e.g., Aramchol), sirtuin stimulators (e.g., MB12066), apoptosis signal-regulating kinase 1 (ASK1) inhibitors (e.g., GS-4997), and immunomodulators (e.g., IMM124E).

[0029] As used herein, the term " inflammatory condition " refers to the medical problem that is directly caused by inflammatory cytokines or cells involved in inflammation.Inflammatory condition includes but is not limited to: non-alcoholic fatty liver disease; arthritis, which is caused by inflammatory cytokines to destroy synovial membrane lesions and joint cartilage and bone destruction; renal failure, which is caused by inflammatory cytokines to restrict blood circulation and damage nephrons; lupus, which is caused by inflammatory cytokines to induce autoimmune attack; asthma, which is caused by inflammatory cytokines to block airways; pulmonary arterial hypertension, which is caused by inflammatory cytokines to induce increased pulmonary arterial pressure; psoriasis, which is caused by inflammatory cytokines to induce dermatitis; pancreatitis, which is caused by inflammatory cytokines to induce pancreatic cell damage; allergy, which is caused by inflammatory cytokines to induce autoimmune reaction; fibrosis, which is caused by inflammatory cytokines to cause traumatic tissue; surgical complications, which are caused by inflammatory cytokines to hinder healing; anemia, which is caused by inflammatory cytokines to hinder erythropoietin production; and fibromyalgia, which is caused by inflammatory cytokines to increase in fibromyalgia patients. Other diseases associated with chronic inflammation include cancer, which is caused by chronic inflammation; heart attacks, in which chronic inflammation contributes to atherosclerosis of the coronary arteries; Alzheimer's disease, in which chronic inflammation destroys brain cells; congestive heart failure, in which chronic inflammation causes myocardial wasting; stroke, in which chronic inflammation promotes thromboembolic events; and aortic stenosis, in which chronic inflammation damages the heart valves. Arteriosclerosis, osteoporosis, Parkinson's disease, infections, inflammatory bowel diseases including Crohn's disease and ulcerative colitis, and multiple sclerosis (a classic autoimmune inflammation-related disease) are also associated with inflammation. Some advanced stages of the disease can be life-threatening. Several methods for treating such inflammatory diseases are available; however, the results are usually unsatisfactory, as evidenced by a lack of efficacy and the associated drug-related side effects.

[0030] As used herein, the term "non-alcoholic fatty liver disease activity score" refers to the results of a histological examination of liver tissue when the tissue is examined for steatosis, ballooning of hepatocytes, and / or lobular inflammation.

[0031] As used herein, the term "liver cancer" refers to hyperproliferative diseases of the liver, including, but not limited to, hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma, secondary or metastatic liver cancer, and hepatoblastoma.

[0032] As used herein, the term "chemotherapeutic agent" refers to a compound that may be useful in treating a disease (e.g., cancer). The chemotherapeutic agent of the present invention may include any suitable chemotherapeutic agent or combination of chemotherapeutic agents (e.g., a cocktail). Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents, platinum compounds, antimetabolites, anthracyclines, taxanes, camptothecin, nitrosoureas, EGFR inhibitors, antibiotics, HER2 / neu inhibitors, angiogenesis inhibitors, kinase inhibitors (e.g., sorafenib), proteasome inhibitors, immunotherapy, hormonal therapy, photodynamic therapy, cancer vaccines, histone deacetylase inhibitors, sphingolipid-modifying agents, oligomers, other unclassified chemotherapeutic agents, and combinations thereof. Exemplary chemotherapeutic agents active against cancer also include, but are not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, taxotere, vincristine, vinblastine, etoposide, teniposide, cisplatin, and diethylstilbestrol (DES).

[0033] The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" mean excipients, diluents, carriers, and / or adjuvants that are useful in preparing pharmaceutical compositions and that are generally safe, non-toxic, and not biologically or otherwise harmful, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary and / or human pharmaceutical use. As used herein and in the claims, "pharmaceutically acceptable excipient, diluent, carrier, and / or adjuvant" includes one or more of such excipients, diluents, carriers, and adjuvants.

[0034] "Pharmaceutically acceptable salts" refers to salts which retain the biological effectiveness and optionally other properties of the free base and which are obtained by reaction with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, and citric acid. The term "pharmaceutically acceptable salts" also refers to compounds which can be combined with the free chloroquine base, such as phosphate and diphosphate salts.

[0035] When embodiments of the disclosed agents form salts, these salts are within the scope of the present disclosure. Reference to any agent of the formula herein is understood to include reference to its salts, unless otherwise specified. As used herein, the term "salt" refers to acidic and / or basic salts formed with inorganic and / or organic acids and bases. Furthermore, when an agent contains both a basic moiety and an acidic moiety, zwitterions ("internal salts") may be formed and are included within the scope of the term "salt" as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, for example, in isolation or purification steps that may be used during preparation.

[0036] As used herein, the term "pharmaceutical composition" is intended to encompass compositions suitable for administration to a subject, e.g., a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants that can elicit an undesired response in a subject.

[0037] The term "prodrug" refers to an inactive precursor of an active substance that is converted in vivo to a biologically active form. Compounds of the present invention include modified forms of R-chloroquine and clemizole, deuterated clemizole, or clemizole metabolites that act as prodrugs. Examples of modifications of compounds of the present invention that can be used to produce prodrugs include, but are not limited to, the addition of esters, glycosides (sugar derivatives), or the addition or removal of other non-toxic chemical groups that are altered by enzymes during metabolism in vivo (e.g., phosphorylation, dephosphorylation, dealkylation, dehydroxylation, or modification of sugar derivatives). Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. For example, prodrugs may be bioavailable by oral administration, whereas the parent compound is not. Prodrugs may also have improved solubility in pharmaceutical compositions compared to the parent drug. Prodrugs can be converted to the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis.

[0038] Abbreviation Abbreviations used in this application include the following: It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Compound A = racemic mixture of R-chloroquine and S-chloroquine Compound B = R-chloroquine Compound C=S-chloroquine Compound D = Clemizole ALT = Alanine aminotransferase HE = Hematoxylin and eosin HFD = High fat diet NAFLD = Nonalcoholic fatty liver disease NASH = Non-alcoholic steatohepatitis SD = Standard deviation SPF = Specific pathogen-free STZ = Streptozotocin

[0039] Compounds of the Invention The formula of R-chloroquine and the synthetic process of R-chloroquine are illustrated in Figure 1. Alternatively, R-chloroquine is commercially available and can be purchased from Chemical Entities of Biological Interest (ChEBI (trademark)); (Catalog number, CHEBI:48811).

[0040] The structures of cimetazole and the main human cimetazole metabolites (M1, M6) and rodent cimetazole metabolites (M12, M14) are shown in Figure 2. Cimetazole hydrochloride is commercially available from APExBIO (trademark) (Catalog number A3316).

[0041] In the human liver, clemizole is first converted to intermediate A, which can be oxidized to M1 by several CYP450 enzymes (CYP3A4, CYP2C19, or CYP2D6). In the presence of CYP2C9 or CYP1A2, M2 is produced, but these enzymes cannot produce M1. CYP2C9 appears to be the sole source of the minor metabolite M4 in humans. CYP3A4 is the most abundantly expressed CYP450 enzyme in the human liver and mediates the majority of this drug biotransformation reaction. Ritonavir, an inhibitor of CYP3A4 activity, can inhibit clemizole metabolism in vitro, suggesting that CYP3A4 plays an important role in clemizole metabolism in humans. In contrast, another type of aromatic oxidation reaction (CYP2C-like) generates the predominant metabolites in rodents (M12, M14, and M15) through the major pathway of clemizole metabolism in rodent liver.

[0042] Synthesis of clemizole hydrochloride Clemizole hydrochloride is commercially available from APExBIO™ (catalog number A3316). Figure 3 illustrates a synthetic scheme for producing clemizole in accordance with Good Manufacturing Practice (GMP). In certain embodiments, deuterated analogs of clemizole can be produced by using deuterated pyrrolidine instead of the pyrrolidiane starting material. Pyrrolidine-2,2,5,5-d4 can be purchased from (Copyright) CDN Isotopes, Inc. (Product No. D-5946).

[0043] Fully substituted pyrrolidine, pyrrolidine-2,2,3,3,4,4,5,5-d8, can be purchased commercially from (Copyright) CDN Isotopes, Inc. (Product No. D-3532).

[0044] Synthesis of R-chloroquine As illustrated in Figure 1, (R)-(-)-chloroquine was prepared by condensing (R)-(-)-4-amino-1-(diethylamino)pentane with the known 4,7-dichloroquinoline. (R)-(-)-4-amino-1-(diethylamino)pentane was prepared by resolving the known racemic (4-amino-1-(diethylamino)pentane via salt formation with the known (D)-(-)-mandelic acid, and separating the two enantiomeric (D)-(-)-mandelate salts by crystallization.

[0045] Preparation of (R)-(-)-4-amino-1-(diethylamino)pentane To a solution of (D)-(-)-mandelic acid (100 g, 658 mmol) in 350 mL of ethyl alcohol was slowly added racemic 4-amino-1-(diethylamino)pentane (98 g, 658 mmol). The mixture was seeded with crystals of the (D)-(-)-mandelate salt of the title compound and allowed to stand overnight. The resulting solid was collected by filtration and quickly washed twice with ice-cold ethanol to give the (D)-(-)-mandelate salt of the title compound as white crystals (Crop 1). The mother liquor was concentrated until turbidity was observed and then reheated until a homogeneous solution formed. After seeding and allowing to stand overnight, the resulting solid was collected by filtration and quickly washed twice with ice-cold ethanol to give a second crop of the desired salt as white crystals (Crop 2). These two salt products (Products 1 and 2) were combined to yield a total of 44 g of the (D)-(-)-mandelate salt of the title compound. The above process was repeated four times. The (D)-(-)-mandelate salt of the title compound (total of 150 g) was combined and then dissolved in ethyl alcohol with heating and sonication. After seeding the solution and allowing it to stand overnight, the resulting solid was collected by filtration and quickly washed twice with ice-cold ethanol to yield the (D)-(-)-mandelate salt of the title compound (recrystallized product 1). The mother liquor was concentrated, and the residue was dissolved in ethyl alcohol with heating and sonication. After seeding the solution and allowing it to stand overnight, the resulting solid was collected by filtration to yield a second crop of the (D)-(-)-mandelate salt of the title compound (recrystallized product 2). The mother liquor was concentrated, and the residue was dissolved in ethyl alcohol with heating and sonication. The solution was then seeded and allowed to stand overnight, after which the resulting solid was collected by filtration to give a third crop of the (D)-(-)-mandelate salt of the title compound (Recrystallized Product 3). These three recrystallized products (Recrystallized Products 1, 2, and 3) were combined and dried under reduced pressure to give a total of 100 g of the (D)-(-)-mandelate salt of the title compound, [α] D=-56.2 (1% in H2O). The salt was suspended in dichloromethane and washed three times with 1 M sodium hydroxide solution. The organic layer was dried (Na2SO4) and concentrated under reduced pressure to give the title compound, which was used directly in the next step.

[0046] Preparation of (R)-(-)-chloroquine A mixture of crude (R)-(-)-4-amino-1-(diethylamino)pentane (approximately 45 g, 285 mmol, 1.00 equiv.), 4,7-dichloroquinoline (56 g, 285 mmol, 1.00 equiv.), and phenol (53.6 g, 570 mmol, 2.0 equiv.) obtained above was heated to 120°C for 18 hours, then cooled to room temperature and diluted with dichloromethane. The resulting mixture was washed with 1.5 M sodium hydroxide solution, and the wash was back-extracted with dichloromethane. The combined organic layers were extracted with 1 M hydrochloric acid. The aqueous extract was basified to pH 12 with saturated sodium carbonate solution and extracted with dichloromethane. The organic extract was dried (NaSO) and concentrated under reduced pressure. The residue was then purified by column chromatography (silica gel, eluting with 2.5% 7N NH3 / MeOH) to give 42.8 g of the title compound, [α] D =-101.3 (1% in EtOH).

[0047] This material was converted to its diphosphate salt by heating a solution of 42.8 g (133 mmol) of the title compound in ethyl alcohol to 90°C for 15 minutes and adding two equivalents (314 g, 267 mmol) of 85% phosphoric acid dropwise. The resulting suspension was heated under reflux (90°C) for 1 hour, then cooled to room temperature. The solid was collected by filtration, washed with ethanol and diethyl ether, and dried under reduced pressure to give 68 g of (R)-(-)-chloroquine diphosphate, [α] D =-82.96 (2.1% in H2O).

[0048] TIFF0007810746000001.tif18138

[0049] Synthesis and purification of R-hydroxychloroquine The process for the synthesis and purification of the enantiomers (R) and (S) hydroxychloroquine is fully described in Blaney, P. et al., "A Practical Synthesis of the Enantiomers of Hydroxychloroquine," Tetrahedron: Asymmetry, 1994, pp. 1815-1822, Vol. 5. A summary of this process is included below.

[0050] As shown in Figure 6, the racemic diamine rac-2 is resolved by crystallizing its salt with S(+)-mandelic acid. Subsequent coupling with 4,7-dichloroquinoline results in S(+)-hydroxychloroquine ((S)-1a). Similarly, the opposite enantiomer of mandelic acid is used to obtain (R)-2 and R(-)-hydroxychloroquine ((R)-1a). Resolution of (R)-2 and (S)-2 involves crystallization of their diastereomeric mandelate salts from isopropanol. Seeding the mixture with the pure diastereomer using 0.5 molar equivalents of S(+)-mandelic acid at 45 °C yields 67% of (S)-3 after a single crystallization, with a diastereomeric excess (de) of 92%. Subsequent hydrolysis of (S)-3 or (R)-3 to the corresponding diamine (S)-2 or (R)-2. Ratios and yields are calculated without taking into account the presence of variable amounts (up to 10%) of water in (S)-2 or (R)-2.

[0051] Preparation of S(+)-5-[N-ethyl-N-(2-hydroxyethyl)amino]-2-pentanamine ((S)-2) A solution of rac-2 (200 g, 1.15 mol) in 2-propanol (350 ml) was added to a solution of (+)-mandelic acid (87.4 g, 0.575 mol) in 2-propanol (500 ml). Additional 2-propanol was added to bring the total volume to 900 ml, and the solution was stirred overnight at room temperature. Filtration afforded white crystals (235 g), which were recrystallized twice more from 2-propanol (1800 nJ and 1600 ml, respectively) to give (S)-3 (145.4 g) as white crystals. The solid was suspended in 35% aqueous sodium hydroxide (350 ml) and extracted with tert-butyl methyl ether (5 × 600 ml). These extracts were combined, dried (MgSO), and concentrated to give (S)-2 (55.5 g, 55%) as a colorless oil. TIFF0007810746000002.tif25142C9H 22 HRMS calculated for N2O: 175.181039; found: 175.180403.

[0052] Preparation of R(-)-5-[N-ethyl-N-(2-hydroxyethyl)amino]-2-pentanamine ((R)-2) The mother liquor from the first crystallization was concentrated. The residue was suspended in 35% aqueous sodium hydroxide (250 mL) and extracted with tert-butyl methyl ether (5 × 550 mL). The combined extracts were dried (MgSO4) and concentrated to give a yellow oil (70.6 g). This was redissolved in 2-propanol (200 mL) and added to a solution of (-)-mandelic acid (64.00 g, 0.421 mol) in 2-propanol (300 mL). Additional 2-propanol was added to bring the total volume to 600 mL, and the solution was stirred overnight at room temperature. Filtration afforded white crystals (111 g), which were recrystallized twice more from 2-propanol (1100 mL and 800 mL, respectively) to give (R)-3 (77.2 g) as white crystals. (R)-3 was suspended in 35% aqueous sodium hydroxide (200 ml) and extracted with tert-butyl methyl ether (5 × 400 ml). The extracts were combined, dried (MgSO), and concentrated to give (R)-2 (29.3 g, 29%) as a colorless oil. TIFF0007810746000004.tif18158C9H 22 HRMS (CL ammonia) calculated for N2O: 175.181039, found: 175.180390.

[0053] Preparation of S(+)-hydroxychloroquine ((S)-1a) A mixture of (S)-2 (55.47 g, 0.32 mol), 4,7-dichloroquinoline (63.03 g, 0.32 mol), and diisopropylethylamine (63.9 mL, 0.37 mol) was heated at reflux under a nitrogen atmosphere at 125°C for 4 days. After cooling, the mixture was transferred to a separatory funnel with 1 M aqueous sodium hydroxide (500 mL) and dichloromethane (500 mL). The organic phase was separated, and the aqueous phase was re-extracted with dichloromethane (2 x 500 mL). The combined organic phases were dried (MgSO4), and concentrated to give a yellow oil (116 g). This yellow oil was chromatographed on silica gel in 95:3:2 dichloromethane:triethylamine:methanol to give (S)-1a (73 g, 78%) as a pale yellow oil. Alternatively, the crude product was chromatographed on alumina in 2:2:1 acetone:hexane:methanol to give (S)-1a as a colorless oil. TIFF0007810746000005.tif61149C 18 H 26 HRMS calculated for ClNO: 335.176440, found: 335.175518.

[0054] Preparation of R(-hydroxychloroquine ((R)-1a) A mixture of (R)-2 (29.34 g, 0.168 mol), 4,7-dichloroquinoline (33.34 g, 0.168 mol), and diisopropylethylamine (33.8 ml, 0.194 mol) was heated at 135 °C under reflux under a nitrogen atmosphere for 3 days. Workup and purification as described for (S)-1a gave (R)-1a (39.8 g, 84%) as a pale yellow oil. Alternative purification procedure: Crude (R)-1a (18.7 g) was dissolved in hydrochloric acid (1 M, 50 ml) and washed with ethyl acetate (2 × 50 ml) to remove 2,7-dichloroquinoline. After neutralization to pH 7.5 with 1 M aqueous sodium hydroxide, the aqueous phase was washed again with ethyl acetate (2 × 50 ml) and then stirred overnight with activated carbon. After filtering through Celite, the mixture was basified to pH 12 and extracted with ethyl acetate (4 x 50 ml), and these extracts were combined, dried (MgSO), and concentrated to give (R)-1a as a pale yellow oil (17.2 g). TIFF0007810746000006.tif34147MS (thermal spray) 338, 336 ([MH]+); 247 (100%, [M-EtNHCH2CH2OH] + ), major fragment ion at 158.

[0055] Procedure for analyzing the enantiomeric purity of (R)-2 and (S)-2 Completely resolved (R)-2 or (S)-2 [α] D is approximately 6. A reliable method for determining the enantiomeric purity is the separation of the diastereomeric derivatives of (R)-2 and (S)-2. 1 Using H-NMR, the addition of 1 molar equivalent of (R)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) to a chloroform solution of (R)-2 and (S)-2 resulted in the formation of the H-NMR spectrum of the two diastereomers. a The resonance linewidth due to H broadens and shifts to higher frequencies, while excess MTPA forms diastereomeric salts. In these salts, H a and H b In the latter example, the resonance due to the H bThis technique allows the detection of as little as 1% of the minor enantiomer. Monoprotonation gives rise to a species that exists in a pseudocyclic form, whereas the diprotonated species is suggested to exist in an acyclic form. Similarly, the diastereoisomeric camphorsulfonamides 1In the H NMR spectrum, the resonances due to the terminal methyl groups of the diamine moiety are completely resolved, even at low field strengths. Conversion of the resolved diamines (R)-2 and (S)-2 to the hydroxychloroquine enantiomers (R)-1a and (S)-1a requires heating (R)-2 and (S)-2 with 4,7-dichloroquinoline in the presence of diisopropylethylamine. The optimum conditions for these two enantiomers are different: at 135 °C, (S)-2 consistently tends to decompose more readily than (R)-2, resulting in a correspondingly less clean conversion. Large-scale purification of (R)-1a and (S)-1a is achieved by acid-base extraction. Excess dichloroquinoline is removed from the aqueous phase below pH 5, and remaining impurities are removed by further extraction between pH 7 and pH 8, followed by treatment of the aqueous phase with charcoal to remove traces of dark material. Pure hydroxychloroquine is extracted at a pH above 8 (most conveniently about pH 12). Both (R)-1a and (S)-1a are oils that require protection from light during storage or turn yellow. After purification, the enantiomers (R)-1a and (S)-1a are converted to the bis(dihydrogen phosphate) salts ((R)-b and ((S)-1b) by treatment with phosphoric acid (2 molar equivalents). Any triethylamine remaining from the preceding chromatography step is removed by trituration with acetone, leaving a deliquescent hydrate. Phosphoric acid (19.7 ml, 0.29 mol) was added to (S)-1a (43.4 g, 0.144 mol) while cooling with ice to moderate the reaction. The resulting gum was then dissolved in acetone. After trituration under acetone, the resulting deliquescent solid was immediately filtered, immediately suspended in fresh acetone (200 ml), and then stirred overnight. Dehydration by heating in ethanolic suspension gave a friable white solid. Rapid filtration gave a white powder, which was immediately transferred to a flask containing ethanol (200 ml). The resulting suspension was refluxed for 4 days, then filtered, and the solid was washed with ethanol. After drying under vacuum to constant weight, the yield of (S)-1b was 52.6 g (69%).(R)-1a was converted to the bis(dihydrogen phosphate) salt in a similar manner. Both the anhydrous sample and the monohydrate melt at 192 °C, substantially higher than the reported melting point of 168-170 °C for the racemate. The enantiomers undergo substantial and reproducible rotation, and as little as 0.5% of the minor enantiomer can be detected by HPLC using a chiral AGP stationary phase. Therefore, both polarimetry and HPLC are suitable for measuring the optical purity of enantiomers. Hydroxychloroquine sulfate is also commercially available and can be purchased from 3B Scientific Corporation™ (catalog number DR001622).

[0056] How to use The present invention also encompasses methods for treating inflammatory conditions, steatohepatitis, and liver cancer associated with inflammation, comprising administering a therapeutically effective amount of clemizole and / or R-chloroquine to a subject in need thereof.

[0057] The present invention also provides a method for treating inflammatory conditions, comprising administering to a subject one or more compounds or a composition comprising one or more compounds of the present invention and a pharmaceutically acceptable vehicle.The present invention also provides a method for treating or preventing inflammatory conditions, in combination with anti-inflammatory therapeutic agents currently used in clinical practice.Examples of anti-inflammatory therapeutic agents include, but are not limited to, nonsteroidal anti-inflammatory drugs such as aspirin, ibuprofen, and naproxen, corticosteroids, anti-inflammatory bioactive compounds such as plumbagin, or immunoselective anti-inflammatory derivatives.

[0058] The present invention provides a method for treating non-alcoholic steatohepatitis, comprising administering to a subject one or more compounds or a composition comprising one or more compounds of the present invention and a pharmaceutically acceptable vehicle. As used herein, the term "non-alcoholic steatohepatitis" refers to a liver disease characterized by liver inflammation accompanied by fat accumulation in the liver. The present invention also provides a method for treating non-alcoholic steatohepatitis in combination with a therapeutic agent currently used in clinical practice to treat conditions commonly associated with non-alcoholic steatohepatitis, such as metabolic syndrome and / or diabetes mellitus. Non-limiting examples of therapeutic agents for metabolic syndrome and / or diabetes mellitus include therapeutic agents for lowering insulin resistance, cholesterol, and triglycerides. Examples of therapeutic agents for lowering insulin resistance include, but are not limited to, metformin, thiazolidinediones, pioglitazone, and rosiglitazone. Examples of therapeutic agents for hypercholesterolemia include, but are not limited to, statins, bile acid sequestrants, cholesterol absorption inhibitors, fibric acid derivatives, or nicotinic acid.

[0059] The present invention provides a method for treating or preventing liver cancer, comprising administering to subject one or more compounds or the composition comprising one or more compounds of the present invention and pharmaceutically acceptable vehicle.As used herein, the term " liver cancer " refers to the hyperproliferative disease of the liver, including but not limited to hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma, secondary liver cancer or metastatic liver cancer and hepatoblastoma.The present invention also provides a method for treating or preventing liver cancer, in combination with the therapeutic agent currently used in clinical practice for treating or preventing liver cancer, including but not limited to chemotherapeutic agents.

[0060] Pharmaceutical compositions of the present invention The clemizole and R-chloroquine of the present invention can be formulated into pharmaceutical compositions. These compositions may contain, in addition to clemizole and / or R-chloroquine, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal.

[0061] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions generally contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included.

[0062] For intravenous, cutaneous, or subcutaneous injection, or injection into the site of pain, the active ingredient is in the form of an orally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability.Those skilled in the art can easily prepare appropriate solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0063] Useful small molecule compounds according to the present invention are administered to an individual preferably in a "therapeutically effective amount" or a "prophylactically effective amount" (as the case may be, although prophylaxis can be considered a treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the aforementioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0064] The compositions may be administered alone or in combination with other therapeutic agents, simultaneously or sequentially, depending on the condition being treated. [Example]

[0065] The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be taken into account.

[0066] Unless otherwise specified, the practice of the present invention employs conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, which are within the skill of one in the art. Such techniques are fully explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0067] Any terms not directly defined herein should be understood to have the meaning commonly associated with them as understood within the technical field of the present invention. Some terms are discussed herein to provide additional guidance to practitioners in describing the compositions, devices, methods, etc. of the present invention, as well as methods for making or using them. It will be recognized that the same thing may be described in multiple ways. Thus, alternative terminology and synonyms may be used for any one or more of the terms discussed herein. Whether a term is detailed or discussed herein should not be considered critical. Some synonyms or alternative methods, materials, etc. are provided. The description of one or a few synonyms or equivalents does not exclude the use of other synonyms or equivalents unless clearly stated otherwise. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the present invention.

[0068] method Induction of nonalcoholic steatohepatitis NASH was induced in 55 male mice by subcutaneous injection of 200 μg streptozotocin (STZ, Sigma-Aldrich, USA) solution on day 2 of age, followed by feeding them a high-fat diet (HFD, 57 kcal% fat, catalog number: HFD32, CLEA Japan, Inc., Japan) at 4 weeks of age. Mice with NASH induced by this method are hereafter referred to as NASH mice.

[0069] Pharmaceutical Compositions and Drug Administration Routes Compounds A, B, C, D, and vehicle were administered orally at a volume of 5 mL / kg. Compound A contained a racemic mixture of R-chloroquine and S-chloroquine.

[0070] Compound B contained R-chloroquine. Compound C contained S-chloroquine.

[0071] Compound D contained clemizole. All test compounds were weighed and dissolved in vehicle (5% DMSO / water).

[0072] Treatment Dose of Drug Compounds A, B, and C were administered orally once daily at a dose of 258 mg / kg for the first two days and 129 mg / kg thereafter. Compound D was administered orally twice daily at a dose of 89 mg / kg. Table 1 below summarizes the treatment schedule.

[0073] Table 1: Treatment schedule for each test group TIFF0007810746000007.tif100148 * 258 mg / kg: First 2 days only.

[0074] animal C57BL / 6 mice (female, 14 days pregnant) were obtained from Japan SLC, Inc. (Shizuoka, Japan). All animals used in this study were housed and cared for in accordance with the Japanese Pharmacological Society's guidelines for animal use.

[0075] Plasma sampling and measurement of whole blood and plasma biochemical properties Non-fasting blood samples were collected by submandibular withdrawal into polypropylene tubes containing an anticoagulant (Novoheparin, Mochida Pharmaceutical, Japan) at 6 weeks of age (before dosing), 7 weeks of age, and 8 weeks of age. The collected blood samples were centrifuged, and the supernatant was collected as heparinized plasma.

[0076] Non-fasting blood glucose was measured in whole blood using a LIFE CHECK (EIDIA Co. Ltd., Japan). Plasma ALT was measured using a FUJI DRI-CHEM 7000 (Fujifilm Corporation, Japan).

[0077] Histopathological analysis For HE staining, sections were cut from paraffin blocks of liver tissue prefixed in Bouin's solution and stained with Lillie-Meyer hematoxylin (Muto Pure Chemicals Co., Ltd., Japan) and eosin solution (Wako Pure Chemical Industries). NAFLD activity scores (NAS) were calculated according to Kleiner's criteria (Kleiner DE. et al., Hepatology, 2005;41:1313). To visualize collagen deposition, Bouin's fixed liver sections were stained with picrosirius red solution (Waldeck, Germany).

[0078] To quantitatively analyze the fibrotic area, bright-field images of Sirius Red-stained sections around the central vein of the liver were acquired using a digital camera (DFC280; Leica, Germany) at 200x magnification, and the positive areas in five fields per section were measured using ImageJ software (National Institutes of Health, USA).

[0079] Macroscopic analysis of the liver The number of tumor nodules formed on the liver surface that were visible to the naked eye was measured, and the maximum diameter of tumor nodules formed on the liver surface that were visible to the naked eye was measured.

[0080] statistical tests Statistical analysis was performed using the Bonferroni multiple comparison test in GraphPad Prism 4 (GraphPad Software Inc., USA). A p value <0.05 was considered statistically significant.

[0081] Example 1: Administration of S-chloroquine reduces liver and body weight in NASH mice Body weight did not change significantly during the treatment period in any group (Table 2). There was no significant difference in mean body weight between the vehicle and all compound groups. During the treatment period, mice died before reaching week 9 as follows: 1 out of 11 mice died in all groups.

[0082] On the day of sacrifice, the mean body weight was significantly reduced in the Compound C group (Table 2). There was no significant difference in mean body weight on the day of sacrifice between the vehicle group and the Compound A, Compound B, and Compound D groups.

[0083] The mean liver weight was significantly reduced in the Compound C group (Table 2). There was no significant difference in mean liver weight between the vehicle group and the Compound A, Compound B, and Compound D groups on the day of sacrifice. There was no significant difference in the mean ratio of liver weight to body weight between the vehicle group and the Compound A, Compound B, Compound C, and Compound D groups on the day of sacrifice.

[0084] Table 2. Body weight and liver weight of NASH mice TIFF0007810746000008.tif21142

[0085] Example 2: Whole blood biochemical properties of mice treated with chloroquine or clemizole in NASH mice There was no significant difference in whole blood glucose levels between the vehicle group and the Compound A, Compound B, Compound C, and Compound D groups during the study period. Plasma ALT levels were measured at the time of sacrifice in the vehicle group and the Compound A, Compound B, Compound C, and Compound D groups (Table 3).

[0086] Table 3. ALT levels in NASH mice treated with test compounds TIFF0007810746000009.tif13142

[0087] Example 3: Decreased NAFLD activity scores are observed in NASH mice treated with R-chloroquine or clemizole Hematoxylin and eosin staining was performed on liver tissue sections obtained from mice treated with the test compound, and the NAFLD activity score was calculated (Table 4).Compound B and Compound D groups showed a significant decrease in NAS compared with the vehicle group.

[0088] Table 4. NAFLD activity scores of NASH mice treated with test compounds TIFF0007810746000010.tif81143

[0089] Example 4: Liver fibrosis area is not affected by administration of test compound in NASH mice Liver Sirius red staining was performed on liver sections of NASH mice treated with test compounds (Table 5). There was no significant difference in fibrotic area at the time of sacrifice between the vehicle group and compound A, compound B, compound C, and compound D groups.

[0090] Table 5: Fibrotic areas in the liver of NASH mice administered the test compound TIFF0007810746000011.tif13142

[0091] Example 5: Body weight and liver weight of mice with NASH-induced hepatocellular carcinoma administered with test compounds The body weight of any test group did not change significantly during the treatment period (Table 6). There was no significant difference in mean body weight between the vehicle group and all compound groups (Table 6). During the treatment period, mice died before reaching the 18th week as follows: in the vehicle and Compound D groups, 1 out of 4 mice died; in the Compound B and Compound C groups, 2 out of 4 mice died; and in the Compound A group, 3 out of 4 mice died. The mean values ​​± SD are shown in Table 6.

[0092] Table 6. Organ and liver weight to body weight ratios of NASH-induced HCC mice treated with test compounds TIFF0007810746000012.tif21142

[0093] Example 6: Administration of clemizole to mice bearing hepatocellular carcinoma reduces liver tumor diameter and the number of tumor nodules The number of tumor nodules formed on the liver surface that were visible to the naked eye was measured. The maximum diameter of the tumor nodules formed on the liver surface that were visible to the naked eye was measured (Table 7). All surviving mice in the vehicle group (n=3) showed visible tumor nodules on the liver surface. Two of the three surviving mice in the Compound D (clemizole) group were observed to have no visible tumor nodules on the liver surface (Figure 8). The mean values ​​± SD are shown in Table 7.

[0094] Table 7. Maximum diameter of visible tumor nodules in HCC mice treated with test compounds. TIFF0007810746000013.tif17142

[0095] Example 7: Human patients with hepatocellular carcinoma (HCC) treated with clemizole A Phase IIa open-label pilot study is underway to test the safety, tolerability, pharmacokinetic, and pharmacodynamic activity of 200 mg, 400 mg, and 500 mg clemizole hydrochloride given orally three times daily in subjects with hepatocellular carcinoma (HCC) who are awaiting liver transplantation or have unresectable disease. The completed study will include up to 40 patients. Initial clinical results from two patients with HCC who received 200 mg clemizole for 3 or 5 months are described below.

[0096] Summary of Patient #1: Patient #1, a 70-year-old man, was diagnosed with HCC in the setting of chronic hepatitis B-induced cirrhosis. The diagnosis was confirmed by dynamic liver CT. Patient #1 underwent radiofrequency ablation therapy for HCC in 2011, but his disease progressed after treatment. Patient #1 also underwent three courses of transarterial chemoembolization (TACE) in April-May 2015 and June 2015. Patient #1 experienced stable disease for approximately 5 months before progression of HCC. The patient then began clemizole treatment, receiving 200 mg of clemizole orally three times daily. After 3 months of clemizole treatment, the patient underwent follow-up dynamic liver CT imaging, which revealed that the HCC remained unchanged, despite a significant increase in tumor size that would have been expected without any treatment. At this time, the patient was in good health and had no complaints or side effects.

[0097] Summary of Patient #2: Patient No. 2 was a 77-year-old man diagnosed with multifocal HCC in the setting of chronic hepatitis B-induced cirrhosis. The diagnosis was confirmed by dynamic liver magnetic resonance imaging. Patient No. 2 failed and / or was unable to tolerate sorafenib treatment and showed disease progression after treatment. Patient No. 2 then began clemizole treatment, receiving 200 mg of clemizole orally three times daily. After 5 months of clemizole treatment, the patient underwent follow-up dynamic liver magnetic resonance imaging, which showed that the HCC remained unchanged, despite a significant increase in tumor size that would have been expected without any treatment. At this time, the patient was in excellent condition and had no complaints or side effects.

[0098] Although only a small number of patients have been treated to date, the efficacy and tolerability observed with the low dose of clemizole used (i.e., 200 mg versus a maximum of 500 mg in subsequent patients) is very exciting in itself, especially when compared with sorafenib, the only approved treatment for HCC. In a phase 2 trial of sorafenib (Abou-Alpha et al. Journal Clinical Oncology 2006, 24(26): 4293-4300), the median time to progression (TTP) was 4.2 months. Toxicity included grade 3 / 4 drug-related toxicities such as fatigue (9.5%), diarrhea (8.0%), and hand-foot skin reaction (HST) (5.1%), with diarrhea occurring in over 40%, hand-foot skin reaction occurring in over 30%, and fatigue occurring in 30%. In a phase 3 trial of sorafenib (Llovet et al. NEJM 2008;359:378-90), the median time to radiological progression was 5.5 months in the sorafenib group and 2.8 months in the placebo group. The overall incidence of treatment-related adverse events was 80% in the sorafenib group. These were primarily gastrointestinal, constitutional, or dermatological in nature (grade 1 or 2 severity), as well as hypophosphatemia (11%, grade 3), and thrombocytopenia (4%, grade 3 or 4).

[0099] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail can be made in these embodiments without departing from the spirit and scope of the invention.

[0100] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A pharmaceutical composition for treating liver inflammation in a subject with non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), comprising an effective amount of R-chloroquine, a deuterated analog of R-chloroquine, an R-chloroquine metabolite, R-hydroxychloroquine, a deuterated analog of R-hydroxychloroquine, or an R-hydroxychloroquine metabolite, the R-chloroquine metabolite is selected from desethyl-chloroquine, bisdesethyl-chloroquine, and 7-chloro-4-aminoquinoline; A pharmaceutical composition, wherein the R-hydroxychloroquine metabolite is selected from desethylchloroquine, desethylhydroxychloroquine, and bisdesethylchloroquine.

2. 10. The pharmaceutical composition of claim 1, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the effective amount comprises a dose of 0.5 mg / kg to 50 mg / kg administered daily, every other day, or weekly.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, administered daily as a double loading dose for the first two days of therapy.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, which significantly reduces the probability of developing liver cancer in a subject.

6. The pharmaceutical composition of any one of claims 1 to 5, which is administered orally.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, which is administered daily, every other day, or weekly.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the inflammation in the subject is characterized by elevated plasma levels of alanine aminotransferase in the subject's plasma compared to levels associated with a normal healthy control population.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the inflammation in the subject is lobular inflammation characterized by multiple concentrations of infiltrating immune cells in histological samples.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the inflammation in the subject is diagnosed from examination of a sample obtained from a liver biopsy.

11. The subject's inflammation is assessed by ultrasound, computed tomography, magnetic resonance imaging, blood tests, or hyperpolarized 13 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the composition is diagnosed by NMR spectroscopy, including C-NMR spectroscopy.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, which significantly reduces alanine aminotransferase plasma levels in a subject.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, administered to a subject in combination with an effective amount of an anti-NASH agent.

14. 14. The pharmaceutical composition of claim 13, wherein the anti-NASH agent comprises an FXR agonist, a LOXL2 inhibitor, a caspase protease inhibitor, cysteamine bitartrate, a galectin-3 inhibitor, a CCR2 and CCR5 pathway inhibitor, a cysteine-depleting agent, an SGLT-2 inhibitor, GLP-1, a bile acid, a synthetic fatty acid and bile acid conjugate, a sirtuin stimulator, an immunomodulator, or a PPAR agonist.

15. 15. The pharmaceutical composition of any one of claims 1-14, administered to a subject in combination with an effective amount of an agent that increases insulin sensitivity in the subject, wherein the agent is selected from metformin, a thiazolidinedione, pioglitazone, or rosiglitazone, thereby also treating insulin resistance in the subject.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, administered to a subject in combination with an effective amount of an agent that lowers cholesterol and / or high triglycerides in the subject, wherein the agent is selected from a statin, a bile acid sequestrant, a cholesterol absorption inhibitor, a fibric acid derivative, or nicotinic acid, thereby also treating high cholesterol and / or high triglycerides in the subject.

17. 17. The pharmaceutical composition of any one of claims 1-16, administered to a subject in combination with an effective amount of clemizole, a deuterated analog of clemizole, or a clemizole metabolite, wherein the clemizole metabolite is selected from M1, M2, M3, M4, M5, M6, M7, M8, M10, M11, M12, M13, M14, M15, M16, M17, or M18: where: M1 is or a pharmaceutically acceptable salt thereof; M2 is or a pharmaceutically acceptable salt thereof; M3 and M11 are or a pharmaceutically acceptable salt thereof, wherein In M3, R1 is H; In M11, R1 is OH; M4, M14, and M15 are or a pharmaceutically acceptable salt thereof, wherein In M4, R1 is OH; In M14, R1 is an O-glucuronide; In M15, R1 is O-gluc (lactam); M5, M8, and M17 are or a pharmaceutically acceptable salt thereof, wherein In M5, R1 and R2 are H; In M8, R1 is OH and R2 is H; In M17, R1 is H and R2 is gluc; M6 is or a pharmaceutically acceptable salt thereof; M7, M16, and M18 are or a pharmaceutically acceptable salt thereof, wherein In M7, R1 and R2 are H; In M16, R1 is H and R2 is O-gluc; In M18, R1 is O-gluc and R2 is OH; M10 and M13 are or a pharmaceutically acceptable salt thereof, wherein In M10, R1 is O − ; In M13, R1 is gluc; M12 is or a pharmaceutically acceptable salt thereof; In M1, M2, M3, M4, M5, M6, M7, M8, M10, M11, M12, M13, M14, M15, M16, M17, or M18, R is is.

Images